Injectable hydrogel for repairing craniocerebral injury and preparation method thereof

Abstract

The invention discloses injectable hydrogel for repairing craniocerebral injury and a preparation method of the injectable hydrogel, and relates to the technical field of biomedicine. The implant is injectable hydrogel loaded with stem cell-derived exosome, carboxymethyl chitosan and sodium alginate are used as base materials of the injectable hydrogel, the stem cell-derived exosome is used as a functional substance, D-glucose-delta lactone is used as a cross-linking agent, protons are released through hydrolysis of the D-glucose-delta lactone, and the stem cell-derived exosome-loaded injectable hydrogel is obtained. Electrostatic interaction is established between carboxymethyl chitosan and sodium alginate, a gelation process is induced, and the stem cell-derived exosome is encapsulated in a cross-linked network of the hydrogel. The injectable hydrogel for repairing the craniocerebral injury can realize local slow release of the exosome, improve retention of the exosome at a target site, reduce the probability of being cleared by a host, prolong the action time, reduce the dosage and improve the bioavailability and the treatment effect.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to an injectable hydrogel for repairing craniocerebral injury and a preparation method thereof. Background Art

[0002] Traumatic brain injury (TBI) is a temporary or permanent brain function injury. There are more than 50 million cases of brain trauma worldwide each year, and the mortality rate of severe TBI patients exceeds 30%. TBI is mainly caused by external forces on the head such as road traffic accidents (motor vehicles, motorcycles and bicycles), penetrating objects, explosions, violent behaviors and sports injuries. The secondary injury caused by TBI has a complex pathological process, including persistent neuroinflammatory response, oxidative stress, nerve cell damage, immune response, ventricular enlargement and edema, white matter and / or gray matter atrophy, blood-brain barrier destruction, increased intracranial pressure, cell apoptosis or autophagy, axonal damage and necrosis, metabolic dysfunction, etc., which ultimately lead to neurological dysfunction. Although the acute condition after trauma can be quickly controlled by surgical means (such as resection of damaged brain tissue, cleaning of cerebral hemorrhage and cranial flap repair), there are still many challenges in the secondary brain injury and long-term neurological dysfunction after surgery. At present, the main treatment for relieving secondary brain injury after surgery is to give neuroprotective drugs (such as gangliosides, vitamin B, etc.) and supplement with rehabilitation training, but the prognosis of patients is still unsatisfactory, and many patients still face varying degrees of cognitive impairment, movement disorders and other long-term neurological impairment. Therefore, studying an auxiliary treatment method for postoperative craniocerebral injury to improve neurological dysfunction after craniocerebral injury has important biomedical research value and broad clinical application prospects.

[0003] In recent years, stem cell therapy has played an important role in neurological diseases, regenerative medicine, and cancer treatment. Stem cell therapy strategies, such as embryonic stem cells, adult stem cells, and induced pluripotent stem cells, have received widespread attention and made great progress in a variety of diseases and disorders. This is because stem cells have the ability to self-renew and differentiate into multiple cell lineages. For example, transplantation of neural stem cells (NSCs) can protect neurons in animal stroke models. However, the success rate of treatment in clinical practice is still limited. At present, stem cell therapy faces several key issues. First, ethical and religious disputes remain an important obstacle. In addition, the supply of stem cells and the difficulty of extended culture also limit its widespread application. At the same time, stem cells may have genetic instability and potential allogeneic immune rejection, which limits their clinical application. Secondly, the administration of stem cells is to apply stem cells directly to the site of tissue injury or intravenous infusion. Studies have found that if stem cells are used directly at the treatment site, their survival time is short and they are prone to cause thrombosis, fever, tumors, etc., thus affecting the treatment effect and the overall safety of patients. For the first problem, the direct reprogramming strategy of somatic cells from one differentiated cell type to another differentiated cell type can solve possible ethical, supply restrictions, and immune rejection issues. At present, several research teams have successfully reprogrammed somatic cells into induced neural stem cells and obtained induced neural stem cells with differentiation potential. For the second problem, compared with stem cell therapy, the therapeutic effect of exosomes secreted by various types of stem cells (mesenchymal, neural, hematopoietic, and embryonic) is more significant. Studies have shown that exosomes can provide neuroprotection against TBI by counteracting cell death, apoptosis, pyroptosis, and ferroptosis through PINK1 / parkin-mediated mitochondrial autophagy. Other studies have shown that exosomes activate the lncRNA TUBB6 / Nrf2 pathway after TBI, inhibit TBI-induced inflammation and ferroptosis, and play a key role in neuroprotection. Therefore, exosomes have application potential in nerve injury repair. At present, the main methods of exosome administration are injection, oral administration, and local administration. However, there are still huge challenges in delivering therapeutic doses of exosomes to the target location. Studies have found that direct injection of exosomes (intravenous, intraperitoneal, subcutaneous injection) can quickly clear exosomes from the blood circulation and accumulate in non-targeted sites. Therefore, in order to increase the retention of exosomes at the target site, reduce the probability of being cleared by the host, and adapt to the wound surface of brain tissue damage, those skilled in the art have proposed an injectable hydrogel for repairing craniocerebral injury and its preparation method to solve the problems raised by the background technology.

[0004] The above information disclosed in this background technology is only used to increase the understanding of the background technology of the present invention and therefore, it may include information that does not constitute the prior art known to a person of ordinary skill in the art. Summary of the invention

[0005] The purpose of the present invention is to provide an injectable hydrogel for repairing craniocerebral injury and a preparation method thereof, so as to solve the problems raised by the above-mentioned background technology.

[0006] To achieve the above purpose, the present invention provides the following technical solution: an injectable hydrogel implant for repairing craniocerebral injury, wherein the implant is an injectable hydrogel loaded with stem cell-derived exosomes, wherein the injectable hydrogel uses carboxymethyl chitosan (CMCS) and sodium alginate (SA) as a substrate, D-glucose-δ lactone (GDL) as a cross-linking agent, and stem cell-derived exosomes as an effective substance. GDL hydrolyzes and releases protons, an electrostatic interaction is established between CMCS and SA, a gelation process is induced, and the stem cell-derived exosomes are encapsulated in the cross-linked network of the hydrogel. The stem cell-derived exosomes are a mixture of one or more of mesenchymal, neural, and somatic cell reprogramming stem cell-derived exosomes.

[0007] Preferably, the carboxymethyl chitosan has a deacetylation degree greater than 90% and a substitution degree of 90% / a deacetylation degree greater than 80% and a substitution degree of 80%, and the sodium alginate has a viscosity of 200-500 mPa.s / 500-1000 mPa.s.

[0008] A method for preparing an injectable hydrogel for repairing craniocerebral injury comprises the following steps:

[0009] Step 1, culturing neural stem cells (NSC) and somatic cell reprogrammed neural stem cells (iNSC), collecting cell culture supernatant, collecting exosomes by gradient centrifugation, and resuspending in PBS to obtain exosome suspension;

[0010] Step 2, respectively preparing carboxymethyl chitosan and sodium alginate solutions, blending them in proportion to form a uniform solution, and uniformly mixing the exosome suspension prepared in step 1 with the uniform solution to obtain a mixed solution, removing bubbles for later use; then preparing a D-glucose-δ-lactone solution, adding the mixed solution to the mixed solution, and mixing uniformly;

[0011] Step 3: The final blend of step 2 is rapidly injected into a suitable part through a syringe at low temperature, and gelation is completed at body temperature of 37°C.

[0012] Preferably, in the step one, first centrifuge at 300g for 10 minutes at 4°C, take the supernatant, and record it as the first supernatant; then, centrifuge the first supernatant at 3000g for 30 minutes at 4°C, take the supernatant, and record it as the second supernatant; then, centrifuge the second supernatant at 10000g for 30 minutes, discard the precipitate, and obtain the third supernatant; finally, centrifuge the third supernatant at 100000g for 120 minutes at 4°C, discard the supernatant, collect the precipitate, resuspend the precipitate with PBS, and prepare an exosome suspension with a concentration of 0.5-50 mg / mL, wherein the exosome suspension contains marker proteins CD63, CD81, and CD9.

[0013] Preferably, in step 1, the protein concentration of the exosomes is 0.5 mg / mL to 50 mg / mL, and the exosomes are stored at -80°C to 4°C.

[0014] Preferably, in step 2, the concentration of the sodium alginate solution is 0.5% to 5%, and the concentration of the carboxymethyl chitosan solution is 1% to 10%.

[0015] Preferably, in step 2, the mass ratio of the sodium alginate solution to the carboxymethyl chitosan solution is 0.3-3.

[0016] Preferably, in step 2, the concentration of the D-glucose-δ-lactone solution is 1% to 10%, and the ratio of the D-glucose-δ-lactone solution in the sodium alginate / carboxymethyl chitosan solution is 0.25 mL / g.

[0017] Preferably, in the step 2, the stem cell-derived exosome suspension and the sodium alginate / carboxymethyl chitosan solution are mixed uniformly and defoamed at 4°C to ensure uniformity and stability. The addition of D-glucose-δ-lactone allows the stem cell-derived exosomes to be encapsulated in the three-dimensional network cross-linked structure of the hydrogel during the gelation process, thereby achieving long-term sustained release of the stem cell-derived exosomes based on the swelling properties of the hydrogel and the erosion of the polymer.

[0018] The present invention also provides the use of the injectable hydrogel prepared by the above preparation method as an injectable hydrogel for repairing craniocerebral injury.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The injectable hydrogel for repairing craniocerebral injury of the present invention can achieve local sustained release of exosomes, improve the retention of exosomes at the target site, reduce the chance of being cleared by the host, prolong the duration of action, reduce the dosage used, and improve its bioavailability and therapeutic effect.

[0021] (2) The injectable hydrogel prepared by the present invention can be applied to exosomes from a variety of different sources and has universal applicability.

[0022] (3) The preparation method of the injectable hydrogel prepared by the present invention is simple and reliable, the equipment is simple, the energy is clean, it is environmentally friendly, and the production can be expanded.

[0023] (4) The injectable hydrogel of the present invention can achieve conformal filling and can also be applied to other clinical needs such as tissue repair, and has broad application prospects.

[0024] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 :Characterization of neural stem cell spheres and their secreted exosomes: (a) optical microscopy; (b) immunofluorescence staining (Nestin, SOX2, DAPI); (c) particle size; (d) transmission electron microscopy; (e) flow cytometry identification of exosome molecular markers; 1 and 2 represent neural stem cells and somatic cell reprogrammed neural stem cells, respectively;

[0026] Figure 2 :Characterization of injectable hydrogels: (a) Schematic diagram of gelation process; (b) FT-IR spectrum; (c) Thermogravimetric diagram; (d) Differential scanning calorimetry diagram;

[0027] Figure 3 :Characterization of injectable hydrogels: (a1-a3) SEM images of Hydrogel-1, Hydrogel-2, and Hydrogel-3; (b1-b3) SEM images of gels prepared at GDL concentrations of 2%, 4%, and 6%; (c) solution transmittance image; (d) hydrogel transmittance image; (e) swelling curve image;

[0028] Figure 4 : Rheological characterization of injectable hydrogels: (ac) Gel kinetics of gels prepared at 2%, 4%, and 6% GDL concentrations at 20°C; (d) Gel kinetics of gels prepared at 2% GDL concentration at 37°C; (e) Statistical graph of sol-gel phase transition time of gels prepared at different GDL concentrations; (f) Statistical graph of sol-gel phase transition time of gels prepared at different temperatures;

[0029] Figure 5 : Release curve of exosomes;

[0030] Figure 6 :In vivo visualization images and fluorescence quantitative statistical graphs of exosome-loaded hydrogels;

[0031] Figure 7 :Evaluation of blood-brain barrier and brain edema repair: (a) Gross appearance of each group after EB injection; (b) Quantitative statistics of each group after EB injection; (c) Quantitative statistics of brain tissue water content of each group;

[0032] Figure 8 :H&E staining of brain tissue structure;

[0033] Fig. 9 :(a) Schematic diagram of the animal experiment; (b) Statistical graph of the rotarod residence time of each group on days 1, 3, 7, 14, 21 and 28; (c) Statistical graph of the missed step rate of each group on days 3, 7, 14, 21 and 28. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Example 1: Cultivation Method of Neural Stem Cells (NSC) and Somatic Cell Reprogrammed Neural Stem Cells (iNSC)

[0036] The method for culturing neural stem cells (NSCs) was as follows: a 16-day-old C57 pregnant mouse was killed by cervical dislocation, and then the abdomen of the mouse was cut open with sterile surgical scissors until the fetus was exposed. The fetus was transferred to 75% ethanol for disinfection with sterile forceps, and then transferred to pre-cooled PBS. The brain of the fetus was removed with forceps and transferred to a clean culture dish. The brain was observed under a stereomicroscope and the meninges on the brain were removed with forceps. The brain was separated and transferred to pre-cooled PBS, and gently blown until no tissue clumps were visible to the naked eye. After passing through a 40μm sterile filter membrane, the cells were centrifuged at 300g for 5min at 4°C, and the culture medium was added to resuspend the cells. The cells were centrifuged at 1×10 5 mL -1 ~10×10 5 mL -1 The cells were inoculated at a density of 100 μg / mL in a T25 culture flask and placed in a 37°C, 5% CO2 incubator for suspension culture. The cells were collected for NSC identification. The supernatant of cell culture at passages 2 to 6 was used for exosome collection.

[0037] Method for culturing somatic cell reprogrammed neural stem cells (iNSC): A 14-day pregnant C57 mouse was killed by cervical dislocation, and then the abdomen of the mouse was cut open with sterile surgical scissors until the fetus was exposed. The fetus was transferred to 75% ethanol for disinfection with sterile forceps, and then transferred to pre-cooled PBS. All internal organs, head and spinal cord of the fetus were removed with forceps. The remaining skin tissue was washed twice with PBS and dissociated with 0.25% trypsin-EDTA solution. Mouse fibroblasts were cultured in a 37°C, 5% CO2 incubator in high glucose medium. The method for directly reprogramming mouse fibroblasts into NSCs is as follows: Mouse fibroblasts were incubated overnight in supernatant containing mixed viruses. 10 μg / mL polybrene was added to facilitate viral infection. One day after infection, the infected fibroblasts were cultured in NSC medium. The NSC medium was changed every 2 days. On the 28th day after retroviral transduction, colonies were formed and cells were manually selected to produce neurospheres. After 4-6 days of culture, the suspended primary neurospheres were collected and re-passaged on poly-D-lysine / fibronectin-coated 6-well plates. After the cell confluence reached 80%, the cells were collected and redispersed into single cells for the second round of neurosphere formation. After 3 rounds of screening and enrichment, the cells were collected for NSC identification, and the cell culture supernatant was used for exosome collection.

[0038] Both NSC and iNSC can grow in suspension to form neurospheres. The morphology of neurospheres was observed under an optical microscope. Neurospheres vary in size and are round or oval in shape, similar to mulberries, with clear edges and strong refractive index, and no obvious protrusions. The cells in the center of the sphere are densely packed, with weak refractive index, and appear dark black ( Figure 1 -a1, a2). By immunofluorescence staining, Nestin and SOX2 (stem cell markers) and DAPI (nuclear staining) were marked. Under a fluorescence microscope, it was observed that the marker proteins of Nestin and SOX2 in the neurospheres were positively expressed ( Figure 1 -b1, b2).

[0039] Example 2: Preparation of exosome suspension derived from NSC or iNSC

[0040] Step 1: After collecting the cell culture medium, first centrifuge at 300g for 10 minutes at 4°C, take the supernatant, and record it as the first supernatant; then, centrifuge the first supernatant at 3000g for 30 minutes at 4°C, take the supernatant, and record it as the second supernatant; then, centrifuge the second supernatant at 10000g for 30 minutes, discard the precipitate, and obtain the third supernatant; finally, centrifuge the third supernatant at 100000g for 120 minutes at 4°C, discard the supernatant, and collect the precipitate.

[0041] Step 2: resuspend the above precipitate with PBS to prepare an exosome suspension with a concentration of 2 mg / mL.

[0042] Nanoparticle tracking technology (NTA), transmission electron microscopy (TEM) and flow cytometry (FCM) were used to identify the particle size, morphology and exosomal protein markers of the isolated particles. NTA results showed that the average diameters of NSC-Exo and iNSC-Exo were 102nm and 150nm, respectively. Figure 1 -c1, c2). Through FCM detection, the expression of exosome marker proteins CD9, CD63 and CD81 all showed strong positive reactions ( Figure 1 -e1, e2). TEM images show that both exosomes present typical membrane structures ( Figure 1 -d1, d2). The above results indicate that the isolated extracellular particles are indeed exosomes.

[0043] Example 3: Preparation and characterization of injectable hydrogels

[0044] The preparation of injectable hydrogel comprises the following steps:

[0045] Step 1: prepare CMCS and SA solutions with a concentration of 2%, stir overnight, and evenly mix the CMCS and SA solutions in a mass ratio of 2:3, 1:1, and 1:3.

[0046] Step 2: prepare GDL solutions with concentrations of 2%, 4%, and 6%, prepare them before use, add the GDL solution to the mixture in step 1 at a concentration of 0.25 mL / g, and mix well.

[0047] Step 3: Rapidly inject the blend described in step 2 into molds of different shapes through a syringe at low temperature to complete gelation at 37°C.

[0048] The prepared gel was characterized by infrared spectrometer (FTIR), thermogravimetric analyzer (TG), differential scanning calorimeter (DSC), rheometer and micro ultraviolet spectrophotometer (UV).

[0049] like Figure 2-a shows the conformality characterization experiment of the injectable hydrogel prepared by the present invention (SA / CMCS mass ratio 1:1; GDL concentration 2%, and an appropriate amount of rhodamine b is added for easy observation). Under neutral conditions, the amino group of CMCS is not protonated and presents a negative charge density, and the carboxyl group of SA is negatively charged. Therefore, the mixture of CMCS and SA is a solution with good fluidity. After adding GDL, GDL can slowly hydrolyze and provide protons to reduce the pH value of the solution. As more amino groups on CMCS are protonated, the strong electrostatic interaction between CMCS and SA molecules will lead to a gelation process. When T<Tgel (Tgel is the gel point time), the mixture after adding GDL has fluidity, and the mixture can be tilted with the bottle. When T>Tgel, the mixture gels into a solid state, and the gel does not fall when the bottle is inverted. Since the hydrolysis rate of GDL is temperature-dependent, the mixture can be injected into molds of different shapes through a syringe with a needle at 20°C. After incubation at 37°C for 10 minutes, the mold is removed to obtain hydrogels of different shapes, indicating that the injectable hydrogel is successfully prepared.

[0050] Figure 2 The FTIR results in -b show that CMCS has a peak at 1626 cm -1 and 1426cm -1 Characteristic peaks appeared at 3416cm -1 The absorption bands nearby are attributed to the stretching vibrations of -OH and -NH groups. SA at 1618 cm -1 and 1413cm -1 Characteristic peaks are also shown at 1035 cm -1 and 3427cm -1 The characteristic peaks near 3443 cm-1 may be caused by the bending and stretching vibrations of the -OH groups in SA. -1 、1631cm -1 and 1454cm -1 The characteristic peaks at show that the characteristic peaks of amino, hydroxyl and carboxyl groups have blue shifted due to the influence of electrostatic interaction.

[0051] TG curve ( Figure 2-c) showed that SA, CMCS and hydrogel experienced three main weight loss stages during the thermal decomposition process: the first stage (about 60-200°C) was the evaporation of water and bound water; the second stage (about 200-300°C) was the decomposition and cleavage of the macromolecular chains; the third stage (about 300-800°C) was the further degradation of the decomposition products. Among these materials, the initial thermal decomposition temperatures of SA, CMCS and hydrogel were 222°C, 237°C and 175°C, respectively. The thermal stabilities of SA and CMCS were similar, and the thermal decomposition rate of hydrogel was slower than that of SA and CMCS. The residual weight percentages of SA, CMCS and hydrogel were 23.2%, 27.8% and 37.5%, respectively. In addition, the DSC curves of CMCS, SA and hydrogel (see Figure 2 -d) shows that the DSA curves of CMCS and SA have endothermic peaks at 248°C and 266°C, respectively, while the hydrogel has only one broad peak at around 218°C. These results indicate that there is a specific intermolecular interaction between SA and CMCS, forming a product different from the raw materials.

[0052] Figure 3 -a, 3-b show the SEM images of the prepared gels at different SA / CMCS mass ratios or GDL contents. Figure 3 a1-a3 is the GDL concentration: 2%; SA / CMCS mass ratio is 2:3 (Hydrogel-1), 1:1 (Hydrogel-2), 3:1 (Hydrogel-3). The microscopic morphology of the hydrogels prepared under these conditions shows that the hydrogels all show a loose porous structure. With the increase of SA content, the pores inside the hydrogel become looser. This phenomenon shows that the increase in the SA ratio leads to a lower cross-linking density, thereby forming larger pores. The size and looseness of these pores directly affect the swelling properties of the hydrogel. In addition, a high SA content may lead to more negative charge distribution and stronger hydrophilicity, which in turn affects the swelling degree of the gel, such as Figure 3 As shown in e. Figure 3 b1-b3 are hydrogels prepared under the conditions of GDL concentration of 2%, 4%, and 6% and SA / CMCS ratio of 1:1. With the increase of GDL, more protonated amino groups in CMCS produce stronger electrostatic interactions with SA molecules, the degree of cross-linking is enhanced, and the pores inside the gel are tighter.

[0053] Figure 3-c, 3-d show the transmittance of the solution for preparing the gel and the gel after cross-linking. The transmittance of CMCS is the highest, the transmittance of SA is the lowest, and the transmittance of the mixed solution is between the two. As the SA content increases, the transmittance gradually decreases, which indicates that there is no interaction between the CMCS and SA molecules in the mixture, and it is a physical mixture. As the SA content gradually increases, the transmittance of the prepared hydrogel gradually increases. The possible reason is that when the SA content is high, the electrostatic interaction of the molecules and the degree of gel cross-linking are weak, which ultimately leads to an increase in transmittance.

[0054] Rheological experiments show that, with other conditions unchanged, as the concentration of GDL increases, the solvent-gel phase transition time decreases from 547±98s, 208±33s, to 128±48s ( Figure 4 -a, b, c, e). Other conditions remain unchanged, the temperature rises from 20℃ to 37℃, and the sol-gel phase transition time decreases from 547±98s to 340±59s ( Figure 4 -a, d, 3-f), indicating that the gel is temperature responsive.

[0055] Example 4: Preparation and release experiment of injectable hydrogel loaded with exosomes

[0056] Step 1: prepare CMCS and SA solutions with a concentration of 4%, stir overnight, mix the CMCS and SA solutions in a mass ratio of 1:1, add an equal mass of exosome suspension and mix evenly, milk exosomes (Milk-Exo) are used as model exosomes, and the protein concentration is 1 mg / mL.

[0057] Step 2: Prepare a 2% GDL solution, prepare it immediately before use, add it to the mixed solution in step 1, 0.25 mL / g, mix well, and the result is Milk-Exo@Gel.

[0058] Step 3: Place the mixture described in step 2 in a 1.5 mL centrifuge tube, add 1 mL of PBS to each centrifuge tube, aspirate 200 μL of PBS at different time points, add new 200 μL of PBS for the next test, and determine the protein amount using a BCA protein assay kit.

[0059] The cumulative release curve was calculated by the following formula:

[0060] Cumulative release (%) = (C1V1+...+C t V t ) / C Exo V Exo ×100%

[0061] C represents the protein concentration determined by the BCA protein assay kit; V represents the volume of the collected supernatant; t represents time; CExo represents the initial exosome protein concentration; V Exo Represents the volume of exosome suspension added.

[0062] Figure 5 The in vitro release of exosomes was recorded. The sustained release of exosomes from hydrogels is mainly based on the following reasons: (1) the swelling property of hydrogels is conducive to the slow release of exosomes by diffusion; (2) the erosion of polymers is conducive to the long-term sustained release of exosomes.

[0063] Example 5: In vivo retention study of injectable hydrogels loaded with exosomes

[0064] In order to facilitate observation under the small animal imaging instrument, the Milk-Exo solution was fluorescently labeled as follows: the Milk-Exo solution was gently mixed with the DIR cell fluorescent probe in PBS (1:1000) and incubated in the dark at room temperature for 30 minutes. The solution was washed once with PBS and ultracentrifuged at 100,000 g for 2 hours at 4°C to remove the unbound dye. The injectable hydrogel loaded with milk exosomes (Milk-Exo-DIR@Gel) was prepared according to the method of Example 4.

[0065] The controlled cortical impact model (CCI) was used to model TBI in mice. The mice were first anesthetized (1.25% tribromoethanol, 0.02 mL / g), the hair on the head was shaved and disinfected with iodine. Subsequently, the epidermis was cut about 1 cm along the midline to expose the skull, and the surface of the skull was gently wiped with a cotton swab to wipe off the vascular membrane on the surface. After drying, a line was drawn between the bregma and the posterior bregma, and then a line was drawn between the midpoint of the raphe and the midpoint of the transverse temporal muscle. The intersection of the two lines was used as the end point, and a 5 mm diameter bone window was drilled on the left skull using a skull drill to expose the brain tissue. Be careful not to damage the brain tissue when opening the bone window. The mice were fixed on a stereotaxic instrument, with their heads kept horizontal. The position of the stereotaxic instrument was adjusted so that the striker was located in the middle of the bone window. The strike depth was adjusted to 1 mm. After the strike was completed and the bleeding stopped, the control group was directly sutured with the epidermis, the injection group was directly injected with Milk-Exo-DIR, and the implantation group was injected with Milk-Exo-DIR@Gel. After the gel was formed, the epidermis was sutured and the mice were returned to the cage after they woke up. The retention of exosomes was observed by a small animal imaging device 0, 7, 14, 21, and 28 days after surgery. Figure 6As shown in the figure, from the fluorescence images of the in vivo imaging system, it can be seen that the fluorescence signal of DIR in the implanted mice is significantly slower than that in the injected mice. The fluorescence of DIR in the injected group gradually decreased from the 7th to the 14th day after injection, and completely disappeared on the 21st day. However, the fluorescence of the implanted group was clear and bright from the 7th to the 14th day, and the fluorescence value reached the maximum on the 14th day due to the sustained release of exosomes; the signal could still be identified until the 28th day. From the statistical data, the retention of Milk-Exo in the Milk-Exo-DIR@Gel group was significantly better than that in the injected group.

[0066] Example 6: Effects of injectable hydrogel loaded with NSC-Exo or iNSC-Exo on relieving brain edema, repairing blood-brain barrier and motor function

[0067] Injectable hydrogels loaded with NSC-Exo (NSC-Exo@Gel) and injectable hydrogels loaded with iNSC-Exo (NSC-Exo@Gel) were prepared according to the method of Example 4. Animal modeling was performed according to the method of Example 4; the control group was not treated; NSC-Exo@Gel and iNSC-Exo@Gel were implanted into the injured site, respectively, and recorded as NSC-Exo@Gel group and iNSC-Exo@Gel group. After surgery, Evans blue dye (2%, 4mL / kg) was injected into the tail vein of each group of mice. When blue color was observed superficially on the whole body of the mice, PBS was perfused into the heart, and the brain tissue was obtained for observation. The brain tissue was then dissolved in formamide, homogenized, and quantified by an enzyme marker, thereby reflecting the degree of damage to the blood-brain barrier. In addition, 7 days after surgery, the mice in each group were killed by cervical dislocation, and the brain tissue was weighed and weighed again after drying to calculate the brain water content of each group. Figure 7 -a, 7-b, the brain tissue of the control group was smooth and undamaged, with almost no blue in the brain, while the blue hematoma in the TBI group was obvious, and the blood-brain barrier was severely damaged. The blue in the two treatment groups was less, and the quantitative detection showed statistical differences between the two treatment groups and the TBI group, indicating that implantation therapy can repair the blood-brain barrier to a certain extent. The results of the brain water content experiment showed ( Figure 7 -c), the water content of rat brain increased significantly after TBI, while the two groups given treatment could significantly reduce the water content of rat brain. Fig. 9-a, balance beam tests were performed on the 7th, 14th, 21st, and 28th days. The mice walked from one end to the other end of a wooden pole with a diameter of 0.5 cm and a length of 1 m, with a walking distance of 80 cm, and entered the black box. Preoperative training was performed 5 times, and the total number of steps and the number of wrong steps of the mice during the wooden walking process after they walked to the end point were observed and recorded on different days after the operation. Rotarod fatigue experiments were performed using a rotarod fatigue instrument on the 1st, 3rd, 7th, 14th, 21st, and 28th days after the operation, and the time when the mice fell from a rotating rod with a certain speed was recorded. Preoperative training was performed 5 times. After the test, the mice were killed by dislocating the neck, and the intact brain was removed, placed in a 4% paraformaldehyde solution at 4°C for fixation overnight, immersed in wax, embedded in paraffin, sliced, and stained with H&E. The results showed that the treatment group could reduce defects and promote brain tissue repair in mice with brain injury models ( Figure 8 ).like Fig. 9 -c, in the balance beam test, the wrong step rate of mice in the treatment group was significantly reduced compared with TBI at 7, 14, 21, and 28 days after injury, and the difference was statistically significant. The effects of the two implantation groups were similar. Fig. 9 -b, the time that the two implantation groups stayed on the rotating rod in the rotating rod fatigue test on days 1, 3, 7, 14, 21, and 28 after modeling was longer than that of the TBI group, and there was a statistical difference after 7 days. The above experiments show that both NSC-Exo@Gel and iNSC-Exo@Gel can improve the motor function impairment after TBI surgery.

[0068] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0069] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An injectable hydrogel implant for repairing craniocerebral injury, characterized in that: The implant is an injectable hydrogel loaded with stem cell-derived exosomes. The injectable hydrogel uses carboxymethyl chitosan and sodium alginate as substrates, stem cell-derived exosomes as functional substances, and D-glucose-δ lactone as a cross-linking agent. Protons are released by hydrolysis of D-glucose-δ lactone, and electrostatic interaction is established between carboxymethyl chitosan and sodium alginate to induce a gelation process. The stem cell-derived exosomes are encapsulated in the cross-linked network of the hydrogel. The stem cell-derived exosomes are a mixture of one or more of mesenchymal, neural, and somatic cell reprogramming stem cell-derived exosomes.

2. A method for preparing an injectable hydrogel for repairing craniocerebral injury, characterized in that: The following steps are included: Step 1, culturing neural stem cells and somatic cell reprogrammed neural stem cells, collecting cell culture supernatant, collecting exosomes by gradient centrifugation, and resuspending in PBS to obtain exosome suspension; Step 2, respectively preparing carboxymethyl chitosan and sodium alginate solutions, blending them in proportion to form a uniform solution, and uniformly mixing the exosome suspension prepared in step 1 with the uniform solution to obtain a mixed solution, removing bubbles for later use; then preparing a D-glucose-δ-lactone solution, adding the mixed solution to the mixed solution, and mixing uniformly; Step 3: The final blend of step 2 is rapidly injected into a suitable part through a syringe at low temperature, and gelation is completed at body temperature of 37°C.

3. The method for preparing an injectable hydrogel for repairing craniocerebral injury according to claim 2, characterized in that: In the step 1, first, centrifuge at 300g for 10 minutes at 4°C, take the supernatant, and record it as the first supernatant; then, centrifuge the first supernatant at 3000g for 30 minutes at 4°C, take the supernatant, and record it as the second supernatant; then, centrifuge the second supernatant at 10000g for 30 minutes, discard the precipitate, and obtain the third supernatant; finally, centrifuge the third supernatant at 100000g for 120 minutes at 4°C, discard the supernatant, collect the precipitate, resuspend the precipitate with PBS, and prepare an exosome suspension with a concentration of 0.5-50 mg / mL, wherein the exosome suspension contains marker proteins CD63, CD81, and CD9.

4. The method for preparing an injectable hydrogel for repairing craniocerebral injury according to claim 2, characterized in that: In the step 1, the protein concentration of the exosomes is 0.5 mg / mL to 50 mg / mL and is stored at -80°C to 4°C.

5. The method for preparing an injectable hydrogel for repairing craniocerebral injury according to claim 2, characterized in that: In the step 2, the concentration of the sodium alginate solution is 0.5% to 5%, and the concentration of the carboxymethyl chitosan solution is 1% to 10%.

6. The method for preparing an injectable hydrogel for repairing craniocerebral injury according to claim 2, characterized in that: In the step 2, the mass ratio of the sodium alginate solution to the carboxymethyl chitosan solution is 0.3-3.

7. The method for preparing an injectable hydrogel for repairing craniocerebral injury according to claim 2, characterized in that: In the step 2, the concentration of the D-glucose-δ-lactone solution is 1% to 10%, and the ratio of the D-glucose-δ-lactone solution in the sodium alginate / carboxymethyl chitosan solution is 0.25 mL / g.

8. The method for preparing an injectable hydrogel for repairing craniocerebral injury according to claim 2, characterized in that: In the step 2, at 4°C, the stem cell-derived exosome suspension and the sodium alginate / carboxymethyl chitosan solution are mixed evenly and defoamed to ensure their uniformity and stability. The addition of D-glucose-δ-lactone allows the stem cell-derived exosomes to be encapsulated in the three-dimensional network cross-linked structure of the hydrogel during the gelation process, thereby achieving long-term sustained release of the stem cell-derived exosomes based on the swelling properties of the hydrogel and the erosion of the polymer.

9. Use of the injectable hydrogel prepared by the preparation method according to any one of claims 2 to 8 as an injectable hydrogel for repairing craniocerebral injury.

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