Magnetic response composite microspheres loaded with stem cell exosomes, and preparation method and application thereof

Abstract

The application discloses a kind of magnetic response composite microspheres loaded stem cell exosome and its preparation method and application, the composite microspheres are biodegradable polymer as matrix, and superparamagnetic iron oxide nanoparticle is enclosed to form magnetic response microsphere, then surface is modified by polydopamine coating and is anchored stem cell-derived exosome.The composite microspheres can be navigated and positioned to the damage site under static magnetic field, and through magnetic mechanical vibration triggered exosome pulse type controllable release under alternating magnetic field, realize positioning-time-active repair, and animal experiment shows that the microsphere can significantly promote nerve function recovery after spinal cord injury.

Description

Technical Field

[0001] This invention relates to a magnetically responsive composite microsphere loaded with stem cell exosomes and its preparation method, and also to the application of the magnetically responsive composite microsphere in the functional repair of central and peripheral nerve injuries. Background Technology

[0002] Central and peripheral nervous system injuries are common and difficult-to-treat diseases in clinical practice, involving multiple pathological processes such as neuronal apoptosis, axonal breakage, demyelination, and glial scar formation. While traditional drug therapy and surgical intervention can alleviate symptoms to some extent, they are insufficient to achieve structural and functional reconstruction of damaged nerve tissue. Stem cell transplantation is considered one of the most promising strategies in the field of nerve repair; however, the low survival rate of transplanted cells in vivo, uncontrollable differentiation, and potential tumorigenic risks severely restrict its clinical translation.

[0003] In recent years, research has revealed that the paracrine effect of stem cells plays a crucial role in tissue repair, and exosomes, as the core carriers of this effect, have attracted widespread attention. Exosomes are lipid bilayer vesicles with a diameter of approximately 40–160 nm secreted by cells, containing abundant bioactive molecules such as proteins, miRNAs, mRNAs, and lipids. Exosomes derived from bone marrow mesenchymal stem cells have been shown to possess multiple biological effects, including anti-inflammatory, anti-apoptotic, pro-angiogenic, and pro-neuroregenerative effects, and can regulate the injury microenvironment towards a repair-friendly direction. However, the short half-life of exosomes, their rapid clearance in vivo, and lack of targeted localization capabilities make it difficult to maintain effective concentrations at the site of injury, severely impacting therapeutic efficacy.

[0004] To address the issue of low exosome delivery efficiency, existing technologies utilize materials such as hydrogels, microspheres, and scaffolds as exosome carriers. For example, CN107582567A discloses an exosome-targeted sustained-release microsphere bioscaffold, its preparation method, and its applications, achieving sustained exosome release through the microsphere structure. CN115040693A discloses a biomaterial containing exosomes derived from CD56+ subcellular populations, using GelMA and decellularized spinal cord matrix as carriers to achieve sustained exosome release. However, in the above-mentioned technical solutions, the release behavior of exosomes relies entirely on the passive degradation of the carrier material, making it impossible to control the release rate as needed according to different stages of damage repair. Furthermore, these carrier systems lack the ability to actively navigate to the injury site, limiting delivery efficiency in deep tissue injury scenarios.

[0005] The application of magnetic nanomaterials in the biomedical field offers a new solution to the aforementioned problems. Superparamagnetic iron oxide nanoparticles can generate directional motion and magnetocaloric effects under the influence of an external magnetic field, and have been widely used in fields such as magnetic resonance imaging, magnetically targeted drug delivery, and magnetocaloric therapy. However, there is currently no technical solution that combines superparamagnetic nanoparticles with exosome-loaded microspheres to achieve precise localization of damaged sites through magnetic field navigation and to trigger on-demand, controllable release of exosomes through alternating magnetic fields. Summary of the Invention

[0006] Purpose of the invention: In view of the shortcomings of existing stem cell exosome delivery systems, which lack active targeting capability and on-demand controllable release function, the purpose of this invention is to provide a magnetically responsive composite microsphere loaded with stem cell exosomes. Another purpose of this invention is to provide a method for preparing the magnetically responsive composite microsphere, and its application in the repair of central and peripheral nerve damage.

[0007] Technical solution: The present invention discloses a magnetically responsive composite microsphere loaded with stem cell exosomes, characterized in that the magnetically responsive composite microsphere loaded with stem cell exosomes comprises a magnetically responsive microsphere containing a biodegradable polymer matrix and superparamagnetic iron oxide nanoparticles dispersed in the biodegradable polymer matrix, the surface of the magnetically responsive microsphere is covered with a polydopamine coating, and stem cell-derived exosomes are anchored on the surface of the polydopamine coating.

[0008] The biodegradable polymer matrix is ​​selected from polylactic-co-glycolic acid copolymer, polycaprolactone, or a blend of polylactic-co-glycolic acid copolymer and polycaprolactone. The molar ratio of lactic acid to glycolic acid in the polylactic-co-glycolic acid copolymer is 50:50–75:25, and the molecular weight is 20,000–80,000 Da. The superparamagnetic iron oxide nanoparticles have a particle size of 8–15 nm, and the mass fraction of superparamagnetic iron oxide nanoparticles in the magnetically responsive microspheres is 10%–25%, with a particle size of 20–80 μm for the magnetically responsive microspheres. The polydopamine coating has a thickness of 5–20 nm and is formed by the self-polymerization of dopamine hydrochloride in a Tris-HCl buffer solution at pH 8.0–8.5. The stem cell-derived exosomes are one or a combination of bone marrow mesenchymal stem cell exosomes or neural stem cell exosomes; when a combination of both is used, the mass ratio of bone marrow mesenchymal stem cell exosomes to neural stem cell exosomes is 2:1–4:1. The loading amount of stem cell-derived exosomes on the surface of magnetically responsive microspheres was 50-200 μg of stem cell-derived exosome protein / mg of magnetically responsive microspheres; the particle size of stem cell-derived exosomes was 40-160 nm, and they expressed CD9, CD63 and CD81 exosome marker proteins.

[0009] The method for preparing magnetically responsive composite microspheres loaded with stem cell exosomes according to the present invention includes the following steps:

[0010] (1) Magnetic responsive microspheres were prepared using a biodegradable polymer matrix and superparamagnetic iron oxide nanoparticles;

[0011] (2) In Tris-HCl buffer, dopamine hydrochloride self-polymerizes on the surface of magnetically responsive microspheres to form a polydopamine coating;

[0012] (3) Stem cell-derived exosomes are anchored to the surface of magnetically responsive microspheres through the synergistic effect of physical adsorption and chemical bonding of polydopamine coating.

[0013] In step (1), the emulsification speed during preparation is 6000~10000 rpm; in step (2), the pH of the ris-HCl buffer is 8.0~8.5, the concentration of dopamine hydrochloride in the Tris-HCl buffer is 0.5 mg / mL-4 mg / mL, and the self-polymerization reaction time is 4-16 h; in step (3), the total protein concentration of exosomes in the stem cell-derived exosome solution is 100~300 μg / mL, the incubation temperature is 4℃, and the incubation time is more than 24 h.

[0014] The present invention also includes the application of the magnetically responsive composite microspheres loaded with stem cell exosomes in the preparation of drugs for the repair of nerve damage.

[0015] Nerve injuries include spinal cord injury, peripheral nerve injury, or traumatic brain injury. In application, the magnetically responsive composite microspheres can be used in combination with protein-based materials or exosomes. The protein-based material is selected from collagen or gelatin, and the exosomes are free exosomes of the same origin as those anchored on the surface of the magnetically responsive composite microspheres. The magnetically responsive composite microspheres are guided to the nerve injury site under a static magnetic field with a strength of 0.3–1.0 T, or an alternating magnetic field is applied to trigger a pulsed release of exosomes. The frequency of the alternating magnetic field is 100–500 kHz, and the field strength is 5–20 kA / m. Each application of the alternating magnetic field lasts 10–30 minutes, and the frequency is 1–3 times daily for 7–28 consecutive days. The medication is administered by injection at a dose of 1–10 mg composite microspheres / kg body weight, using physiological saline or a buffer solution containing 0.5%–2% sodium hyaluronate.

[0016] This invention relates to composite microspheres using a biodegradable polymer matrix and superparamagnetic iron oxide nanoparticles as the magnetically responsive core. A biomimetic dopamine coating anchors stem cell exosomes on the microsphere surface, achieving precise navigation and positioning under an applied static magnetic field and pulsed, controllable release triggered by an alternating magnetic field. This constructs a complete closed loop of localization-timing-active repair, overcoming the technical shortcomings of existing exosome delivery systems, such as passive release and poor targeting. This invention constructs magnetically responsive microsphere carriers by combining superparamagnetic nanoparticles with a biodegradable polymer matrix. Biomimetic dopamine chemically anchors stem cell-derived exosomes on their surface, achieving the dual functions of magnetic field navigation and controllable release triggered by an alternating magnetic field, providing a novel cell-free therapeutic strategy for nerve injury repair. Under the action of an applied alternating magnetic field, the superparamagnetic nanoparticles inside the composite microspheres generate magnetomechanical vibrations, triggering the controllable release of surface-anchored exosomes. This invention also provides the application of the aforementioned magnetically responsive composite microspheres in the preparation of drugs for the functional repair of nerve injuries.

[0017] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: The magnetically responsive composite microspheres provided by this invention endow the microsphere carrier with magnetic field responsiveness through superparamagnetic iron oxide nanoparticles. Under an applied static magnetic field, the microspheres can be guided to precisely locate the injury site, solving the problem of poor targeting in existing exosome delivery systems. Pulsed, on-demand release of exosomes is achieved through a magnetomechanical vibration mechanism triggered by an alternating magnetic field, breaking the limitations of passive degradation and release by traditional carriers. The timing and dosage of exosome release can be flexibly adjusted according to different stages of injury repair. The polydopamine biomimetic coating provides both exosome anchoring points and antioxidant and neuroprotective functions, forming a synergistic repair effect with the exosomes. The dual-source combination strategy of bone marrow mesenchymal stem cell exosomes and neural stem cell exosomes fully leverages the complementary advantages of anti-inflammatory immune regulation and promotion of neural differentiation and regeneration. Experimental results show that in a spinal cord injury animal model, the magnetically responsive composite microspheres combined with an alternating magnetic field treatment regimen of this invention can improve the BBB motor function score by at least 3 points and shorten the latency of motor evoked potentials by more than 25%, significantly better than the control group of simple exosome injection and non-magnetic microsphere-loaded exosomes. The composite microspheres of this invention can be navigated and located to the damaged site under a static magnetic field, and under an alternating magnetic field, they can trigger the pulsed and controllable release of exosomes through magnetomechanical vibration, realizing localization-timed-active repair, which can significantly promote the recovery of nerve function after spinal cord injury. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the preparation process of the magnetically responsive composite microspheres of the present invention;

[0019] Figure 2 Transmission electron microscope image of the superparamagnetic iron oxide nanoparticles prepared in Example 1;

[0020] Figure 3 The XRD pattern of the superparamagnetic iron oxide nanoparticles prepared in Example 1;

[0021] Figure 4 Scanning electron microscope image of the magnetically responsive composite microspheres prepared in Example 2;

[0022] Figure 5 Transmission electron microscope image of the cross-section of the magnetically responsive composite microspheres prepared in Example 2;

[0023] Figure 6 Transmission electron microscope image of bone marrow mesenchymal stem cell exosomes extracted in Example 4;

[0024] Figure 7 This is a confocal laser scanning microscope image of the microspheres loaded with exosomes in Example 5;

[0025] Figure 8 The kinetic curve for the controlled release of exosomes triggered by the alternating magnetic field in Example 6;

[0026] Figure 9 The results of neuronal survival rate detection in each group in the in vitro oxygen-glucose deprivation-reperfusion model of Example 7;

[0027] Figure 10 The curves showing the changes in BBB motor function scores over time in each group of the rat spinal cord injury model in Example 8 are shown.

[0028] Figure 11 Immunofluorescence staining images of the spinal cord tissue of the injured segment in rats of each group 8 weeks after surgery in Example 8. Detailed Implementation

[0029] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings.

[0030] Example 1: Preparation of superparamagnetic iron oxide nanoparticles

[0031] (1) The overall preparation process of magnetically responsive composite microspheres is as follows: Figure 1 As shown, the process mainly includes four stages: synthesis of superparamagnetic nanoparticles, preparation of composite microspheres, modification with polydopamine coating, and exosome anchoring.

[0032] Superparamagnetic iron oxide nanoparticles were prepared by co-precipitation method:

[0033] 2.35 g of ferric chloride hexahydrate and 0.86 g of ferrous chloride tetrahydrate were dissolved in 100 mL of deionized water and stirred thoroughly in an 80°C water bath under nitrogen protection. 25% ammonia was added dropwise to the solution until the pH reached 10.0, and the reaction was continued for 30 min with stirring. After the reaction was complete, the precipitate was collected by magnetic separation using an external permanent magnet and washed three times each with deionized water and anhydrous ethanol to remove unreacted precursors and byproducts. The washed product was dispersed in a solution containing 100 mL of 0.1 mol / L sodium citrate and reacted at 90°C for 60 min to modify the surface with citric acid, imparting good water dispersibility and colloidal stability to the nanoparticles. After magnetic separation, washing, and lyophilization, citric acid-modified superparamagnetic iron oxide nanoparticles were obtained.

[0034] The citric acid-modified superparamagnetic iron oxide nanoparticles prepared in this embodiment were characterized by transmission electron microscopy, and the results are as follows: Figure 2 As shown, the prepared citric acid-modified superparamagnetic iron oxide nanoparticles are spherical with an average particle size of 10-12 nm and a uniform particle size distribution.

[0035] X-ray diffraction analysis was performed on the citric acid-modified superparamagnetic iron oxide nanoparticles prepared in this embodiment, and the results are as follows: Figure 3 As shown in the figure, characteristic diffraction peaks consistent with those of standard iron(III) oxide appeared in the spectrum, confirming that the product has an inverse spinel-type iron(III) oxide crystal structure.

[0036] The citric acid-modified superparamagnetic iron oxide nanoparticles prepared in this embodiment were vibrated. The results of the sample magnetometer measurement showed that the saturation magnetization of the citric acid-modified superparamagnetic iron oxide nanoparticles was 62 emu / g, and the coercivity was close to zero. They have typical superparamagnetic characteristics, that is, no residual magnetism is retained after the external magnetic field is removed. This is crucial for preventing the nanoparticles from agglomerating in vivo.

[0037] Thermogravimetric analysis of the citric acid-modified superparamagnetic iron oxide nanoparticles prepared in this embodiment showed that the mass fraction of the citric acid modification layer was about 8%, indicating that the surface modification layer was complete and appropriate.

[0038] Example 2: Preparation of magnetically responsive composite microspheres

[0039] (1) Polylactic acid-glycolic acid copolymer microspheres loaded with superparamagnetic nanoparticles were prepared by emulsification solvent evaporation method:

[0040] A polylactic acid-glycolic acid copolymer (polylactic acid-glycolic acid copolymer, purchased from Sigma-Aldrich, USA, catalog number P2191) with a molecular weight of approximately 40,000 Da and a lactic acid to glycolic acid molar ratio of 75:25 was selected as the matrix material. 500 mg of the polylactic acid-glycolic acid copolymer was dissolved in 5 mL of dichloromethane, and a dichloromethane suspension containing 100 mg of citric acid-modified superparamagnetic iron oxide nanoparticles prepared in Example 1 (concentration 20 mg / mL) was added. The mixture was ultrasonically dispersed for 15 min to ensure uniform distribution of the citric acid-modified superparamagnetic iron oxide nanoparticles in the polymer solution. The above organic phase was added dropwise to 50 mL of an aqueous phase containing 1% polyvinyl alcohol, and emulsified using a high-speed homogenizer at 8000 rpm for 3 min to form an oil-in-water emulsion. The oil-in-water emulsion was transferred to 400 mL of an aqueous phase containing 0.3% polyvinyl alcohol and stirred continuously at 300 rpm for 4 h at room temperature to allow complete evaporation of the organic solvent. After stirring, the emulsion was further stirred at 3000 rpm. The microspheres were collected by centrifugation at rpm, washed three times with deionized water to remove residual polyvinyl alcohol and free nanoparticles, and then freeze-dried to obtain dry magnetically responsive composite microsphere powder.

[0041] The magnetically responsive composite microsphere powder prepared in this embodiment was characterized by transmission electron microscopy, and the results are as follows: Figure 4 As shown, the prepared magnetically responsive composite microspheres are regular spherical in shape, with a smooth and dense surface and an average particle size of 45 μm.

[0042] like Figure 5 As shown, after embedding the magnetically responsive composite microspheres in epoxy resin and then observing the sections under a transmission electron microscope, it can be seen that the superparamagnetic iron oxide nanoparticles are uniformly dispersed inside the polymer matrix without obvious agglomeration.

[0043] Energy dispersive X-ray spectroscopy analysis of the magnetically responsive composite microsphere powder prepared in this embodiment confirmed the presence of iron element signals inside the microspheres, further verifying the successful encapsulation of nanoparticles.

[0044] Thermogravimetric analysis of the magnetically responsive composite microsphere powder prepared in this embodiment showed that the mass fraction of superparamagnetic nanoparticles in the microspheres was approximately 15%. Measurements using a vibrating sample magnetometer indicated that the saturation magnetization of the composite microspheres was approximately 9.3 emu / g, maintaining superparamagnetic characteristics.

[0045] The magnetically responsive composite microspheres prepared in this embodiment were suspended in phosphate buffer. Under the action of an external permanent magnet (surface field strength 0.5 T), the magnetically responsive composite microspheres could be completely enriched on the magnet side within 60 s. After the magnet was removed, the magnetically responsive composite microspheres could be redispersed by slight oscillation, indicating that they have good magnetic responsiveness and redispersibility.

[0046] (2) In order to investigate the effect of preparation parameters on the performance of microspheres, a series of experiments were conducted by adjusting the amount of citric acid-modified superparamagnetic iron oxide and the emulsification speed according to the preparation process of step (1) of this embodiment.

[0047] The results showed that when the mass fraction of nanoparticles in the microspheres increased from 5% to 25%, the saturation magnetization of the microspheres increased from 3.1 emu / g to 15.6 emu / g, and the magnetic field response time decreased from 180 s to 30 s. Specifically, at mass fractions of 5%, 10%, 15%, 20%, and 25%, the saturation magnetization of the composite microspheres were 3.1, 6.2, 9.3, 12.4, and 15.6 emu / g, respectively, and the times required for complete enrichment under the action of a 0.5 T external permanent magnet were 180, 90, 60, 45, and 30 s, respectively. The basal release rates without magnetic field after 24 h of exosome anchoring were 7.2%, 5.1%, 3.8%, 3.2%, and 3.0%, respectively. However, when the mass fraction of nanoparticles exceeded 25%, the sphericity of the microspheres decreased significantly, pits and cracks appeared on the surface, and the mechanical strength decreased. This is because the excessive inorganic filling amount disrupted the continuity of the polymer matrix. Therefore, the preferred range for the mass fraction of nanoparticles is 10% to 25%, with an optimal value of 15%.

[0048] The results of the effect of emulsification speed showed that when the emulsification speed increased from 5000 rpm to 12000 rpm, the average particle size of the microspheres decreased from 85 μm to 18 μm. Specifically, at emulsification speeds of 5000, 6000, 8000, 10000, and 12000 rpm, the average particle sizes of the prepared microspheres were 85, 72, 45, 28, and 18 μm, respectively, corresponding to injection throughput (using a 22G needle) of 65%, 85%, 98%, 95%, and 82%, and exosome cumulative release over 72 h (without magnetic field) of 28%, 22%, 18%, 19%, and 31%, respectively. Excessively large microspheres are not conducive to injection administration, while excessively small microspheres result in a high surface area to volume ratio, leading to excessively rapid exosome release. Considering both injectability and release kinetics, the preferred microsphere particle size range is 20–80 μm, corresponding to an emulsification speed of 6000–10000 rpm, with 8000 rpm being the preferred value.

[0049] Example 3: Polydopamine coating modification on microsphere surface

[0050] (1) Take 100 mg of the magnetically responsive composite microspheres prepared in step (1) of Example 2 and disperse them in 50 mL of 10 mmol / L Tris-HCl buffer at pH 8.5 to obtain a suspension. Add dopamine hydrochloride to the suspension to make a final concentration of 2 mg / mL, and shake at 150 rpm for 12 h at room temperature. Under alkaline conditions, dopamine molecules undergo oxidative self-polymerization, and their catechol groups are oxidized to o-benzoquinone intermediates, which then form a dense polydopamine coating on the surface of the microspheres through intermolecular crosslinking. After the reaction is completed, collect the microspheres by centrifugation at 3000 rpm and wash them five times with deionized water to completely remove unreacted dopamine monomers and oligomers. After lyophilization, obtain the magnetically responsive microspheres modified with polydopamine coating.

[0051] In this embodiment, the magnetically responsive microspheres modified with a polydopamine coating changed color from white to light brown, visually confirming the successful coating. X-ray photoelectron spectroscopy analysis revealed characteristic peaks of nitrogen on the microsphere surface, further confirming the presence of the polydopamine coating.

[0052] Transmission electron microscopy (TEM) measurements of the polydopamine-coated magnetically responsive microspheres prepared in this embodiment showed that the thickness of the polydopamine coating was approximately 10–15 nm. Water contact angle measurements of the polydopamine-coated magnetically responsive microspheres prepared in this embodiment showed that the water contact angle of the coated microspheres decreased from 78° to 42°, indicating a significant improvement in hydrophilicity. This is beneficial for the dispersion of the microspheres in body fluids and subsequent anchoring of exosomes. The catechol and amino functional groups of the polydopamine coating provide abundant active sites for the subsequent covalent binding of amino- or thiol-containing biomolecules via Schiff base reactions or Michael addition reactions.

[0053] (2) In order to optimize the coating conditions, the effects of dopamine concentration and reaction time on coating thickness and uniformity were investigated according to the preparation process of this embodiment.

[0054] As the dopamine concentration increased from 0.5 mg / mL to 4 mg / mL, the coating thickness increased from 3 nm to 25 nm. Specifically, at dopamine concentrations of 0.5, 1, 2, and 4 mg / mL, the coating thicknesses after 12 h of reaction were 3, 7, 12, and 25 nm, respectively, with exosome anchoring efficiencies of 28%, 45%, 60%, and 48% (the efficiencies decreased at 4 mg / mL due to coating particle aggregation leading to burying of active sites). A coating that is too thin has insufficient active sites, resulting in low exosome anchoring efficiency. A coating that is too thick may clog the microporous structure of the microsphere surface, affecting the degradation of the internal polymer matrix and drug release kinetics.

[0055] When the reaction time was extended from 4 h to 24 h, the coating thickness increased with time, but after 16 h, the coating growth rate decreased significantly, and an uneven structure of polydopamine particle aggregation began to appear on the surface. Specifically, at reaction times of 4, 8, 12, 16, and 24 h, the coating thicknesses obtained under a dopamine concentration of 2 mg / mL were 4, 9, 12, 15, and 18 nm, respectively, and the coating uniformity scores were 3, 4, 5, 4, and 2 (out of 5, with 5 being the best). The score decreased after 16 h due to the aggregation of polydopamine particles on the surface.

[0056] Considering the coating thickness, uniformity, and exosome anchoring efficiency, the optimal dopamine concentration is 2 mg / mL and the reaction time is 12 h. Under these conditions, the coating thickness is approximately 10-15 nm, and the surface is uniform without particle aggregation.

[0057] Example 4: Extraction and Identification of Stem Cell Exosomes

[0058] The extraction process of exosomes from bone marrow mesenchymal stem cells is as follows: Human bone marrow mesenchymal stem cells (BMSCs) from passages 3-5 were cultured in low-glucose DMEM medium containing 10% fetal bovine serum at 37 °C in a 5% CO2 incubator. When cell confluence reached 80%, the serum-containing medium was discarded, and the cells were washed twice with phosphate-buffered saline (PFS). The culture was then replaced with low-glucose DMEM medium containing 1% exosome-free PFS and cultured for another 48 h. After collecting the conditioned medium, differential centrifugation was performed sequentially: first, centrifugation at 300 g for 10 min removed suspended cells; the supernatant was then centrifuged at 2000 g for 20 min to remove dead cell debris; and finally, the supernatant was centrifuged at 10000 g for 30 min to remove large vesicles and cell debris. The resulting supernatant was filtered through a 0.22 μm filter and then ultracentrifuged at 100000 g for 70 min to precipitate the exosomes. The supernatant was discarded, the precipitate was resuspended in phosphate-buffered saline, and washed again by ultracentrifugation at 100000 g for 70 min. Finally, the exosome precipitate was resuspended with an appropriate amount of phosphate buffer, aliquoted, and stored at -80 °C for later use.

[0059] Nanoparticle tracking analysis showed that the average particle size of the extracted exosomes was 95 nm, the peak particle size was 82 nm, and the particle size distribution was concentrated in the range of 40~160 nm, which is consistent with the typical size characteristics of exosomes.

[0060] Transmission electron microscopy analysis was performed on the bone marrow mesenchymal stem cell exosomes prepared in this embodiment, and the results are as follows: Figure 6As shown, transmission electron microscopy with negative staining revealed that exosomes exhibited a typical saucer-like or cup-shaped morphology with a clear lipid bilayer membrane structure. Western blotting analysis showed that exosomes highly expressed exosome marker proteins such as CD9, CD63, CD81, and TSG101, while not expressing the endoplasmic reticulum marker protein Calnexin, ruling out contamination by organelle debris. BCA protein quantification determined the exosome protein concentration to be approximately 0.8–1.2 mg / mL.

[0061] The extraction of neural stem cell exosomes was performed using a similar method as described above. Neural stem cell spheres from generations 2-4 of mice were suspended in DMEM / F12 serum-free medium containing B27 supplementation, 20 ng / mL epidermal growth factor, and 20 ng / mL basic fibroblast growth factor. After digesting the neurospheres into single cells, they were seeded at an appropriate density and cultured for 48 h. The conditioned medium was then collected, and extraction and purification were performed using the same differential ultracentrifugation protocol as for bone marrow mesenchymal stem cell exosomes. The obtained neural stem cell exosomes had an average particle size of 88 nm, and Western blotting confirmed the expression of marker proteins such as CD63, CD81, and Alix. Notably, the neural stem cell exosomes were rich in microRNAs closely related to neural development and myelin formation, such as miR-124, miR-21, and miR-219, providing a molecular basis for their specific function in neural repair.

[0062] Example 5: Anchoring of exosomes on the surface of microspheres

[0063] Exosomes were anchored to the microsphere surface via a synergistic effect of physical adsorption and chemical bonding, utilizing the active catechol and amino groups of the polydopamine coating: 50 mg of the polydopamine-coated magnetically responsive microspheres prepared in Example 3 were dispersed in 5 mL of phosphate buffer to obtain a microsphere suspension. Exosomes from bone marrow mesenchymal stem cells and neural stem cells extracted in Example 4 were mixed at a mass ratio of 3:1 and added to the microsphere suspension to achieve a total exosome protein concentration of 200 μg / mL. The mixture was incubated at 4 °C with low-speed shaking at 50 rpm for 24 h. The polydopamine coating achieves stable anchoring of exosomes through the following mechanisms: the catechol groups on the polydopamine surface undergo Schiff base reactions or Michael addition reactions with the amino groups of exosome membrane surface proteins to form covalent bonds; the phenolic hydroxyl groups of the polydopamine coating achieve physical adsorption with the lipid bilayer of the exosome membrane through hydrogen bonding; and electrostatic interactions occur between the positively charged regions on the polydopamine surface and the negatively charged phosphatidylserine residues on the exosome surface. The aforementioned multiple anchoring mechanisms enable exosomes to achieve a stable binding state on the surface of microspheres that can be dissociated under magnetomechanical vibration, which is the key basis for realizing controllable release triggered by alternating magnetic fields.

[0064] After incubation, microspheres were collected by centrifugation at 3000 rpm, and the supernatant was retained for determining the amount of unbound exosomes. The microspheres were gently washed twice with phosphate-buffered saline (PBS) to remove loosely adsorbed exosomes. The exosome anchoring efficiency was calculated by measuring the amount of exosome protein in the supernatant and washing solution. BCA protein quantification results showed that the amount of exosome protein loaded per milligram of microsphere was approximately 120 μg, with an anchoring efficiency of approximately 60%. To verify the presence and integrity of exosomes on the microsphere surface, the exosome-loaded microspheres were subjected to immunofluorescence staining. Immunofluorescence labeling of the microspheres with FITC-labeled anti-CD63 antibody was performed, and confocal laser scanning microscopy showed a uniform green fluorescence signal on the microsphere surface, such as... Figure 7 As shown, this confirms that exosomes are uniformly distributed on the surface of the microspheres. Furthermore, after labeling the exosomes with PKH26 red fluorescent dye and performing an anchoring experiment, confocal microscopy revealed uniform red fluorescence on the microsphere surface, further confirming the integrity of the exosome membrane structure.

[0065] To investigate the effect of exosome addition on loading and anchoring efficiency, following the anchoring process of this embodiment and keeping other conditions constant (50 mg of polydopamine-coated magnetically responsive microspheres, 5 mL of phosphate buffer, and incubation at 4 °C with shaking for 24 h), a series of experiments were conducted with the total exosome protein concentration in the exosome solution set to 50, 100, 150, 200, 300, and 400 μg / mL, respectively. BCA protein quantification results showed that the corresponding exosome protein loading per milligram of microsphere was 35, 70, 95, 120, 150, and 180 μg, with anchoring efficiencies of 70%, 70%, 63%, 60%, 50%, and 45%, respectively. The exosome particle size distribution (nanoparticle tracking analysis) and immunofluorescence intensity (CD63 labeling) remained basically consistent, confirming that the integrity and function of the exosomes were not affected during the anchoring process. When the total exosome protein concentration exceeds 300 μg / mL, the marginal effect of further increasing the feed amount on improving the loading capacity is significantly weakened due to the gradual saturation of the active sites on the polydopamine coating surface, and the raw material utilization rate decreases significantly. Considering the loading capacity, anchoring efficiency, and raw material economy, the preferred total exosome protein concentration is 100~300 μg / mL, corresponding to a loading capacity of 50~200 μg exosome protein / mg magnetically responsive microspheres. Within this preferred range, the prepared magnetically responsive exosome composite microspheres exhibit good controllable release performance and therapeutic effects in subsequent alternating magnetic field-triggered release and in vivo and in vitro nerve repair experiments.

[0066] Example 6: Performance Test of Controlled Exosome Release Triggered by Alternating Magnetic Field

[0067] (1) 5 mg of the magnetically responsive composite microspheres loaded with exosomes prepared in Example 5 were dispersed in 1 mL of phosphate buffer and placed in the center of the coil of an alternating magnetic field generator. A pulsed magnetic field-triggered release experiment was conducted under the conditions of an alternating magnetic field frequency of 300 kHz and a field strength of 15 kA / m. The experimental protocol involved applying the alternating magnetic field three times a day, each time for 20 min, with a 6 h interval between each application. Samples were taken and an equal volume of fresh buffer was added during the intervals without the application of the alternating magnetic field and after each application. The concentration of exosome protein and the number of exosome particles in the release solution at each time point were detected by BCA protein quantification and nanoparticle tracking analysis. The results showed that during the resting period without the application of the alternating magnetic field, the exosomes exhibited a very low basal release rate, such as... Figure 8 As shown, the cumulative release over 24 hours was only 3%–5% of the total load, indicating that the polydopamine coating has a strong anchoring effect on exosomes. Each 20-minute application of an alternating magnetic field triggered the rapid release of approximately 8%–12% of the load of exosomes, with the release amount showing a predictable decreasing trend with increasing application frequency. This pulsed release pattern is highly compatible with the phased treatment needs of nerve injury repair, where high concentrations of anti-inflammatory factors are required during the acute inflammatory phase and regenerative factors are needed during the subacute phase.

[0068] (2) In order to clarify the mechanism of exosome release triggered by alternating magnetic field, the release behavior of magnetic response composite microspheres loaded with exosomes prepared in Example 5 under different magnetic field parameters was systematically studied according to the test process of step (1) of this embodiment.

[0069] The results showed that at a fixed field strength of 15 kA / m, increasing the frequency from 100 kHz to 500 kHz increased the single-trigger release rate from 5% to 14%; at a fixed frequency of 300 kHz, increasing the field strength from 5 kA / m to 20 kA / m increased the single-trigger release rate from 4% to 16%. This indicates that the release rate is positively correlated with both the magnetic field frequency and field strength. Infrared thermal imaging revealed that under conditions of 300 kHz and 15 kA / m, the temperature of the microsphere suspension increased by only about 2–3°C within 20 min, far below the temperature threshold that causes protein denaturation, thus ruling out the adverse effects of magnetocaloric effects on exosome activity. Therefore, the release of exosomes is mainly attributed to the micromechanical vibrations generated by the Brownian and Nillelian relaxations of the superparamagnetic nanoparticles in an alternating magnetic field. This nanoscale vibration is transmitted to the microsphere surface through the polymer matrix, perturbing the non-covalent bonding forces (including hydrogen bonds and electrostatic interactions) between the polydopamine coating and the exosomes, causing some exosomes to dissociate and release from the microsphere surface. Covalently bound exosomes can also be gradually released under strong magnetostrictive vibrations due to the dynamic reversibility of Schiff base bonds, explaining the decreasing release rate with increasing application frequency—covalently bound exosomes require stronger or longer vibrations to dissociate.

[0070] Comparative Example 1

[0071] The preparation process of exosomes loaded on pure polylactic acid-glycolic acid copolymer microspheres without superparamagnetic nanoparticles is the same as in Examples 2-5, except that the superparamagnetic iron oxide modified with citric acid prepared in Example 1 is not added during the preparation of microspheres in Example 2.

[0072] Following the testing process in step (1) of Example 6, under the same conditions, after loading exosomes onto pure polylactic acid-glycolic acid copolymer microspheres without superparamagnetic nanoparticles, the cumulative release amount after 24 h under the applied alternating magnetic field was not significantly different from that without the applied magnetic field group, and the cumulative release amount after 7 days was about 35%, confirming that the magnetic field-triggered release mechanism is entirely dependent on the superparamagnetic nanoparticles in the microspheres.

[0073] Comparative Example 2

[0074] The preparation process of the magnetically responsive microspheres without polydopamine coating is the same as in Examples 1-5, except that the modification process with polydopamine coating as in Example 3 is not performed.

[0075] Following the testing procedure in step (1) of Example 6, after loading exosomes onto magnetically responsive microspheres without a polydopamine coating through physical adsorption, the exosome release rate reached over 60% within 24 hours, even without the application of an alternating magnetic field. This indicates that physical adsorption alone cannot achieve stable anchoring and controllable release of exosomes. The above comparative experiments fully demonstrate that the synergistic effect of superparamagnetic nanoparticles and the polydopamine coating is a necessary condition for achieving magnetic field-triggered controllable release.

[0076] Example 7 Evaluation of in vitro neuroprotective and regeneration-promoting effects

[0077] To verify the neuroreparative effect of magnetically responsive microspheres loaded with exosomes, a rat cortical neuronal oxygen-glucose deprivation-reperfusion model was established to simulate ischemic neuronal injury. Primary cortical neurons were isolated from the cerebral cortex of newborn SD rats aged 1-3 days and cultured for 7 days until maturity in Neurobasal medium supplemented with 2% B27. Mature neurons were then subjected to oxygen-glucose deprivation treatment: the medium was replaced with glucose-free DMEM, and the neurons were cultured in a hypoxic incubator at 37°C containing 95% N2 and 5% CO2 for 2 h to simulate ischemia. Subsequently, normal medium and normoxic conditions were restored to simulate reperfusion. Immediately after oxygen-glucose deprivation reperfusion, the following treatments were applied: magnetically responsive microspheres loaded with dual-source exosomes under alternating magnetic field (magnetically responsive microspheres loaded with dual-source exosomes prepared in Example 5 + treatment in step (1) of Example 6), magnetically responsive microspheres loaded with dual-source exosomes without magnetic field (i.e., magnetically responsive composite microspheres loaded with exosomes prepared in Example 5, without alternating magnetic field treatment) (magnetically responsive microspheres loaded with dual-source exosomes prepared in Example 5), free exosomes, blank magnetically responsive microspheres, and a blank control group (where the free exosomes group only added 200 μg / mL of dual-source exosomes extracted in Example 4; the blank magnetically responsive microspheres group added magnetically responsive composite microspheres without exosomes prepared in Example 2; and the blank control group only added serum-free culture medium). The alternating magnetic field application protocol was 300 kHz, 15 kA / m, 20 min each time, twice a day. Evaluation was performed at 24 h and 72 h after oxygen-glucose deprivation reperfusion.

[0078] The results of CCK-8 cell viability testing are as follows: Figure 9As shown, oxygen-glucose deprivation-reperfusion treatment reduced neuronal survival to 42% of the normal control. The neuronal survival rate of the magnetically responsive microsphere-loaded exosome group combined with an alternating magnetic field recovered to 68% and 79% at 24 h and 72 h, respectively, significantly higher than the 56% and 63% of the free exosome group and the 60% and 67% of the microsphere-exosome group without a magnetic field. The neuronal survival rate of the blank magnetically responsive microsphere group showed no significant difference from the blank control, ruling out the cytotoxicity of the microsphere material itself. TUNEL apoptosis assays showed that the neuronal apoptosis rate in the combined alternating magnetic field group was 18%, which was 12 and 8 percentage points lower than that in the free exosome group and the microsphere-without-magnetic-field group, respectively. Real-time quantitative polymerase chain reaction analysis showed that the expression level of the anti-apoptotic gene Bcl-2 in neurons of the combined magnetic field group was 3.5 times that of the oxygen-glucose deprivation model group, the expression level of the pro-apoptotic gene Bax was reduced to 0.3 times that of the model group, and the mRNA expression levels of brain-derived neurotrophic factor and nerve growth factor were upregulated by 4.2 times and 3.8 times, respectively. Enzyme-linked immunosorbent assay (ELISA) of the culture medium supernatant showed that the secretion of pro-inflammatory cytokines interleukin-1β and tumor necrosis factor-α decreased by 62% and 55%, respectively, while the secretion of the anti-inflammatory cytokines interleukin-10 increased by 2.8 times in the combined magnetic field group. These results indicate that pulsed exosome release triggered by an alternating magnetic field, compared to passive release and free exosomes, can maintain the effective concentration of exosomes in the damaged microenvironment for a longer period, thus more fully exerting their anti-inflammatory, anti-apoptotic, and neurotrophic effects.

[0079] Example 8: Evaluation of the in vivo therapeutic effect in a rat spinal cord injury model

[0080] A complete T10 segment spinal cord transection injury model was established using adult female SD rats. Sixty rats were randomly divided into five groups of 12 each: the sham-operated group (only the lamina was opened without spinal cord injury), the model group (no treatment was given after spinal cord injury was established), the free exosome injection group (100 μg of dual-source free exosomes extracted in Example 4, prepared at a 3:1 mass ratio of bone marrow mesenchymal stem cell exosomes to neural stem cell exosomes) injected at the injury site, the non-magnetic microsphere exosome group (injected with the same amount of non-magnetic polylactic-co-glycolic acid copolymer microspheres prepared in Comparative Example 1 with an equal amount of exosome loading), and the magnetically responsive microsphere combined with magnetic field group (injected with the same amount of exosome-loaded magnetically responsive composite microspheres prepared in Example 5, and treated with an alternating magnetic field daily post-surgery). Injection was performed using a microsyringe, injecting 10 μL of microsphere suspension (containing 5 mg of microspheres) into the injury center at a depth of approximately 1.5 mm. The alternating magnetic field therapy regimen involved placing the rat at the center of a coil and applying an alternating magnetic field of 300 kHz and 15 kA / m for 20 minutes each time, twice a day, for 28 consecutive days. Simultaneously, a small permanent magnet sheet (5 mm in diameter, 1 mm thick, with a surface field strength of approximately 0.5 T) was implanted subcutaneously above the injury site to provide a continuous static magnetic field to guide the microspheres toward the injury site.

[0081] The recovery of hindlimb motor function in rats was assessed weekly using the BBB motor function scoring system post-surgery. Figure 10 As shown, the BBB score of the model group rats remained below 2 points throughout the observation period, indicating complete hind limb paralysis. At 4 weeks post-surgery, the BBB scores of the free exosome group and the non-magnetic microsphere exosome group were 5.8 and 6.5 points, respectively, indicating incomplete hind limb joint movement. The magnetically responsive microsphere combined with magnetic field group achieved a BBB score of 9.2 points at 4 weeks post-surgery, demonstrating coordinated hind limb movement and occasional weight-bearing gait, representing improvements of 3.4 and 2.7 points compared to the free exosome group and the non-magnetic microsphere exosome group, respectively, with statistically significant differences. At 8 weeks post-surgery, the BBB score of the magnetically responsive microsphere combined with magnetic field group further increased to 12.5 points, exhibiting sustained coordinated hind limb gait, while the scores of the free exosome group and the non-magnetic microsphere exosome group were 7.8 and 8.6 points, respectively.

[0082] In terms of electrophysiological testing, motor evoked potentials (MEPs) were measured in rats of each group 8 weeks post-surgery. In the sham-operated group (where only the lamina was opened without spinal cord injury), the MEP latency was 5.2 ms, while no identifiable MEP waveforms were detected in the model group. The MEP latencies in the free exosome group and the non-magnetic microsphere exosome group were 11.8 ms and 10.5 ms, respectively, with significantly reduced amplitudes. In the magnetically responsive microsphere combined with magnetic field group, the MEP latency was 7.6 ms, with amplitude recovering to 55% of the sham-operated group. The latency was 35.6% shorter than that in the free exosome group, indicating a significant improvement in neural electrical signal transmission function.

[0083] Eight weeks post-surgery, rats were sacrificed, and spinal cord tissue from the injured segment was collected for histological analysis. Hematoxylin-eosin staining revealed large areas of cystic cavities and dense glial scar tissue in the model group, with almost no residual nerve tissue. The cavity area was reduced in the free exosome group and the non-magnetic microsphere exosome group, but clear scar boundaries were still visible. The magnetically responsive microsphere combined with a magnetic field group had the smallest cavity area, with more new tissue filling the cavity, and indistinct scar boundaries. Figure 11 As shown, immunofluorescence staining revealed that the density of NF-200-positive nerve fibers in the damaged area of ​​the magnetically responsive microspheres combined with the magnetic field group was 4.8 times that of the model group, the area of ​​MBP-positive myelin sheath was 3.2 times that of the model group, and the area of ​​reactive proliferation of GFAP-positive astrocytes was reduced by 42%. Furthermore, CD31-positive neovascular structures were detected in the damaged area of ​​the combined magnetic field group, with a vessel density 2.6 times that of the model group, indicating that the pro-angiogenic effect of exosomes was effectively exerted.

[0084] To visually verify the enrichment effect of microspheres at the injury site under the guidance of a static magnetic field, an in vivo distribution tracking experiment was conducted using fluorescently labeled magnetically responsive composite microspheres. Rhodamine B fluorescent dye was coupled to the surface of the microspheres and administered via tail vein injection to rats with spinal cord injury. Immediately after injection, a permanent magnet was placed above the injury site to maintain static magnetic field guidance for 6 hours. In vivo fluorescence imaging showed that the fluorescence signal intensity at the injury site in the static magnetic field-guided group was 4.2 times that of the group without static magnetic field guidance, confirming that the static magnetic field can effectively guide the circulating magnetically responsive microspheres to the injury site for directional enrichment. Twenty-four hours post-surgery, in vitro fluorescence imaging of major organs was performed. The accumulation of microspheres in the liver and spleen of the static magnetic field-guided group was reduced by 38% and 45%, respectively, compared to the group without static magnetic field guidance, indicating that the magnetic navigation strategy not only increased the microsphere concentration at the injury site but also reduced the non-specific retention of microspheres in the reticuloendothelial system, improving overall delivery efficiency. Prussian blue staining further confirmed that the injury area in the static magnetic field-guided group contained more iron-containing microsphere particles, consistent with the fluorescence imaging results.

[0085] The in vivo degradation behavior of magnetically responsive microspheres is crucial for their safety and long-term efficacy. Scanning electron microscopy was performed on tissue samples from the damaged area at different postoperative time points. At 2 weeks post-operation, superficial erosion marks appeared on the surface of the microspheres, with the spherical outline remaining largely intact. At 4 weeks, significant cracks and fragmentation appeared on the surface of the microspheres, with some microspheres disintegrating into several fragments. At 8 weeks, most microspheres degraded into fragments smaller than 5 μm, with only a few larger fragments remaining. At 12 weeks, almost no intact microsphere structure was observed, as the degradation products had been absorbed by the surrounding regenerating tissue. Inductively coupled plasma mass spectrometry (ICP-MS) was used to detect changes in iron content in tissues and blood at each time point. The results showed that the iron content in the injection site reached a peak at 4 weeks post-operation and then gradually decreased, returning to near-normal levels by 12 weeks. Whole blood iron content and serum ferritin levels remained within the normal physiological range throughout the observation period, with no signs of iron overload. These results indicate that the superparamagnetic iron tetroxide nanoparticles released during microsphere degradation can be cleared by the body through normal iron metabolism pathways and do not cause iron accumulation toxicity.

[0086] The therapeutic effect of this invention was also verified in a peripheral nerve injury model. A sciatic nerve compression injury model was established using SD rats. Magnetic-responsive composite microsphere suspension was injected into the nerve surrounding the injured segment, and treated with an alternating magnetic field for 28 days. Neurophysiological testing and histological analysis were performed 4 weeks post-surgery. Results showed that the sciatic nerve conduction velocity in the magnetic-responsive microsphere combined with magnetic field group recovered to 72% of the normal side, significantly higher than the 53% in the free exosome group and the 31% in the model control group. Toluidine blue stained semi-thin sections showed that the regenerated nerve fiber density in the combined magnetic field group was 812 fibers / mm², the myelinated fiber ratio reached 68%, and the myelin sheath thickness was approximately 0.8 μm, which were superior to the 586 fibers / mm², 52%, and 0.5 μm in the free exosome group, respectively. Gastrocnemius muscle wet weight ratio analysis showed that the combined magnetic field group had the mildest muscle atrophy, with an affected / healthy side wet weight ratio of 0.76, while the model group was only 0.42. The above results confirm that the magnetically responsive composite microspheres of the present invention also exhibit excellent therapeutic effects in the repair of peripheral nerve injuries, and verify the wide applicability of this technical solution.

[0087] Example 9: Comparative Experiment of Exosomes from Different Sources

[0088] To verify the superiority of the dual-source combination strategy of bone marrow mesenchymal stem cell exosomes and neural stem cell exosomes, the magnetically responsive microspheres combined with a magnetic field group in Example 8 were further subdivided into three subgroups for comparison: a group loaded only with bone marrow mesenchymal stem cell exosomes (prepared according to the procedure in Example 5, except that the 3:1 mass ratio of bone marrow mesenchymal stem cell exosomes and neural stem cell exosomes was replaced with the same amount of bone marrow mesenchymal stem cell exosomes), a group loaded only with neural stem cell exosomes (prepared according to the procedure in Example 5, except that the 3:1 mass ratio of bone marrow mesenchymal stem cell exosomes and neural stem cell exosomes was replaced with the same amount of stem cell exosomes), and a dual-source exosome combination group prepared in Example 5 (mass ratio 3:1). In a rat spinal cord injury model, the BBB scores of the three subgroups at 4 weeks post-surgery were 7.8, 7.2, and 9.2, respectively. Histological analysis showed that the single-source bone marrow mesenchymal stem cell exosome group had a better anti-inflammatory effect, less inflammatory cell infiltration in the damaged area, and higher interleukin-10 levels, but relatively lower density of newly generated nerve fibers. The single-source neural stem cell exosome group had better nerve regeneration effect, with higher NF-200 positive fiber density and MBP positive myelin sheath area, but relatively insufficient early anti-inflammatory effect. Example 5, the dual-source combination group, combined the advantages of both, showing the best performance in both anti-inflammatory regulation and nerve regeneration promotion. This result is consistent with the functional complementarity of the bioactive molecules contained in the two types of exosomes: bone marrow mesenchymal stem cell exosomes are rich in anti-inflammatory-related miRNAs such as miR-146a and miR-21, which mainly play an immunomodulatory and anti-inflammatory role; neural stem cell exosomes are rich in pro-neural differentiation-related miRNAs such as miR-124 and miR-219, which mainly promote neural progenitor cell differentiation and myelin sheath regeneration. When the two types of exosomes are combined in an optimal ratio, they exert a temporally complementary effect at different stages of damage repair—in the early stage, bone marrow mesenchymal stem cell exosomes dominate the remodeling of the anti-inflammatory microenvironment, while in the later stage, neural stem cell exosomes dominate neural regeneration and functional recovery.

[0089] Example 10 Safety Evaluation

[0090] The biocompatibility of the magnetically responsive composite microspheres was systematically evaluated. In vitro cytotoxicity assays were performed using the CCK-8 assay to detect the toxicity of the microsphere extract to L929 fibroblasts and PC12 neurons. Extracts were prepared by co-incubating microspheres at different concentrations (0.1–10 mg / mL) with complete culture medium at 37 °C for 72 h. After treatment with each concentration of extract for 24 h and 72 h, cell viability was higher than 90%, and according to GB / T 16886.5, the cells were deemed non-cytotoxic (grade 0–1). Hemolysis assays showed that the hemolysis rate of the microsphere suspension was less than 2% within the concentration range of 0.1–5 mg / mL, meeting the hemolysis performance requirements for materials used in medical devices. In acute systemic toxicity experiments, the microsphere suspension was administered to ICR mice via tail vein injection, and no deaths or abnormal reactions were observed within 72 h. In the subchronic toxicity experiment, microspheres were injected locally into SD rats via lesions. Rats were sacrificed at 4 and 8 weeks post-surgery for tissue sampling. Hematoxylin-eosin staining of major organs (heart, liver, spleen, lung, and kidney) showed no pathological changes. Blood biochemistry tests showed no significant differences in alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, and blood urea nitrogen among the groups. Iron content analysis showed that at 8 weeks post-surgery, the iron content in the tissue surrounding the injection site was slightly higher than in normal tissue, but far below the iron overload toxicity threshold. Furthermore, the iron content in the liver and spleen was not significantly different from the control group, indicating that the released iron ions can be cleared through normal metabolic pathways and will not cause systemic iron accumulation. The degradation behavior of the microspheres in vivo was tracked by observing tissue sections. At 4 weeks post-surgery, the microspheres maintained a relatively intact outline but showed surface erosion and fragmentation. At 8 weeks post-surgery, most microspheres had degraded into small fragments and were absorbed by surrounding tissues. At 12 weeks post-surgery, the microspheres were almost completely degraded. The degradation products, lactic acid and glycolic acid, can be metabolized normally through the tricarboxylic acid cycle and will not have adverse effects on the body. In addition, immunohistochemical analysis of the tissue surrounding the injection site was performed to evaluate the local immune response. CD68-positive macrophages peaked one week post-surgery and gradually declined to near-normal tissue levels by four weeks, representing a normal acute inflammatory response after foreign body implantation. No adverse immune responses such as chronic granulomas or fibrous encapsulation were observed. Immunogenicity testing of exosomes showed that microsphere-loaded stem cell exosomes did not cause a significant increase in specific IgG antibody titers after repeated injections, confirming the low immunogenicity advantage of exosomes as a cell-free therapeutic agent. Regarding the safety evaluation of the alternating magnetic field, after 28 days of continuous magnetic field exposure, histological examination of normal tissues and organs surrounding the injury area revealed no magnetic field-related thermal damage or structural abnormalities. Temperature monitoring showed that the core body temperature of the animals increased by no more than 0.5 °C during alternating magnetic field treatment, which was within the safe range.

[0091] The magnetically responsive composite microspheres of this invention achieve efficient repair of nerve damage through the following multi-level synergistic mechanisms. At the carrier level, the polylactic acid-glycolic acid copolymer matrix provides the physical support space and controllable degradation framework for exosomes, with a degradation rate matching the nerve repair cycle. Superparamagnetic iron oxide nanoparticles dispersed within the matrix endow the microspheres with dual magnetic functions: providing navigational driving force under a static magnetic field, allowing the microspheres to be guided to the core damage area after injection; and generating nanoscale mechanical vibrations through Brownian and Niehr relaxation mechanisms under an alternating magnetic field, providing a controllable physical driving force for the triggered release of exosomes. At the coating level, the polydopamine biomimetic coating performs three functions: acting as a chemical and physical anchoring layer to firmly bind exosomes to the surface of the microspheres, achieving an ideal release mode of low basal release and high triggered release; providing antioxidant activity with its abundant phenolic hydroxyl groups and catechol structure, scavenging excess reactive oxygen free radicals in the damaged microenvironment, and reducing secondary damage to residual nerve tissue caused by oxidative stress; and synergistically enhancing the neuroprotective effect with neurotrophic factors in exosomes. At the bioactivity level, the time-complementary release strategy of dual-source exosomes precisely matches the pathophysiological process of nerve injury repair: Bone marrow mesenchymal stem cell exosomes released during the acute phase of injury regulate the NF-κB and TLR4 signaling pathways via miR-146a and miR-21, inhibiting microglia M1 polarization and promoting M2 polarization transition, thus reshaping the anti-inflammatory repair microenvironment; neural stem cell exosomes continuously released during the subacute and chronic repair phases promote neural progenitor cell differentiation into neurons via miR-124 targeting SOX9, and promote oligodendrocyte precursor cell differentiation and myelin regeneration via miR-219. The organic integration of these multi-level synergistic mechanisms enables the magnetically responsive composite microsphere system of this invention to exhibit significantly superior therapeutic effects compared to existing technologies in nerve function repair.

Claims

1. A magnetically responsive composite microsphere loaded with stem cell exosomes, characterized in that, The magnetically responsive composite microspheres loaded with stem cell exosomes comprise magnetically responsive microspheres containing a biodegradable polymer matrix and superparamagnetic iron oxide nanoparticles dispersed within the biodegradable polymer matrix. The surface of the magnetically responsive microspheres is coated with a polydopamine coating, and stem cell-derived exosomes are anchored on the surface of the polydopamine coating.

2. The magnetically responsive composite microspheres loaded with stem cell exosomes according to claim 1, characterized in that, The biodegradable polymer matrix is ​​selected from polylactic acid-glycolic acid copolymer, polycaprolactone, or a blend of polylactic acid-glycolic acid copolymer and polycaprolactone.

3. The magnetically responsive composite microspheres loaded with stem cell exosomes according to claim 1, characterized in that, The superparamagnetic iron oxide nanoparticles have a particle size of 8–15 nm, and the mass fraction of superparamagnetic iron oxide nanoparticles in the magnetically responsive microspheres is 10%–25%, with the particle size of the magnetically responsive microspheres being 20–80 μm.

4. The magnetically responsive composite microspheres loaded with stem cell exosomes according to claim 1, characterized in that, The polydopamine coating has a thickness of 5-20 nm and is formed by the self-polymerization of dopamine hydrochloride in Tris-HCl buffer.

5. The magnetically responsive composite microspheres loaded with stem cell exosomes according to claim 1, characterized in that, The stem cell-derived exosomes are one or a combination of bone marrow mesenchymal stem cell exosomes or neural stem cell exosomes; when it is a combination of the two, the mass ratio of bone marrow mesenchymal stem cell exosomes to neural stem cell exosomes is 2:1 to 4:

1.

6. The magnetically responsive composite microspheres loaded with stem cell exosomes according to claim 1, characterized in that, The loading amount of stem cell-derived exosomes on the surface of magnetically responsive microspheres was 50–200 μg of stem cell-derived exosome protein / mg of magnetically responsive microspheres; the particle size of stem cell-derived exosomes was 40–160 nm.

7. The method for preparing magnetically responsive composite microspheres loaded with stem cell exosomes according to any one of claims 1 to 6, characterized in that, Includes the following steps: (1) Magnetic responsive microspheres were prepared using a biodegradable polymer matrix and superparamagnetic iron oxide nanoparticles; (2) In Tris-HCl buffer, dopamine hydrochloride self-polymerizes on the surface of magnetically responsive microspheres to form a polydopamine coating; (3) Stem cell-derived exosomes are anchored to the surface of magnetically responsive microspheres through the synergistic effect of physical adsorption and chemical bonding of polydopamine coating.

8. The preparation method according to claim 7, characterized in that, In step (1), the rotation speed during emulsification is 6000~10000 rpm. In step (2), the pH of the Tris-HCl buffer is 8.0~8.5, the concentration of dopamine hydrochloride in the Tris-HCl buffer is 0.5 mg / mL-4 mg / mL, and the self-polymerization reaction time is 4-16 h. In step (3), the total protein concentration of exosomes in the stem cell-derived exosome solution is 100~300 μg / mL, the incubation temperature is 4 ℃, and the incubation time is more than 24 h.

9. The use of the magnetically responsive composite microspheres loaded with stem cell exosomes as described in any one of claims 1 to 6 in the preparation of drugs for the repair of nerve damage.

10. The application according to claim 9, characterized in that, The magnetically responsive composite microspheres can be used in combination with protein-based materials or exosomes. The protein-based materials are selected from collagen or gelatin, and the exosomes are free exosomes of the same origin as the exosomes anchored on the surface of the magnetically responsive composite microspheres.

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