A coaxial core-shell fiber sustained-release system based on PVA core layer encapsulating exosomes and a preparation method and application thereof

Abstract

The application belongs to the technical field of biomedical materials and bone tissue engineering, and particularly relates to a coaxial core-shell fiber sustained-release system based on PVA core layer loaded with exosomes and a preparation method and application thereof. The coaxial core-shell fiber is composed of a core layer and a shell layer. The core layer contains polyvinyl alcohol and exosomes, and the shell layer contains an in-vivo degradable polymer material. The coaxial electrospinning is used to construct the core-shell fiber structure, so that the exosomes are loaded in the hydrophilic core layer, and the shell layer is used for physical coating and release regulation of the exosomes. By adjusting the material composition and preparation parameters of the core layer and the shell layer, the stable loading and continuous delivery of the exosomes in the fiber are realized, and the retention time of the exosomes in the local application process is improved. The coaxial core-shell fiber sustained-release system based on the PVA core layer loaded with the exosomes has good biocompatibility, structural support and continuous delivery capacity, and can be used for preparing biomedical materials for promoting bone tissue repair and regeneration.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and bone tissue engineering technology, and particularly relates to a coaxial core-shell fiber sustained-release system based on PVA core layer-encapsulated exosomes, its preparation method and application. Background Technology

[0002] Bone regeneration disorders are a core technological challenge that urgently needs to be overcome in the fields of bone tissue engineering and regenerative medicine. Among them, bone regeneration disorders caused by gene defects have become a key focus and difficulty in clinical repair treatment due to their complex pathogenesis and unique pathological state. The pathological characteristics of this type of gene defect-related bone regeneration disorder are not only manifested by a significant decrease in the differentiation potential of endogenous osteoblasts and insufficient synthesis and deposition of bone matrix, leading to a severely delayed bone defect healing process, but also accompanied by the disorder and imbalance of the local bone regeneration microenvironment, including cytokine balance, nutrient supply and tissue compatibility. This makes it difficult for traditional bone repair materials, such as hydroxyapatite and tricalcium phosphate, and simple osteopromoting strategies, such as local injection of growth factors, to specifically address the above-mentioned pathological problems, and thus cannot achieve the ideal bone defect repair effect, failing to meet the needs of clinical treatment.

[0003] In research on bone regeneration disorders, addressing the core challenge of limited endogenous osteogenic potential due to gene defects, there is an urgent need to introduce exogenous bioactive signals to continuously and precisely intervene in and compensate for the impaired bone regeneration process, thereby reconstructing a normal osteogenic metabolism and regenerative microenvironment. Exosomes, as naturally occurring bioactive vesicles secreted by cells, can carry various functional biomolecules such as proteins, lipids, and nucleic acids. They can efficiently regulate the proliferation, differentiation, and apoptosis of target cells, while effectively improving the local tissue microenvironment and promoting intercellular signal transduction. They have shown broad application prospects in bone tissue engineering and bone injury repair, and have become a research hotspot in recent years.

[0004] However, exosomes face significant technical bottlenecks in practical applications: free exosomes have a short retention time in local defect areas and are prone to diffusion and loss; furthermore, their bioactivity is rapidly diminished by factors such as enzymatic degradation and oxidation, preventing them from generating a sustained and stable biological stimulus at bone defect sites, severely limiting their clinical efficacy. For gene defect-related bone regeneration disorders, the inherent defects in the endogenous osteogenic system necessitate even higher requirements for sustained exosome delivery efficiency and activity maintenance. Existing exosome delivery methods, such as direct injection and simple mixed delivery, cannot simultaneously address the core needs of exosome activity protection, sustained local delivery, and efficient bone repair promotion, making effective intervention for gene defect-related bone regeneration disorders difficult.

[0005] To address the technical challenges in exosome delivery and improve the utilization efficiency of exosomes at bone defect sites, constructing sustained-release exosome delivery systems using scaffold materials has become an important research direction in this field. Core-shell fibers, with their unique layered spatial configuration, can encapsulate bioactive substances such as exosomes in the core layer. Simultaneously, the shell structure achieves physical encapsulation, protection, and diffusion rate regulation of the bioactive substances in the core layer, offering significant potential advantages in protecting the integrity of bioactive components, reducing burst release effects, and achieving long-term sustained release of bioactive substances. Compared to traditional methods that directly adsorb exosomes onto the surface of scaffold materials or simply mix them into a matrix, the core-shell structure can achieve stable and controllable local delivery of exosomes at bone defect sites while effectively maintaining their bioactivity. This characteristic is irreplaceable for gene-related bone regeneration disorders that require long-term biological stimulation to reconstruct bone regeneration function. However, existing core-shell fiber exosome encapsulation technologies still have the following limitations: First, it is difficult to balance exosome encapsulation and activity retention: the preparation of core-shell fibers mostly relies on high-voltage electrospinning. The high-voltage electric field, organic solvent volatilization, and high-speed stretching during the process can easily cause damage to the exosome membrane structure and inactivation of internal nucleic acids / proteins. If the process intensity is reduced to protect the activity, it will lead to instability of the core layer structure and a decrease in encapsulation efficiency.

[0006] Second, the release behavior is difficult to control precisely: the degradation of the shell material and the release of exosomes from the core layer are often asynchronous, which can easily lead to early burst release or late interruption of release; and the fluctuations in the microenvironment of bone defects can further interfere with the release dynamics, making it difficult to achieve a stable, uniform, and long-term supply of exosomes, which cannot match the slow repair cycle of gene-deficient bone regeneration.

[0007] Third, the materials themselves lack biocompatibility and bone integration: commonly used core-shell fiber substrates are mostly synthetic polymers, such as PLGA, PELA, PVA, etc., which have weak osteoconductivity and cell adhesion, and slow integration with bone tissue.

[0008] In summary, existing technologies still have significant shortcomings in addressing the clinical needs of bone regeneration disorders related to gene defects. There is an urgent need to develop novel exosome delivery materials that combine exosome activity protection, effective loading, sustained release capability, excellent biocompatibility, and bone repair adaptability. Based on this, this invention constructs an exosome sustained-release system based on a coaxial core-shell fibrous membrane and its preparation method, providing a superior technical means to improve bone regeneration disorders related to gene defects caused by impaired osteogenic function. This has significant clinical application value and research significance. Summary of the Invention

[0009] The purpose of this invention is to provide a coaxial core-shell fiber sustained-release system based on PVA core layer-encapsulated exosomes, its preparation method and application, to solve the problems of easy diffusion and loss of free exosomes in bone defect areas, short local retention time, difficulty in maintaining activity and difficulty in continuous release, especially to solve the technical problems of impaired osteogenic capacity, lack of continuous biological activity stimulation and effective delivery carrier support in gene defect-related bone regeneration disorders.

[0010] In view of this, the present invention provides a method for preparing a coaxial core-shell fiber sustained-release system based on PVA core layer-loaded exosomes, comprising the following steps: (1) Disperse polyvinyl alcohol and exosomes in water to obtain a core spinning solution; (2) Dissolve biodegradable polymer materials in an organic solvent to obtain shell spinning solution; (3) The core spinning solution is injected into the inner needle of the coaxial needle in the coaxial electrospinning device, and the shell spinning solution is injected into the outer needle of the coaxial needle. Coaxial electrospinning is performed under the action of a high voltage electric field to collect a coaxial core-shell fiber sustained-release system based on PVA core layer loaded exosomes.

[0011] Furthermore, in step (1): The exosomes are derived from mesenchymal stem cells; and / or, The concentration of exosomes in the core spinning solution is 5-20 mg / mL; and / or, The nuclear spinning solution after adding exosomes was stored at 1-6℃ for later use.

[0012] Furthermore, in step (1), the exosomes are derived from ectodermal mesenchymal stem cells.

[0013] Furthermore, in step (2): The biodegradable polymer material is selected from silk fibroin and polycaprolactone, and the mass ratio of silk fibroin to polycaprolactone in the shell spinning solution is 1:6 to 6:1; and / or, The total mass concentration of silk fibroin and polycaprolactone in the shell spinning solution is 5 wt% to 10 wt%; and / or, The organic solvent is selected from one or more of hexafluoroisopropanol, chloroform, dichloromethane, toluene, xylene, ethyl acetate, propyl acetate, ethyl propionate, carbon disulfide, ethanol, and acetone.

[0014] Furthermore, in step (3), the distance between the needle and the receiver in the coaxial electrospinning device is set to 10~20cm; the voltage is set to 12~20kV; the flow rate of the shell spinning solution is set to 0.3~1.0 mL / h; and the flow rate of the core spinning solution is set to 0.1~0.5 mL / h.

[0015] Furthermore, in step (1), the preparation process of the core spinning solution is as follows: Polyvinyl alcohol is dispersed in water and stirred until dissolved. Then, exosomes are added and dispersed evenly to obtain a core spinning solution. The mass concentration of polyvinyl alcohol in the core spinning solution is 5 wt% to 10 wt%.

[0016] Furthermore, in step (1), the preparation process of the core spinning solution is as follows: Polyvinyl alcohol is dissolved in water and stirred until completely dissolved. The resulting polyvinyl alcohol aqueous solution is then frozen at -20℃ to -10℃ for 6 to 24 hours, followed by thawing at 2℃ to 8℃ for 2 to 6 hours. This process is repeated 1 to 3 times to obtain a polyvinyl alcohol solution with a physically cross-linked micro-region structure. After the polyvinyl alcohol solution naturally returns to room temperature, a modifier at 0.3 to 0.8 wt% of polyvinyl alcohol is added and stirred until dissolved. Then, exosomes are added and stirred until evenly dispersed to obtain the pretreated core spinning solution.

[0017] Furthermore, the modifier is selected from one or more of fatty alcohol polyoxyethylene ether, Tween, poloxamer, and polyethylene glycol; and / or, The degree of alcoholysis of the polyvinyl alcohol is 85-92%; and / or, The degree of polymerization of the polyvinyl alcohol is 500~1700; and / or, The mass concentration of polyvinyl alcohol in the core spinning solution is 5 wt% to 8 wt%.

[0018] The present invention also provides a coaxial core-shell fiber sustained-release system based on PVA core layer-encapsulated exosomes, which is prepared by the above-described preparation method.

[0019] The present invention also provides the application of the above-mentioned coaxial core-shell fiber sustained-release system based on PVA core layer-encapsulated exosomes in the preparation of materials that improve gene defect-related bone regeneration disorders.

[0020] Compared with existing technologies, the coaxial core-shell fiber sustained-release system based on PVA core layer-encapsulated exosomes, its preparation method, and its application described in this invention have the following advantages: 1. This invention uses polyvinyl alcohol as the core layer material and a blend of silk fibroin and polycaprolactone as the shell layer material to construct exosome-loaded coaxial core-shell fibers. This allows the exosomes to be mainly loaded in the core layer, with the shell layer physically encapsulating the core layer. This helps reduce the impact of the external environment on the stability of exosomes, thereby improving the stability of exosomes in local applications and providing a structural basis for their continuous biological function in bone defect areas.

[0021] 2. This invention regulates the microstructure and release behavior of fibers by controlling the mass ratio of silk fibroin to polycaprolactone in the shell. For gene defect-related bone regeneration disorders, this tunable shell structure helps maintain local bioactive signal input for a longer period of time, making it more suitable for improving insufficient bone regeneration caused by impaired osteogenic capacity.

[0022] 3. The coaxial core-shell fibers loaded with exosomes constructed in this invention can achieve exosome release over a longer period. The sustained-release system exhibits superior long-acting and linear slow-release performance of exosomes. This invention does not merely provide an exosome carrier, but rather provides a relatively stable biological activity stimulus to the local bone defect site. Therefore, it is more suitable for improving the problems of insufficient osteogenic signal and delayed repair in gene defect-related bone regeneration disorders.

[0023] 4. This invention selects silk fibroin and polycaprolactone as shell materials. Silk fibroin is beneficial for improving the biocompatibility and cell-cell interaction of the material, while polycaprolactone is beneficial for providing the necessary structural stability and slow degradation characteristics. Therefore, the shell formed by the combination of the two can take into account both the biological adaptability and scaffold support function required for bone repair. Thus, the exosome-loaded coaxial core-shell fibers constructed in this invention not only have a sustained-release effect, but also provide a more suitable local repair microenvironment for gene defect-related bone regeneration disorders.

[0024] 5. This invention pre-treats the core spinning solution through freeze-thaw cycles, utilizing ice crystal compression to induce local aggregation of polyvinyl alcohol (PVA) molecular chains, forming stable microcrystalline regions as physical crosslinking points, thus constructing a mildly physically crosslinked network. This network offers the following synergistic advantages: (a) extremely low crosslinking point density, preserving sufficient hydrophilicity and swelling capacity of the core fibers; (b) insoluble microcrystalline regions effectively anchor the PVA molecular chains, significantly reducing the dissolution rate of the core fibers in an aqueous environment; (c) during electrospinning, this network effectively binds exosomes within the core fibers, reducing exosome leakage and improving encapsulation efficiency. In use, this mildly physically crosslinked network inhibits the burst release of exosomes, achieving slow and continuous release from the core layer.

[0025] 6. In this invention, after the freeze-thaw cycle and before adding exosomes, an appropriate amount of modifier is added to the core spinning solution. This modifier can reduce the strong hydrogen bonding between polyvinyl alcohol molecular chains, improve the fluidity and electrospinning formability of the core spinning solution, and impart good flexibility to the core fibers. At the same time, the addition of the modifier can improve the dispersion uniformity and loading of exosomes in the core spinning solution, ensuring that the exosomes are uniformly distributed in the fibers, thereby ensuring the stability and repeatability of the release behavior. Attached Figure Description

[0026] Figure 1 Scanning electron microscopy images of coaxial core-shell fiber sustained-release systems based on PVA core-layer-loaded exosomes prepared under different silk fibroin / polycaprolactone mass ratios, used to characterize the effect of shell ratio on fiber micromorphology and fiber diameter. Figure 2 This is a fluorescence verification image of the core-shell structure of a coaxial core-shell fiber sustained-release system based on PVA core-layer-encapsulated exosomes. Different fluorescent dyes were added to the core and shell layers to verify whether the fiber formed a core-shell structure after coaxial electrospinning. Figure 3 The graphs show the exosome release curves of the PVA core-shell fiber sustained-release system with different concentrations of exosomes loaded in PBS, used to characterize the sustained-release behavior of the fibers and the effect of different exosome loading concentrations on release performance. Figure 4 In vitro cumulative release curves of coaxial core-shell fiber membranes (both loaded with 10 mg / mL exosomes) in PBS from unmodified (control group) and freeze-thaw modified groups. Figure 5 The images show the micro-CT three-dimensional reconstruction images and quantitative analysis results of the skull defect areas in gene-deficient mice 8 weeks after surgery: A represents the micro-CT three-dimensional reconstruction images of the skull defect areas in each group; B represents the quantitative analysis results of BS in the defect areas in each group; and C represents the quantitative analysis results of BV / TV in the defect areas in each group. Detailed Implementation

[0027] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0028] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0029] This invention aims to provide a coaxial core-shell fiber sustained-release system based on PVA core layer-encapsulated exosomes for improving gene defect-related bone regeneration disorders. By encapsulating exosomes in a hydrophilic core layer and utilizing the shell layer for physical encapsulation and release regulation, the system can improve the stability of exosomes during local application and prolong their duration of action in bone defect areas. This makes it more suitable for the needs of continuous bioactive stimulation and local repair microenvironment regulation in gene defect-related bone regeneration disorders.

[0030] Specifically, this invention provides a method for preparing a coaxial core-shell fiber sustained-release system based on PVA core layer-encapsulated exosomes, characterized by comprising the following steps: (1) Disperse polyvinyl alcohol and exosomes in water to obtain a core spinning solution; (2) Dissolve biodegradable polymer materials in an organic solvent to obtain shell spinning solution; (3) The core spinning solution is injected into the inner needle of the coaxial needle in the coaxial electrospinning device, and the shell spinning solution is injected into the outer needle of the coaxial needle. Coaxial electrospinning is performed under the action of a high voltage electric field to collect a coaxial core-shell fiber sustained-release system based on PVA core layer loaded exosomes.

[0031] As some specific examples of the present invention, in step (1), the exosomes are derived from mesenchymal stem cells, and more preferably from exosomes derived from ectodermal mesenchymal stem cells.

[0032] As a preferred example of the present invention, in step (1), the concentration of exosomes in the core spinning solution is 5~20 mg / mL, specifically 5, 10, or 20 mg / mL, and particularly preferably 10 mg / mL.

[0033] As a preferred example of the present invention, in step (1), polyvinyl alcohol is stirred and dissolved at 35~40°C, preferably 37°C.

[0034] As a preferred example of the present invention, the nuclear layer spinning solution after adding exosomes in step (1) is stored at 1~6°C, preferably 4°C, for later use.

[0035] As some examples of the present invention, in step (1), the preparation process of the core spinning solution is as follows: Polyvinyl alcohol is dispersed in water and stirred until dissolved. Then, exosomes are added and dispersed evenly to obtain a core spinning solution. The mass concentration of polyvinyl alcohol in the core spinning solution is 5 wt% to 10 wt%, more preferably 7 wt%.

[0036] As a preferred example of the present invention, in step (1), the preparation process of the core spinning solution is as follows: Polyvinyl alcohol is dissolved in water and stirred until completely dissolved. The resulting polyvinyl alcohol aqueous solution is then frozen at -20℃ to -10℃ for 6 to 24 hours, followed by thawing at 2℃ to 8℃ for 2 to 6 hours. This process is repeated 1 to 3 times to obtain a polyvinyl alcohol solution with a physically cross-linked micro-region structure. After the polyvinyl alcohol solution naturally returns to room temperature, a modifier at 0.3 to 0.8 wt% of polyvinyl alcohol is added and stirred until dissolved. Then, exosomes are added and stirred until evenly dispersed to obtain the pretreated core spinning solution.

[0037] As some specific examples of the present invention, the modifier is selected from one or more of fatty alcohol polyoxyethylene ether, Tween, poloxamer, and polyethylene glycol, preferably fatty alcohol polyoxyethylene ether or Tween.

[0038] As a preferred example of the present invention, the polyvinyl alcohol is a medium-low degree of alcoholysis PVA with a degree of alcoholysis of 85-92%.

[0039] As a preferred example of the present invention, the degree of polymerization of the polyvinyl alcohol is 500~1700, more preferably 500~800.

[0040] As a preferred example of the present invention, when pretreating the core spinning solution, the mass concentration of polyvinyl alcohol in the core spinning solution is 5 wt% to 8 wt%.

[0041] In this invention, the core layer spinning solution is pretreated through freeze-thaw cycles. The ice crystal compression effect promotes the local aggregation of polyvinyl alcohol (PVA) molecular chains, forming stable microcrystalline regions as physical crosslinking points, thus constructing a mildly physically crosslinked network. This network has an extremely low crosslinking density, significantly reducing the dissolution rate of the core layer fibers in an aqueous environment while retaining a certain degree of hydrophilicity. Simultaneously, during electrospinning, this network effectively binds exosomes within the core layer fibers, reducing exosome leakage and improving coating efficiency. In use, this mildly physically crosslinked network also prevents the rapid dissolution of the core layer PVA in an aqueous environment, inhibiting the burst release of exosomes and achieving a slow, continuous release of the core layer. Based on this, by adding an appropriate amount of modifier after the freeze-thaw cycle and before adding exosomes, the strong hydrogen bonding between polyvinyl alcohol molecular chains can be reduced, the fluidity and electrospinning formability of the core spinning solution can be improved, the flexibility of the core fiber can be increased, and the dispersion uniformity and loading of exosomes in the core spinning solution can be improved, ensuring that the exosomes are uniformly distributed in the fiber, thereby ensuring the stability and repeatability of the release behavior.

[0042] As some specific examples of the present invention, the biodegradable polymeric material is selected from one or more of the following: silk fibroin, polycaprolactone, polylactic acid-glycolic acid copolymer, polyhydroxyacetic acid, polyhydroxy fatty acid ester, polypropylene carbonate, polydioxanone, polyurethane, gelatin, chitosan, collagen, etc.

[0043] As a preferred example of the present invention, the biodegradable polymeric material is selected from silk fibroin and polycaprolactone.

[0044] As some specific examples of the present invention, the organic solvent is selected from one or more of hexafluoroisopropanol, chloroform, dichloromethane, toluene, xylene, ethyl acetate, propyl acetate, ethyl propionate, carbon disulfide, ethanol, and acetone.

[0045] In the selection of bone regeneration scaffold materials, this invention utilizes the excellent properties and synergistic effects of silk fibroin (SF) and polycaprolactone (PCL) as shell materials for constructing an exosome sustained-release delivery system. Silk fibroin is widely available and has excellent biocompatibility. Its molecular structure contains active groups such as amino and hydroxyl groups, which can significantly improve the hydrophilicity of the material, promote osteoblast adhesion, proliferation, and differentiation, and provide a favorable bioactive environment for bone regeneration. Polycaprolactone (PCL), as a biodegradable polymer, has excellent film-forming properties, mechanical strength, and slow degradation characteristics, providing stable structural support for the scaffold, ensuring the spatial configuration required during bone repair, and avoiding support failure due to rapid material degradation. Using silk fibroin (SF) and polycaprolactone (PCL) as the shell layer of the core-shell structured fiber in this invention can achieve a synergistic balance between the construction of a bioactive environment and structural support function. At the same time, this invention also selects polyvinyl alcohol (PVA) as a hydrophilic core layer carrier to load exosomes, which can effectively improve the uniformity of exosome loading, provide a stable internal reservoir for the subsequent continuous release of exosomes, and further optimize the delivery efficiency and activity maintenance effect of exosomes.

[0046] As some specific examples of the present invention, in step (2), the total mass concentration of silk fibroin and polycaprolactone in the shell spinning solution is 5 wt% to 10 wt%, more preferably 7 wt%.

[0047] As some specific examples of the present invention, in step (2), the mass ratio of silk fibroin to polycaprolactone in the shell spinning solution is 1:6 to 6:1, more preferably, the mass ratio of silk fibroin to polycaprolactone in the shell spinning solution is 2:5.

[0048] As a preferred example of the present invention, in step (2), the organic solvent is hexafluoroisopropanol.

[0049] As some examples of the present invention, in step (3), the distance between the needle and the receiver in the coaxial electrospinning device is set to 10~20cm; the voltage is set to 12~20kV; the flow rate of the shell spinning solution is set to 0.3~1.0 mL / h; and the flow rate of the core spinning solution is set to 0.1~0.5 mL / h.

[0050] As a preferred example of the present invention, in step (3), the distance between the needle and the receiver in the coaxial electrospinning device is set to 15 cm; the voltage is set to 16 kV; the shell flow rate is set to 0.6 mL / h; and the core flow rate is set to 0.3 mL / h.

[0051] As a preferred example of the present invention, in step (3), the diameter of the coaxial core-shell fiber output by the coaxial electrospinning device is in the nanometer or micrometer range; more preferably, the diameter of the coaxial core-shell fiber is 50nm~500nm.

[0052] In addition, the present invention also provides a coaxial core-shell fiber sustained-release system based on PVA core layer-loaded exosomes, which is prepared by the above-described preparation method.

[0053] In addition, the present invention also provides the application of the above-mentioned coaxial core-shell fiber sustained-release system based on PVA core layer-encapsulated exosomes in the preparation of materials that improve gene defect-related bone regeneration disorders.

[0054] Specifically, as some examples of the present invention, the coaxial core-shell fiber sustained-release system based on PVA core layer encapsulated exosomes described in the present invention can be used to prepare implantable continuous delivery materials for bone tissue defect repair.

[0055] As some examples of the present invention, the coaxial core-shell fiber sustained-release system based on PVA core layer-encapsulated exosomes described in the present invention can be used to prepare bone defect repair materials caused by impaired osteogenic function.

[0056] As some examples of the present invention, the coaxial core-shell fiber sustained-release system based on PVA core layer-encapsulated exosomes described in the present invention can be used to prepare cranial critical defect repair materials related to gene defects.

[0057] As some examples of the present invention, the gene defect-related bone regeneration disorder is a bone defect repair disorder caused by impaired osteogenic function.

[0058] As some examples of the present invention, the gene defect-related bone regeneration disorder is p75NTR defect-related bone regeneration disorder.

[0059] In summary, the coaxial core-shell fiber sustained-release system based on PVA core layer encapsulated exosomes described in this invention utilizes coaxial electrospinning technology to encapsulate exosomes in the fiber core layer, and uses silk fibroin and polycaprolactone as the shell layer, thereby forming an exosome fiber sustained-release system with a core-shell structure through the gradual degradation of the shell layer.

[0060] The coaxial core-shell fiber sustained-release system based on PVA core layer encapsulated exosomes described in this invention can achieve efficient encapsulation and stable sustained release of exosomes, and can well preserve the biological activity of exosomes loaded in the fiber. Compared with direct injection of exosomes, it can delay the release rate of exosomes, prolong their residence time at the target site, avoid burst release, and achieve long-term, linear release of exosomes.

[0061] The coaxial core-shell fiber sustained-release system based on PVA core layer-encapsulated exosomes described in this invention can regulate the shell structure, fiber morphology, and exosome release behavior by adjusting the ratio of silk fibroin to polycaprolactone in the shell, thereby controlling the exosome release cycle. The prepared coaxial core-shell fibers can utilize the shell as a physical barrier to protect the exosomes in the core layer, reducing the impact of organic solvents and external environmental factors on their activity during preparation. Furthermore, the gradual degradation and diffusion regulation of the shell material enables sustained exosome release, prolonging their local action time at the defect site, thus providing a continuous bioactive signal for improving gene defect-related bone regeneration disorders.

[0062] This invention uses polyvinyl alcohol as a hydrophilic core layer carrier and silk fibroin and polycaprolactone as shell materials. Silk fibroin helps improve the biocompatibility and cell interaction of the material, while polycaprolactone helps provide the necessary structural stability and slow degradation characteristics. Thus, the coaxial core-shell fiber has the dual functions of exosome activity protection, continuous release and bone repair applicability, providing a new technical solution for improving gene defect-related bone regeneration disorders.

[0063] This invention improves the stability of exosomes during local application and prolongs their duration of action in bone defect areas by encapsulating exosomes in a hydrophilic core layer and using a shell layer for physical encapsulation and release regulation. This makes it more suitable for the needs of continuous bioactive stimulation and local repair microenvironment regulation in gene defect-related bone regeneration disorders.

[0064] Furthermore, the application described in this invention emphasizes improving the bone regeneration-related microenvironment and promoting bone repair through localized and sustained delivery of exosomes, rather than directly repairing the pathogenic genes themselves.

[0065] The following specific embodiments further illustrate the implementation of the present invention: Example 1

[0066] Preparation of a coaxial core-shell fiber sustained-release system based on PVA core layer-loaded exosomes: First, 0.7 g of polyvinyl alcohol (PVA) was added to 10 mL of deionized water; the mixture was then magnetically stirred at 37 °C to obtain a 7 wt% PVA aqueous solution for later use. Next, exosomes derived from ectodermal mesenchymal stem cells were added to 10 mL of 7% PVA aqueous solution, respectively, to achieve final concentrations of 5, 10, and 20 mg / mL of exosomes in the nuclear layer. After thorough mixing, the solutions were stored at 4 °C for later use.

[0067] Silk fibroin (SF) and polycaprolactone (PCL) were dissolved in 10 mL of hexafluoroisopropanol solvent at mass ratios of 1:6, 2:5, 3:4, 1:1, 5:2, and 6:1 to prepare SF / PCL blended shell solutions with a total concentration of 7 wt% in order to screen for the optimal shell ratio.

[0068] The core layer spinning solution and the shell layer spinning solution are injected into the inner and outer needles of a coaxial needle, respectively. Spinning is performed under the action of a high-voltage electric field to collect exosome-loaded SF / PCL / PVA coaxial core-shell fibers, which is the coaxial core-shell fiber sustained-release system based on PVA core layer encapsulated exosomes described in this invention. The distance between the needle and the receiver in the coaxial electrospinning device is set to 15 cm; the voltage is set to 16 kV; the shell layer flow rate is set to 0.6 mL / h; and the core layer flow rate is set to 0.3 mL / h.

[0069] Example 2

[0070] Preparation of a coaxial core-shell fiber sustained-release system based on PVA core layer-loaded exosomes: First, 0.7 g of polyvinyl alcohol (PVA) was added to 10 mL of deionized water. After obtaining a 7 wt% PVA aqueous solution by magnetic stirring at 37 °C, the PVA aqueous solution was frozen at -20 °C to -10 °C for 12 hours, and then thawed at 4 °C for 4 hours. This was repeated twice to obtain a PVA solution with a physically cross-linked microregion structure. After the PVA solution naturally returned to room temperature, 0.5 wt% of Tween-80 was added to it and stirred until completely dissolved. Then, exosomes derived from ectodermal mesenchymal stem cells were added to make the final concentration of exosomes in the mixture 10 mg / mL. After stirring and mixing, the pretreated nuclear spinning solution was obtained and stored at 4 °C for later use.

[0071] Silk fibroin (SF) and polycaprolactone (PCL) were dissolved in 10 mL of hexafluoroisopropanol solvent at a mass ratio of 5:2 to prepare an SF / PCL blended shell solution with a total concentration of 7 wt%.

[0072] The core layer spinning solution and the shell layer spinning solution are injected into the inner and outer needles of a coaxial needle, respectively. Spinning is performed under the action of a high-voltage electric field to collect exosome-loaded SF / PCL / PVA coaxial core-shell fibers, which is the coaxial core-shell fiber sustained-release system based on PVA core layer encapsulated exosomes described in this invention. The distance between the needle and the receiver in the coaxial electrospinning device is set to 17 cm; the voltage is set to 12 kV; the shell layer flow rate is set to 0.5 mL / h; and the core layer flow rate is set to 0.4 mL / h.

[0073] Experimental Example 1 To investigate the effect of the mass ratio of silk fibroin (SF) to polycaprolactone (PCL) in the shell on the fiber microstructure and diameter, scanning electron microscopy was used to observe the microstructure of the coaxial core-shell fiber sustained-release system based on PVA core-shell exosomes prepared with different SF / PCL mass ratios (1:6, 2:5, 3:4, 1:1, 5:2, 6:1) in Example 1. The results are as follows. Figure 1 The results are shown.

[0074] Experimental Example 2 To verify the formation of the core-shell structure, 0.01 g of calcein (a green fluorescent dye) was added to the core solution, and 0.01 g of rhodamine B (a red fluorescent dye) was added to the shell solution. The fibers were spun in the dark for 3 seconds under the conditions of Example 1, collected on a glass slide, and immediately observed under an inverted fluorescence microscope. The results are as follows. Figure 2 As shown, red fluorescence is distributed around the periphery of the fiber, while green fluorescence is concentrated in the center of the fiber, exhibiting clear spatial layering characteristics. This indicates that the core-shell structure was successfully constructed, providing a structural basis for the encapsulation of exosomes in the core layer.

[0075] Experimental Example 3 A PVA-based coaxial core-shell fiber sustained-release system for exosomes loaded with different concentrations (5, 10, 20 mg / mL) of ectodermal mesenchymal stem cells was placed in PBS solution and incubated at 37°C. PBS solutions were collected on days 1, 3, 5, 7, 10, 14, 21, and 28. The exosome concentration in the solution was detected using a BCA kit, and exosome release curves were plotted. Fresh PBS was used after each exosome collection. The results are shown below. Figure 3As shown, the lines of different colors represent the release curves for different exosome concentrations: black line represents an exosome concentration of 5 mg / mL, blue line represents an exosome concentration of 10 mg / mL, and yellow line represents an exosome concentration of 20 mg / mL. In SF / PCL / PVA coaxial core-shell fibers loaded with different concentrations of exosomes, the total release period of exosomes (Exo) lasted approximately 28 days. Although the 20 mg / mL group showed a relatively linear release curve during the sustained release phase, its initial burst release effect was more pronounced, and it reached the release plateau earlier than the 10 mg / mL group. Considering these results, the PVA core-layer-based coaxial core-shell fiber sustained-release system loaded with 10 mg / mL Exo exhibited the best long-acting, linear, slow-release performance.

[0076] Experiment 4: Evaluation of skull defect repair in gene-deficient mice To evaluate the repair effect of the coaxial core-shell fiber sustained-release system based on PVA core layer loaded with exosomes in gene defect-related bone regeneration disorders, a skull defect model was established using gene defect mice, and micro-CT observation and quantitative analysis were performed 8 weeks after surgery. p75NTR gene defect mice were selected to establish a skull defect model. Based on the different implanted materials, the experimental animals were divided into the following groups: (1) p75NTR gene defect control group (Control group); (2) Free exosome group (Exo group); (3) Blank fiber membrane group: SF / PCL / PVA coaxial core-shell fiber membrane without exosome loading was implanted into the defect area (SF / PCL / PVA group); (4) Exosome-loaded SF / PCL / PVA coaxial core-shell fiber membrane group (SF / PCL / PVA group). Exo The exosomes are divided into groups (n=5), with each group having n=5; preferably, the exosomes are exosomes derived from ectodermal mesenchymal stem cells. The exosomes loaded with SF / PCL / PVA coaxial nucleo-shell fibrous membranes are prepared using the method described in Example 1 above.

[0077] Under aseptic conditions, a skull defect modeling surgery was performed on mice with the aforementioned gene deficiency. Standardized bone defects were prepared at specific locations on the skull, and corresponding experimental materials were implanted into each defect. After surgery, the mice were routinely fed for 8 weeks. Skull specimens were then collected for micro-CT scanning to reconstruct three-dimensional images of the defect area. Quantitative morphological analysis of the defect area was performed, and the results are shown below. Figure 5The detection indicators included BS (Bone Surface Area) and BV / TV (Bone Volume / Total Volume). BS reflects the surface characteristics of newly formed bone tissue in the defect area, while BV / TV evaluates the degree of new bone formation and bone mass recovery within the defect area. Micro-CT three-dimensional reconstruction results at 8 weeks post-surgery showed that new bone formation in the defect area was limited in the gene-deficient control group, and the defect area remained relatively obvious. In contrast, the exosome-loaded SF / PCL / PVA coaxial core-shell fibrous membrane group showed more newly formed bone tissue filling the defect area, and improved defect edge continuity, suggesting that this material can promote cranial defect repair in the context of gene defects. Further quantitative analysis showed that the BS and BV / TV of the exosome-loaded SF / PCL / PVA coaxial core-shell fibrous membrane group were higher than those of the control group and superior to those of the unloaded exosome fibrous membrane group, with statistically significant differences (P<0.05). This indicates that the material can improve the level of bone formation and bone mass recovery in the defect area, thereby improving gene-deficient-related bone regeneration disorders.

[0078] Experimental Example 5: Comparison of Exosome Sustained-Release Effects Following the method described in Example 3 above, exosome sustained-release experiments were conducted on the exosome-loaded SF / PCL / PVA coaxial core-shell fibers prepared in Examples 1 (unmodified) and 2 (modified), and the results were plotted as follows. Figure 4 The exosome release curve shown is based on Figure 4 It can be seen that the release rate of the modified group is slowed down and the burst release rate at 24h is significantly reduced, indicating that the microcrystalline physical cross-linking network constructed by freeze-thaw cycle can effectively inhibit the burst release of exosomes and achieve sustained release.

[0079] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A method for preparing a coaxial core-shell fiber sustained-release system based on PVA core layer-encapsulated exosomes, characterized in that, Including the following steps: (1) Disperse polyvinyl alcohol and exosomes in water to obtain a core spinning solution; (2) Dissolve biodegradable polymer materials in an organic solvent to obtain shell spinning solution; (3) The core spinning solution is injected into the inner needle of the coaxial needle in the coaxial electrospinning device, and the shell spinning solution is injected into the outer needle of the coaxial needle. Coaxial electrospinning is performed under the action of a high voltage electric field to collect a coaxial core-shell fiber sustained-release system based on PVA core layer loaded exosomes.

2. The method for preparing a coaxial core-shell fiber sustained-release system based on PVA core layer-loaded exosomes according to claim 1, characterized in that, In step (1): The exosomes are derived from mesenchymal stem cells; and / or, The concentration of exosomes in the core spinning solution is 5-20 mg / mL; and / or, The nuclear spinning solution after adding exosomes was stored at 1-6℃ for later use.

3. The method for preparing a coaxial core-shell fiber sustained-release system based on PVA core layer-loaded exosomes according to claim 2, characterized in that, In step (1), the exosomes are derived from ectodermal mesenchymal stem cells.

4. The method for preparing a coaxial core-shell fiber sustained-release system based on PVA core layer-loaded exosomes according to claim 1, characterized in that, In step (2): The biodegradable polymer material is selected from silk fibroin and polycaprolactone, and the mass ratio of silk fibroin to polycaprolactone in the shell spinning solution is 1:6 to 6:1; and / or, The total mass concentration of silk fibroin and polycaprolactone in the shell spinning solution is 5 wt% to 10 wt%; and / or, The organic solvent is selected from one or more of hexafluoroisopropanol, chloroform, dichloromethane, toluene, xylene, ethyl acetate, propyl acetate, ethyl propionate, carbon disulfide, ethanol, and acetone.

5. The method for preparing a coaxial core-shell fiber sustained-release system based on PVA core layer-encapsulated exosomes according to claim 1, characterized in that, In step (3), the distance between the needle and the receiver in the coaxial electrospinning device is set to 10~20cm; the voltage is set to 12~20kV; the flow rate of the shell spinning solution is set to 0.3~1.0 mL / h; and the flow rate of the core spinning solution is set to 0.1~0.5 mL / h.

6. The method for preparing a coaxial core-shell fiber sustained-release system based on PVA core layer-encapsulated exosomes according to claim 1, characterized in that, In step (1), the preparation process of the core spinning solution is as follows: Polyvinyl alcohol is dispersed in water and stirred until dissolved. Then, exosomes are added and dispersed evenly to obtain a core spinning solution. The mass concentration of polyvinyl alcohol in the core spinning solution is 5 wt% to 10 wt%.

7. The method for preparing a coaxial core-shell fiber sustained-release system based on PVA core layer-loaded exosomes according to claim 1, characterized in that, In step (1), the preparation process of the core spinning solution is as follows: Polyvinyl alcohol is dissolved in water and stirred until completely dissolved. The resulting polyvinyl alcohol aqueous solution is then frozen at -20℃ to -10℃ for 6 to 24 hours, followed by thawing at 2℃ to 8℃ for 2 to 6 hours. This is one cycle, and the cycle is repeated 1 to 3 times to obtain a polyvinyl alcohol solution with a physically cross-linked microregion structure. After the polyvinyl alcohol solution naturally returns to room temperature, a modifier of 0.3~0.8 wt% of polyvinyl alcohol is added to it, stirred and dissolved, and then exosomes are added and stirred and dispersed evenly to obtain the pretreated core spinning solution.

8. The method for preparing a coaxial core-shell fiber sustained-release system based on PVA core layer-encapsulated exosomes according to claim 7, characterized in that, The modifier is selected from one or more of fatty alcohol polyoxyethylene ether, Tween, poloxamer, and polyethylene glycol; and / or, The degree of alcoholysis of the polyvinyl alcohol is 85-92%; and / or, The degree of polymerization of the polyvinyl alcohol is 500~1700; and / or, The mass concentration of polyvinyl alcohol in the core spinning solution is 5 wt% to 8 wt%.

9. A coaxial core-shell fiber sustained-release system based on PVA core layer-loaded exosomes, characterized in that, It is prepared by any one of the preparation methods of claims 1 to 8.

10. The application of the PVA core-layer-encapsulated exosome coaxial core-shell fiber sustained-release system according to claim 9 in the preparation of materials that promote bone tissue regeneration.

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