A cerebrospinal fluid-enriched silk fibroin hydrogel scaffold system loaded with neural stem cell exosomes, preparation method and application thereof

Abstract

The present application relates to a cerebrospinal fluid-enriched silk fibroin hydrogel support system loaded with neural stem cell exosomes, a preparation method and applications thereof, in particular, a cerebrospinal fluid-enriched silk fibroin hydrogel loaded with neural stem cell-derived exosomes to promote the repair of neurons after spinal cord injury, belonging to the technical field of biological medicine. The present application designs engineered neural stem cell exosomes targeting neuron cells, which can improve the ischemic environment after spinal cord injury, promote vascular regeneration and inhibit neuron aging, and is more beneficial to local nerve repair and regeneration. By combining with the cerebrospinal fluid-enriched silk fibroin hydrogel to form an exosome sustained-release system applied to the local spinal cord injury, the treatment efficiency of the exosomes is improved, rapid release is realized, the cumulative release amount is large, the action time is long, the functional recovery after spinal cord injury is obviously improved, and good practical application value is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes, a preparation method and applications thereof. Background Art

[0002] Spinal cord injury (SCI) is a common condition in spinal surgery. It refers to a central nervous system disorder characterized by physiological dysfunction or loss of sensory and motor functions, resulting from complete or incomplete damage to the spinal cord tissue following direct or indirect trauma. According to statistics, approximately 10.4 to 83 cases of SCI occur per million people worldwide each year, with morbidity and mortality rates rising annually. Mild SCI can lead to loss of productivity, while severe SCI can cause limb paralysis, incontinence, and loss of the ability to care for oneself, severely impacting patients' quality of life. Unfortunately, due to the poor plasticity of the central nervous system and limited neural regeneration, the regenerative capacity of the spinal cord is extremely weak after injury. Currently, there is no effective treatment to fully restore neurological function after SCI.

[0003] Exosomes are subcellular components secreted by cells. They are essentially nanoscale membrane vesicles with a diameter ranging from approximately 30 to 100 nm and a double-layer membrane structure. Their contents primarily consist of proteins, lipids, and nucleic acids. Due to their high biocompatibility and low immunogenicity, exosomes often offer superior delivery capabilities compared to artificial liposome-based drug delivery systems, making them useful as drug delivery vehicles. However, natural exosomes lack targeting, resulting in rapid clearance and uptake by non-target cells or interactions with cell membranes, resulting in a short half-life for exosome-derived drugs. To address these limitations of natural exosomes, researchers have developed a variety of strategies for engineering exosomes, including surface engineering of the exosome membrane to enhance exosome targeting and stability, and engineering the exosome's contents to allow for drug loading. These modified exosomes are collectively referred to as engineered exosomes. Numerous studies have demonstrated the potential of engineered exosomes for the treatment of spinal cord injury.

[0004] Patent CN 113046316 B discloses the use of exosomes-hydrogels to promote local angiogenesis after spinal cord injury. This invention establishes a mouse spinal cord injury model and observes the therapeutic effect of exosomes derived from M2 bone marrow macrophages on spinal cord injury. The therapeutic effect of the sustained-release system formed by the combination of exosomes and hydrogels on spinal cord injury is evaluated by observing various indicators after spinal cord injury. Immunofluorescence staining is also used to observe the regeneration of blood vessels at the injured site of the mouse tail spinal cord.

[0005] The internal environment after spinal cord injury is primarily characterized by low oxygen, low nutrition, and high acidity. Impaired oxygen clearance leads to metabolic disorders in nerve cells, significantly reducing repair efficiency and ultimately causing them to age. Related studies have confirmed that injury can trigger the aging of nerve cells in the spinal cord tissue and the disruption of the extracellular matrix, further confirming the importance of cellular senescence in the development and progression of spinal cord injury. Currently, there are no exosome products specifically designed to inhibit neuronal aging after spinal cord injury.

[0006] Cerebrospinal fluid (CSF) is a clear fluid that surrounds the brain and provides mechanical support, transfers micronutrients and signaling molecules to neurons, and participates in the removal of unnecessary metabolites. CSF directly interacts with the extracellular space of the brain and can therefore reflect the occurrence of pathophysiological changes. Studies have found that CSF from young mice can reduce cellular senescence in the cerebral cortex of Alzheimer's mice and improve memory impairment in Alzheimer's mice. This mechanism may be related to CSF ​​inhibiting neuronal senescence by regulating complement C3 / C3aR. Currently, there are no reports on the use of CSF to prevent and treat neuronal senescence after spinal cord injury, thereby repairing spinal cord damage.

[0007] The regenerative capacity of the central nervous system is limited, so effective treatments are urgently needed to promote the repair of damaged nerves. Summary of the Invention

[0008] The purpose of the present invention is to solve the above problems and provide a cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes, a preparation method and its application. The cerebrospinal fluid-rich silk protein hydrogel and the neural stem cell-derived exosomes are used to repair neurons after spinal cord injury. The present invention designs engineered neural stem cell exosomes targeting neuronal cells, which can improve the local ischemic environment after spinal cord injury, promote angiogenesis and inhibit neuronal aging, and are more conducive to the repair and regeneration of local nerves. By combining with a silk protein hydrogel rich in mouse cerebrospinal fluid to form an exosome sustained-release system for application to the local spinal cord injury, the exosome treatment efficiency is improved, sustainable sustained release is achieved, and long-term action is achieved, significantly improving the functional recovery after spinal cord injury in mice, and has good practical application value.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] In a first aspect, the present invention provides an engineered exosome, which is a neural stem cell exosome transfected with RVG-LAMP2B and secreted by a neural stem cell.

[0011] Furthermore, the engineered exosomes contain specific markers including CD63, CD9 and TSG101.

[0012] Furthermore, the engineered exosomes, namely RVG-LAMP2B-Exos, are obtained by isolating and culturing primary neural stem cells, then incubating them with complete medium (DMEM complete medium) containing exosome-free serum, and collecting them by differential centrifugation.

[0013] Furthermore, the engineered exosomes are prepared into a local administration (injection) preparation or a local external use preparation.

[0014] The second aspect of the present invention provides a cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes, the hydrogel scaffold system comprising: a composite hydrogel scaffold, and engineered exosomes loaded on the composite hydrogel scaffold; the preparation method of the composite hydrogel scaffold comprises the following steps: after the silk protein is modified by methacrylation, cerebrospinal fluid is added, and the composite hydrogel scaffold is formed under light conditions.

[0015] Furthermore, the engineered exosomes are extracted from neural stem cells after transfection of the RVG-LAMP2B plasmid, and are used to increase the senescence phenotype of neuronal cells that appear after spinal cord injury, and to target and inhibit neuronal cell senescence.

[0016] Furthermore, the composite hydrogel scaffold is prepared by the following method: after the silk protein is modified by methacrylation, cerebrospinal fluid is added, and the composite hydrogel scaffold is formed under 405nm light conditions.

[0017] Furthermore, the cerebrospinal fluid is the cerebrospinal fluid of young mice. The cerebrospinal fluid can also be commercially available artificial cerebrospinal fluid.

[0018] A third aspect of the present invention provides a method for preparing a cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes. The method for preparing the hydrogel scaffold system comprises:

[0019] Preparation of composite hydrogel scaffolds and engineered exosomes;

[0020] The engineered exosomes are added to a composite hydrogel scaffold to prepare a hydrogel scaffold system.

[0021] Furthermore, the method for constructing a hydrogel scaffold system by adding the engineered exosomes to a composite hydrogel scaffold comprises the following steps: mixing the engineered exosomes with the composite hydrogel scaffold, wherein the concentration of the engineered exosomes in the composite hydrogel scaffold is 100-200 ug / mL, co-culturing for 1-2 hours under dark conditions to form an aqueous exosome-hydrogel preparation, namely the hydrogel scaffold system, which is stored at 4°C in the dark.

[0022] Furthermore, the composite hydrogel scaffold is an aqueous photo-crosslinked hydrogel.

[0023] Furthermore, the hydrogel scaffold system is a topical hydrogel scaffold system. The preparation method of the topical hydrogel scaffold system includes an exosome-hydrogel preparation using a photo-crosslinked cerebrospinal fluid-rich silk protein hydrogel as a carrier. The specific method for preparing the hydrogel scaffold includes: methacrylating the silk protein, adding it to the cerebrospinal fluid of young mice, and irradiating it with 405nm light to form a composite hydrogel scaffold.

[0024] The fourth aspect of the present invention also provides the use of the hydrogel scaffold system in the preparation of spinal cord injury repair drugs (spinal cord injury treatment preparations).

[0025] Furthermore, the engineered exosomes were mixed with the composite hydrogel scaffold, the concentration of the engineered exosomes in the composite hydrogel scaffold was 100-200 ug / mL, and co-cultured for 1-2 hours under dark conditions to form an exosome-hydrogel sustained-release preparation, i.e., the spinal cord injury repair drug, which was stored at 4°C in the dark.

[0026] Furthermore, the hydrogel scaffold system was implanted in situ into the spinal cord injury defect.

[0027] Furthermore, when the exosome-gel sustained-release preparation is used, it is applied as a dressing to the injured spinal cord at a rate of 80-120ul / cm 2 Covered exosome-gel sustained-release preparation, at 405nm 60mW / cm 2 Under the given conditions, the hydrogel is exposed to light for 30s-1min to transform from liquid phase to solid phase, and the wound is sutured layer by layer.

[0028] Furthermore, the spinal cord injury repair drug includes one or more of the following uses:

[0029] a) Enhance neuronal angiogenesis after spinal cord injury;

[0030] b) inhibiting neuronal senescence after spinal cord injury;

[0031] c) Improve motor function recovery after spinal cord injury.

[0032] The technical concept of this application is as follows:

[0033] Exosomes derived from neural stem cells (NSC) have been shown to have specific neurorepair effects. A large number of studies have shown that engineered exosomes can promote the treatment of spinal cord injury. The engineered modification of exosomes can be given powerful therapeutic capabilities by targeted delivery through transfection of plasmid fragments. Based on this, RVG-LAMP2B is transfected into exosomes to achieve targeted neurons and repair damage. The present invention overcomes the existing technical defects and prepares a neuron-targeted neural stem cell exosome, namely RVG-LAMP2B-Exos, which can promote angiogenesis after spinal cord injury and inhibit neuronal aging, which is more conducive to nerve repair and regeneration. By combining with a photo-crosslinked cerebrospinal fluid-rich silk protein hydrogel to form an exosome scaffold system for application to local spinal cord injury, the therapeutic efficiency of exosomes is improved, and sustainable sustained release is achieved to achieve long-term action, significantly improving functional recovery after spinal cord injury in mice.

[0034] This study found that the cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes can alleviate spinal cord injury and play a repair role. In cell experiments, the material has the effect of reducing lipopolysaccharide (LPS)-induced neuronal cell inflammatory response and inhibiting neuronal cell aging. The specific mechanism of action is: engineered exosomes inhibit the decomposition of glutamine and reduce the level of neuronal cell aging, thereby alleviating spinal cord injury. At the same time, the present invention also found through animal experiments that injecting the material into the damaged spinal cord of mice can effectively repair the damage.

[0035] Compared with the prior art, the present invention has the following advantages:

[0036] 1) The present invention provides a cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes for in situ treatment of spinal cord injury, which can inhibit the decomposition of glutamine and reduce the aging level of neuronal cells, thereby alleviating spinal cord injury.

[0037] 2) The present invention uses cerebrospinal fluid-rich silk protein hydrogel as an exosome carrier. Generally, hydrogels are easily affected by the environment, which can easily change the gel state and affect the culture effect of exosomes, making it difficult for the exosomes to be evenly distributed in the gel. Silk protein hydrogels will only become solid phases under irradiation within a specific wavelength range, and have good biocompatibility, allowing exosomes to better combine with hydrogels in the aqueous phase, greatly increasing their effect. The addition of cerebrospinal fluid to the hydrogel can inhibit neuronal aging to a greater extent.

[0038] 3) The present invention uses a cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes to repair spinal cord injury, providing a new use for the material, greatly improving its application value, expanding the application range of the exosome-hydrogel, and opening up new areas of application, which has broad application prospects and commercial value.

[0039] 4) The present invention utilizes a hydrogel rich in cerebrospinal fluid of young mice to provide a sustained-release environment for exosomes. The cerebrospinal fluid in the cerebrospinal fluid-rich silk protein hydrogel serves as a carrier to delay the degradation of exosomes, relying on the exosomes to exert a therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Figure 2 is the flow cytometry test result for the identification of neural stem cells, including (a) the expression of Nestin and (b) the expression of SOX2.

[0041] Figure 2 These are test images of engineered exosomes, including (a) transmission electron microscopy image, which shows that the exosomes have a spherical morphology; (b) particle size-concentration distribution diagram of nanoparticle tracking analysis (NTA); (c) volume particle size distribution diagram of nanoparticle tracking analysis. NTA measurement shows that the average diameter of exosomes is around 100 nm.

[0042] Figure 3 These are the Western blot images of the NSCs group, NSCs-Exos group, Exos-NC group, and RVG-LAMP2B-Exos group. Western blot verified the expression of engineered exosome markers including CD63, CD9, TSG101, and LAMP2B.

[0043] Figure 4 These are the staining results of RVG-LAMP2B-Exos binding to mouse spinal cord neurons, where (a) the phalloidin staining results show that RVG-LAMP2B-Exos bind most tightly to neurons, and (b) the quantitative expression of immunofluorescence staining.

[0044] Figure 5 Comparison of the exosome release rates from silk hydrogels before and after the addition of cerebrospinal fluid. (a) The composite hydrogel with cerebrospinal fluid releases more exosomes per day. Furthermore, compared to the silk hydrogel-exosomes group alone, the exosome release from the cerebrospinal fluid-exosomes group is more sustained. (b) The composite hydrogel with cerebrospinal fluid exhibits a higher cumulative exosome release efficiency.

[0045] Figure 6 The figures show the results of validating the effects of RVG-LAMP2B-Exos on endothelial cell and neuronal aging, including: (a) angiogenesis diagram of the Control group, LPS, and LPS+RVG-LAMP2B-Exos group; (b) Western blot diagram of the Control group, LPS, and LPS+RVG-LAMP2B-Exos group.

[0046] Figure 7 These are the staining results of cerebrospinal fluid-rich silk protein hydrogel-exosomes in alleviating spinal cord injury in vivo, including: (a) HE staining results, (b) quantitative expression of HE staining, (c) NF, GFAP, DAPI, and Merge staining results, and (d) quantitative expression of NF, GFAP, DAPI, and Merge staining.

[0047] Figure 8 Diagram of the mechanism of action of cerebrospinal fluid-rich silk protein hydrogel loaded with engineered exosomes derived from neural stem cells in repairing spinal cord injury. DETAILED DESCRIPTION

[0048] The present invention is described in detail below with reference to the accompanying drawings and examples. The following examples will help those skilled in the art further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that those skilled in the art may make various modifications and improvements without departing from the scope of the present invention. All of these modifications and improvements are protected by the present invention.

[0049] The present invention relates to a cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes, a preparation method and its application, specifically a cerebrospinal fluid-rich silk protein hydrogel loaded with neural stem cell-derived exosomes to promote the repair of neurons after spinal cord injury, belonging to the field of biomedicine. The present invention designs engineered neural stem cell exosomes targeting neuronal cells, which can improve the local ischemic environment after spinal cord injury, promote angiogenesis and inhibit neuronal aging, and are more conducive to the repair and regeneration of local nerves. By combining with cerebrospinal fluid-rich silk protein hydrogel to form an exosome sustained-release system for application to the local spinal cord injury, the exosome treatment efficiency is improved, rapid release is achieved, the cumulative release amount is large, the action time is long, and the functional recovery after spinal cord injury is significantly improved, with good practical application value.

[0050] Any features such as preparation methods, materials, structures or composition ratios that are not clearly described in this technical solution shall be deemed to be common technical features disclosed in the prior art.

[0051] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0052] Example 1

[0053] Neural stem cell isolation and culture:

[0054] Primary neural stem cells were extracted using 6-week-old C57BL / 6 mice. The specific steps are as follows: First, put the 6-week-old pregnant female SPF C57BL / 6 mice purchased from the Experimental Animal Department of Fudan University into the animal incubator, and place the cell extraction related articles in the ultraclean table and irradiate under ultraviolet lamp for half an hour. All instruments are high-pressure steam sterilized. The mice were first anesthetized by inhaling 3% isoflurane (RWD, Shenzhen, Guangdong), and then maintained in an anesthetized state with 1.8% isoflurane. Then perform a cesarean section operation, carefully dissect the uterus and placenta to extract the pups. The pregnant and pup mice were killed by excessive anesthesia. The pregnant and pup mice were killed by excessive anesthesia. The pups were immersed in 75% ethanol, made dorsal incisions on the skin and soft tissues, and obtained the spinal cord. Then, the excised spinal cord was washed in pre-cooled medium (Gibco, Gardena, CA), cut into small pieces, and transferred to a 100-micron pore size screen (Falcon, Corning, NY, USA). The obtained cell suspension was centrifuged at 300 rpm for 5 minutes at 4°C. The precipitated cells were resuspended in medium (Gibco, Gardena, CA) supplemented with 2% B27 (Gibco), glutamine (0.5 mM, Gibco), penicillin (100 IU / mL), streptomycin (100 mg / mL, Gibco), epidermal growth factor (20 ng / mL, Peprotech), and basic fibroblast growth factor (20 ng / mL, Peprotech) and cultured at 37°C with 5% CO2 for 48 hours to generate neural spheres. The culture medium was collected. Flow cytometry experiments verified the surface markers of neural stem cells. The results showed that Nestin and SOX2 were highly expressed in the cells Figure 1

[0055] RVG-LAMP2B-Exos acquisition:

[0056] ​The RVG-LAMP2B plasmid was constructed by GeneChem (Shanghai). To obtain engineered exosomes, neural stem cells were further transfected with the RVG-LAMP2B plasmid using Lipofectamine 2000 (Invitrogen, USA). The cells were centrifuged sequentially: 37°C, 300 rpm for 10 min, 4°C, 2000 rpm for 10 min, and 4°C, 10,000 rpm for 60 min. After centrifugation, the supernatant was passed through a 0.22 μm sterile filter (Falcon) to remove cellular debris. The filtrate was ultracentrifuged at 120,000 rpm for 70 min, the supernatant discarded, and the pellet resuspended in 200 μL of phosphate-buffered saline (PBS, Gibco) to isolate and obtain RVG-LAMP2B-Exos (engineered exosomes). Characterization was performed by electron microscopy, flow cytometry, and Western blotting. Transmission electron microscopy and NTA measurements showed that the isolated exosomes exhibited a double-membrane spherical morphology with a diameter in the range of approximately 100 nm (see Figure 2 Western blot was used to verify the expression of exosome-specific markers CD63, CD9, and TSG101 as well as transfected LAMP2B (see Figure 3 ), DAPI, PKH26 and Phalloidine staining were used to verify that RVG-LAMP2B-Exos had the most obvious binding to mouse spinal cord neurons compared with NSCs-Exos (exosomes obtained by the same method as RVG-LAMP2B-Exos without transfection of neural stem cells with RVG-LAMP2B plasmid) and Exos-NC (natural exosomes collected from neural stem cells without transfection of any plasmid). Figure 4 ).

[0057] Preparation of cerebrospinal fluid-rich silk protein hydrogel-exosomes (cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes):

[0058] A certain amount of silk cocoons were weighed using an electronic balance, and silk protein fibers were extracted by degumming. The silk was dissolved with lithium bromide and then methacrylated (40 g of silkworm cocoons were placed in 8 L of 0.05 M Na2CO3 solution and boiled at 100 ° C for 45 min to remove serine. The cocoons were washed with warm water, cold water, and deionized water in sequence, and dried in an oven at 40 ° C for 24 h to obtain degummed silk fibroin. Subsequently, 20 g of degummed silk fibroin was dissolved in 9.3 M The solution was dissolved in a LiBr solution at 60°C for 1 hour with stirring at 300 rpm. Then, 6 mL of glycidyl methacrylate (GMA) was added to the solution and stirred at 60°C and 300 rpm for 4 hours. The resulting solution was dialyzed against deionized water using a dialysis membrane (14,000 Da cutoff) for 5 days to remove salts and unreacted GMA. For further measurements, a portion of the SilMA solution was frozen in liquid nitrogen for 30 minutes, lyophilized for 3 days, and stored at -20°C. The solution was concentrated to produce a silk protein hydrogel precursor solution with a mass fraction of 5% to 30% (30% in this example). To obtain cerebrospinal fluid from mice, the mice were anesthetized and their heads were fixed in an orienting device. The hair on the head and neck was clipped and disinfected. A longitudinal incision (approximately 1 cm) was made along the longitudinal axis with a scalpel. The dorsal muscles of the neck were bluntly dissected with scissors to expose the foramen magnum. Cerebrospinal fluid was directly aspirated through the foramen magnum. After aspiration, the outer layers of muscle and skin were sutured. Then, cerebrospinal fluid was added to the precursor solution at a mass ratio of 100:1 to 1:100 (100:1 in this embodiment), 405 nm, 60 mW / cm 2 Under the conditions of illumination for 30s-1min (1min in this embodiment), RVG-LAMP2B-Exos were then mixed into the liquid hydrogel at a ratio of 500μg:50μL to form a suspension to obtain CSF-SilkMA-RVG-LAMP2B-Exos. In addition, RVG-LAMP2B-Exos were added to the simple silk protein hydrogel (cerebrospinal fluid was not added to the precursor solution) at the same ratio to obtain SilkMA-RVG-LAMP2B-Exos as a comparative example. Compared with the cerebrospinal fluid-rich hydrogel used in this embodiment, it was found through this example that the composite hydrogel of the present invention after adding cerebrospinal fluid had a higher single-day release of exosomes and a higher cumulative release efficiency. At the same time, compared with the simple silk protein hydrogel-exosome group, the exosome release time of the cerebrospinal fluid-exosome group was longer ( Figure 5 ), it can be seen that the addition of cerebrospinal fluid can release more exosomes faster and at the same time achieve a longer release time, prolong the release time of exosomes, and increase the release amount of exosomes.

[0059] Effects of RVG-LAMP2B-Exos on endothelial cell and neuronal aging:

[0060] The present embodiment provides the experimental process of RVG-LAMP2B-Exos reducing the aging level of nerve cells in vitro and promoting angiogenesis. In the present embodiment, three experimental groups are set, which are: control group (spinal cord neuron cells without any treatment), LPS group (LPS, mouse spinal cord neuron cells are adjusted to a concentration of 2x10 7 / L of Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (10%, FBS) and penicillin-streptomycin solution (100 U / ml and 100 mg / ml, respectively), and the prepared cell suspension is inoculated in a 96-well plate at 100 μL per well, and is placed for 24 h, and is intervened with 5 μg / ml LPS for 12 h), LPS+RVG-LAMP2B-Exos group (LPS+RVG-LAMP2B-Exos, mouse spinal cord neuron cells are adjusted to a concentration of 2x10 7 / L of cell suspension, and the prepared cell suspension is inoculated in a 96-well plate at 100 μL per well, and is placed in a cell incubator for 24 h, and is replaced with a medium containing RVG-LAMP2B-Exos at a concentration of 10 μmol / L for 48 h, and then is intervened with 5 μg / ml LPS at a concentration of 5 μg / ml for 12 h). It is found through the present embodiment that, compared with the control group and the LPS group, the blood vessel formation experiment shows that RVG-LAMP2B-Exos has the effect of promoting angiogenesis Figure 6 a), and the WB result shows that RVG-LAMP2B-Exos reduces LPS-induced aging of neuron cells Figure 6 b).

[0061] Detection of cerebrospinal fluid-enriched silk fibroin hydrogel-exosomes in reducing spinal cord injury in mice:

[0062] This example provides an experimental process in which cerebrospinal fluid-rich silk protein hydrogel-exosomes reduce the level of neuroinflammation and inhibit neurofibrosis in vivo. In order to study the in vivo therapeutic effect, 40 6-week-old SD rats were taken. In this example, five experimental groups (examples or comparative examples) were set up, namely: Sham group (sham operation group), SCI group (simple spinal cord injury group), SCI + cerebrospinal fluid-rich silk protein hydrogel group (SCI + CSF-SilkMA, injected with 1 μL of 200 μg / mL CSF-SilkMA), SCI + silk protein hydrogel-exosome group (SCI + SilkMA-RVG-LAMP2B-Exos, injected with 1 μL of 200 μg / mL SilkMA and 1 μL of 20 μM / LRVG-LAMP2B-Exos mixture), SCI + cerebrospinal fluid-rich silk protein hydrogel-exosome group (SCI + CSF-SilkMA-RVG-LAMP2B-Exos, injected with 1 μL of 200 μg / mL CSF-SilkMA and 1 μL of a 20 μM / LRVG-LAMP2B-Exos mixture) (Example). After 4 weeks, the long-term recovery effect of the mice was evaluated. HE and NF / GFAP staining were performed. HE was used to observe neuroinflammation in spinal cord neurons. Our results showed that the SCI group had the strongest neuroinflammatory response, while the simple silk protein hydrogel-exosome group (SCI+silk protein hydrogel-exosome group) and the cerebrospinal fluid-rich hydrogel-exosome group (SCI+cerebrospinal fluid-rich silk protein hydrogel-exosome group) both had significantly reduced neuroinflammation (P<0.001). Among them, the cerebrospinal fluid-rich hydrogel-exosome group had the mildest inflammatory response, with almost no significant difference from the control group (Sham group); NF fluorescence staining was used to observe the expression and growth of neuronal axons, reflecting the repair and regeneration ability of neurons. Similarly, the results showed that the cerebrospinal fluid-rich hydrogel-exosome group had the highest NF expression, indicating that this group had the strongest axon repair effect (P<0.001); GFAP staining is a major marker for astrocytes and is used to assess the degree of fibrosis after nerve injury. The higher the degree of fibrosis, the worse the prognosis of scar repair will be. The experimental results showed that the SCI group had the most obvious neurofibrosis, while the cerebrospinal fluid-rich silk protein hydrogel-exosomes could significantly reverse this phenomenon compared with the other groups. In particular, compared with the simple silk protein hydrogel-exosomes group, the addition of cerebrospinal fluid further promoted the recovery of neurons (P<0.001). The simple cerebrospinal fluid-rich silk protein hydrogel group (SCI+cerebrospinal fluid-rich silk protein hydrogel group) was weaker than the cerebrospinal fluid-rich hydrogel-exosomes group (SCI+cerebrospinal fluid-rich silk protein hydrogel-exosomes group) in inhibiting neuroinflammatory response, neurofibrosis and promoting axonal growth. Figure 7 ).

[0063] A mouse spinal cord injury model was constructed and the silk fibroin hydrogel-exosome was implanted into the spinal cord injury site with (or without) cerebrospinal fluid enrichment. The silk fibroin hydrogel-exosome was applied in the form of a dressing according to 80-120ul / cm 2 The exosome-hydrogel preparation was irradiated at 405nm 60mW / cm 2 for 30s-1min under the condition, so that the hydrogel was converted from liquid phase to solid phase, and the wound was sutured layer by layer, as shown in Figure 8 .

[0064] The above description of the embodiments is to facilitate the understanding and use of the invention by those of ordinary skill in the art. Those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without having to undergo creative labor. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art within the scope of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes, characterized in that: The hydrogel scaffold system includes: a composite hydrogel scaffold, and engineered exosomes loaded on the composite hydrogel scaffold; The preparation method of the composite hydrogel scaffold comprises the following steps: after the silk protein is modified by methacrylation, cerebrospinal fluid is added to form the composite hydrogel scaffold under light conditions; The engineered exosomes are extracted after RVG-LAMP2B plasmid is transfected into neural stem cells.

2. The cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes according to claim 1, characterized in that: The composite hydrogel scaffold is prepared by the following method: after the silk protein is modified by methacrylation, cerebrospinal fluid is added, and the composite hydrogel scaffold is formed under 405nm light conditions.

3. A method for preparing a cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes according to any one of claims 1 to 2, characterized in that: The preparation method of the hydrogel scaffold system comprises: Preparation of composite hydrogel scaffolds and engineered exosomes; The engineered exosomes are added to a composite hydrogel scaffold to prepare a hydrogel scaffold system.

4. The preparation method according to claim 3, characterized in that The method for constructing a hydrogel scaffold system by adding the engineered exosomes to a composite hydrogel scaffold comprises the following steps: mixing the engineered exosomes with the composite hydrogel scaffold, wherein the concentration of the engineered exosomes in the composite hydrogel scaffold is 100-200 ug / mL, and co-culturing for 1-2 hours under light-proof conditions to form an aqueous exosome-hydrogel preparation, namely the hydrogel scaffold system.

5. Use of the hydrogel scaffold system according to any one of claims 1 to 2 in preparing a drug for repairing spinal cord injury.

6. Use of the hydrogel scaffold system according to claim 5 in preparing a spinal cord injury repair drug, characterized in that: The hydrogel scaffold system was implanted in situ into the spinal cord injury defect.

7. The use according to claim 6, characterized in that When the hydrogel scaffold system is used, it is applied as a dressing on the surface of the injured spinal cord at a rate of 80-120ul / cm 2 Covered hydrogel scaffold system, 60mW / cm at 405nm 2 Under the given conditions, the hydrogel is exposed to light for 30s-1min to transform from the aqueous phase to the solid phase, and the wound is sutured layer by layer.

8. The use according to claim 6, characterized in that The hydrogel scaffold system is administered as a local injection formulation.

9. The use according to claim 5, characterized in that The spinal cord injury repair drug includes one or more of the following uses: a) Enhance neuronal angiogenesis after spinal cord injury; b) inhibiting neuronal senescence after spinal cord injury; c) Improve motor function recovery after spinal cord injury.

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