Multifunctional hydrogel, drug-loaded hydrogel, and preparation method and application thereof

Abstract

The present invention provides a multifunctional hydrogel, a drug-loaded hydrogel, and a preparation method and application thereof, and belongs to the field of biomedical technology. The multifunctional hydrogel provided by the present invention uses gelatin, transglutaminase and thiolated hyaluronic acid as raw materials for preparation, which solves the problem of limited performance of hydrogel products in the prior art. The hydrogel product prepared by the present invention can provide cell adhesion through the gelatin network cross-linked by transglutaminase, and at the same time provide ROS scavenging effect through the hyaluronic acid network cross-linked by dynamic disulfide bonds. The combination of the two networks can regulate the degradation of the hydrogel; the drug-loaded hydrogel provided by the present invention is through the encapsulation of exosomes derived from neural stem cells in the hydrogel, and the two together achieve the effect of enhancing the inhibition of glial scar and anti-inflammatory. The material provided by the present invention can promote the repair and regeneration of neurons and myelin, and can then be used to prepare drugs for the treatment of cerebral hemorrhage, providing an effective means for the treatment of related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to multifunctional hydrogel, drug-loaded hydrogel and preparation methods and applications thereof. Background Art

[0002] Intracerebral hemorrhage is a disease with high morbidity and poor prognosis, causing immeasurable damage to human life and health. However, the treatment options for intracerebral hemorrhage are very limited, especially in improving the lesion microenvironment, promoting nerve cell survival and nerve regeneration function.

[0003] Hydrogels are a class of cross-linked polymers with a three-dimensional network structure that has a large number of hydrophilic groups or domains. These networks have a high affinity for water but do not dissolve due to the presence of chemical or physical bonds formed between the polymer chains. Hydrogels are cross-linked polymer networks that not only absorb and retain large amounts of water, but also mimic the biological fluids in the natural extracellular matrix. Hydrogels extracted from natural polymers mimic many features of the extracellular matrix and therefore may guide cell migration and growth during tissue regeneration and wound healing, and have the potential to stably encapsulate and transplant cells. Over the years, hydrogels have been widely studied and applied to various biomedical applications, including tissue engineering, drug delivery, wound healing, cell culture, etc.

[0004] Injectable hydrogels as members of hydrogels are very attractive, they can fill defects of any shape, allowing uniform incorporation of therapeutic molecules and / or cells without surgical intervention. Injectable hydrogels exhibit low viscosity in the sol state, so they can be arbitrarily shaped. In particular, due to the unique advantages of in situ drug delivery applications, injectable hydrogels have attracted widespread attention as carrier materials due to their advantages such as good injectability and minimally invasive administration procedures. In situ injection has the advantages of non-invasiveness and in situ shaping compared to preformed hydrogels that need to be surgically implanted in the body, so it has attracted great attention. In addition, injectable hydrogels can be used as a scaffold material to culture cells, and its advantages are high water content and adjustable biocompatibility, so that complex cell interactions and in vivo microenvironmental conditions can be simulated. Therefore, constructing a hydrogel system as a 3D in vitro cell culture system has become a hot spot in tissue engineering and regenerative medicine research.

[0005] Chinese patent CN105148322B discloses an injectable hydrogel and a preparation method thereof, comprising a chitosan solution with a concentration of 0.5-2%, a sodium glycerophosphate solution with a concentration of 10%, and a sodium hyaluronate solution with a concentration of 0.2-1%, and the volume ratio thereof is 10%-60%:30%:10%-60%. The total volume of the injectable hydrogel is 100%, and the moisturizing effect of the hydrogel can be improved by adding sodium hyaluronate, and the biocompatibility is good. The antibacterial effect and transparency of the hydrogel can be improved by adding chitosan, so that the performance of the hydrogel product is improved.

[0006] However, the performance of hydrogel products in the existing technology still needs to be further improved. Summary of the invention

[0007] The purpose of the present invention is to provide a multifunctional hydrogel, a drug-loaded hydrogel and a preparation method and application thereof, which have excellent therapeutic effects.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a multifunctional hydrogel, which is made from raw materials comprising the following components:

[0010] Gelatin, transglutaminase, and thiolated hyaluronic acid.

[0011] Preferably, in the multifunctional hydrogel, the final concentration of gelatin is 5-15%;

[0012] and / or, in the multifunctional hydrogel, the final concentration of the transglutaminase is 10-40 μg / mL;

[0013] And / or, in the multifunctional hydrogel, the final concentration of the thiolated hyaluronic acid is 0.5-1.5%.

[0014] The present invention also provides a method for preparing the multifunctional hydrogel, comprising the following steps:

[0015] The gelatin, transglutaminase and thiolated hyaluronic acid are mixed and stirred, and placed under the condition of 35-39° C. to obtain the multifunctional hydrogel.

[0016] Preferably, the gelatin participates in the reaction in the form of a gelatin solution;

[0017] The transglutaminase participates in the reaction in the form of a transglutaminase solution;

[0018] The thiolated hyaluronic acid participates in the reaction in the form of a thiolated hyaluronic acid solution.

[0019] Preferably, the solvent used in the gelatin solution, transglutaminase solution or thiolated hyaluronic acid solution is a buffer solution;

[0020] The buffer is PBS buffer.

[0021] The present invention also provides a drug-loaded hydrogel, comprising the multifunctional hydrogel and an active ingredient dispersed in the multifunctional hydrogel.

[0022] Preferably, the active ingredient is exosomes.

[0023] Preferably, the concentration of the exosomes in the multifunctional hydrogel is 10 7 ~10 8 Pieces / mL.

[0024] The present invention also provides the use of the multifunctional hydrogel, the multifunctional hydrogel prepared by the preparation method, or the drug-loaded hydrogel in the preparation of a drug for treating cerebral hemorrhage.

[0025] The present invention also provides the use of the multifunctional hydrogel, the multifunctional hydrogel prepared by the preparation method, or the drug-loaded hydrogel in any of the following:

[0026] A. Prepare products that promote the proliferation of neural stem cells;

[0027] B. Preparation of ROS inhibitors;

[0028] C. Preparation of glial scar inhibitors;

[0029] D. Preparation of anti-inflammatory products;

[0030] E. Prepare products that promote neuron / myelin / axon / brain tissue repair;

[0031] F. Preparation of products that promote neuron / myelin / axon / brain tissue regeneration;

[0032] G. Prepare products that promote vascular reconstruction;

[0033] H. Preparation of products that promote neuronal differentiation.

[0034] Beneficial effects of the present invention:

[0035] The multifunctional hydrogel provided by the present invention uses gelatin, transglutaminase and thiolated hyaluronic acid as preparation raw materials. The prepared hydrogel product can provide cell adhesion through the gelatin network cross-linked by transglutaminase, and at the same time provide ROS scavenging effect through the hyaluronic acid network cross-linked by dynamic disulfide bonds. The combination of the two networks can regulate the degradation of the hydrogel.

[0036] The drug-loaded hydrogel provided by the present invention further encapsulates exosomes derived from neural stem cells in the hydrogel, and the two together achieve enhanced inhibition of glial scar and anti-inflammatory effects. The material provided by the present invention can promote the repair and regeneration of neurons and myelin sheaths, and can be used to prepare drugs for the treatment of cerebral hemorrhage, providing an effective means for the treatment of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the composition of the exosome-carrying hydrogel;

[0038] Figure 2 This is the result of detecting cell proliferation using CCK8 method;

[0039] Figure 3 This is the result of DHE staining to detect ROS scavenging effect;

[0040] Figure 4 Figure 2 is the result of H&E staining of rat brain tissue samples, where: A and A1 (the area in the dotted box in A is enlarged) are H&E staining of the model group; B and B1 (the area in the dotted box in B is enlarged) are H&E staining of the hydrogel group; C and C1 (the area in the dotted box in C is enlarged) are H&E staining of the hydrogel + exosome group;

[0041] Figure 5 Figure 2 is the result of the detection of GFAP and NeuN expression in rat brain tissue samples, where: A and B are the immunofluorescence staining of GFAP and NeuN in the model group, respectively; C and D are the immunofluorescence staining of GFAP and NeuN in the hydrogel group, respectively; E and F are the immunofluorescence staining of GFAP and NeuN in the hydrogel + exosome group, respectively; G and H are the immunofluorescence staining statistics of GFAP and NeuN in each group, respectively;

[0042] Figure 6 Figure 2 is the result of the detection of MAP-2, GAP-43 and CD31 expression in rat brain tissue samples, where: A and D are the immunofluorescence staining of MAP-2, GAP-43 and CD31 in the model group, respectively; B and E are the immunofluorescence staining of MAP-2, GAP-43 and CD31 in the hydrogel group, respectively; C and F are the immunofluorescence staining of MAP-2, GAP-43 and CD31 in the hydrogel + exosome group, respectively; G, H and I are the immunofluorescence staining statistics of MAP-2, GAP-43 and CD31 in each group, respectively;

[0043] Figure 7The results of detecting the protein levels of TNF-α, IL-1β and IL-10 in brain tissue, among which: A is the Western Blot detection result; B is the statistical result comparison chart of the protein level of TNF-α; C is the statistical result comparison chart of the protein level of IL-1β; D is the statistical result comparison chart of the protein level of IL-10. DETAILED DESCRIPTION

[0044] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] The method for preparing the secretion body used in the present invention is as follows:

[0046] Preparation of exosomes from neural stem cells

[0047] 1. Collect the culture medium of neural stem cells (NSCs) in the logarithmic growth phase, centrifuge at 300g for 10 min at 4°C, and discard the cell components;

[0048] 2. Collect the supernatant and centrifuge at 2000g for 20 min at 4°C to remove dead cells.

[0049] 3. Filter the culture solution obtained in the previous step through a 0.22 μm filter, transfer the filtrate to an ultrafiltration tube using a Pasteur tube, and centrifuge at 4000 g for 50 min at 4°C;

[0050] 4. Add the supernatant obtained in the previous step to a new ultracentrifuge tube and centrifuge at 100,000 g at 4 °C.

[0051] At 70 minutes, the precipitate obtained on the bottom wall of the centrifuge tube is neural stem cell-derived exosomes (NSC-Exos);

[0052] 5. Gently aspirate the supernatant, resuspend with PBS, pipette gently repeatedly and centrifuge;

[0053] 6. To further purify NSC-Exos, the supernatant was added to a new ultracentrifuge tube and centrifuged again at 100,000 g for 70 min at 4°C.

[0054] 7. Gently discard the supernatant, then add 100ul PBS to dissolve the NSC-Exos precipitate. Resuspend the obtained precipitate in PBS, pipette it into an EP tube, seal it, and store it in a -80℃ refrigerator for later use.

[0055] Gelatin, TGs, and thiolated hyaluronic acid were dissolved in PBS, respectively, and then mixed and stirred thoroughly to obtain final concentrations of 10%, 25 μg / mL, and 1%, respectively, which could form gel at 37°C.

[0056] Gelatin, TGs, and thiolated hyaluronic acid were dissolved in PBS and then mixed thoroughly to a final concentration of 10%, and 1%, respectively. 7 / mL), mix well and form gel at 37℃.

[0057] Schematic diagram of the composition of the exosome-loaded hydrogel Figure 1 shown.

[0058] Effect of hydrogel on cell proliferation

[0059] Neural stem cells were inoculated onto the surface of hydrogels and exosome-loaded hydrogels, and the cell proliferation was detected using the CCK8 method. Figure 2 shown.

[0060] After 3 days of culture, the cell proliferation in the hydrogel and exosome-loaded hydrogel groups was significantly higher than that in the blank control group. After continued culture until the 7th day, all three groups of cells proliferated, and the exosome-loaded hydrogel group was significantly higher than the other two groups. This indicates that the exosome-loaded hydrogel has the effect of promoting the proliferation of neural stem cells.

[0061] Therapeutic application of hydrogel in rats with cerebral hemorrhage

[0062] Collagenase was used to establish a rat model of cerebral hemorrhage. Three weeks later, hydrogels and hydrogel-loaded exosomes were injected into the lesions. The therapeutic effect was detected by tissue staining, immunofluorescence, and protein blotting. On the third day after hydrogel implantation, the ROS scavenging effect was detected by DHE staining. Figure 3 As shown in the figure, the ROS levels in the hydrogel group and the hydrogel-loaded exosome group were significantly lower than those in the model group, indicating that the hydrogel has a ROS scavenging effect.

[0063] Eight weeks after surgery, H&E staining ( Figure 4 ) showed that the model group had a larger cavity volume, unclear structure, increased intercellular space, a large amount of white matter and gray matter loss, and cavities in the gray and white matter areas. In contrast, the hydrogel and hydrogel-exosome groups promoted the reduction of cavities, capillaries formed in the cavities, and the regenerated tissue showed a well-ordered structure.

[0064] The expression of GFAP and NeuN was detected. Figure 5As shown: A large number of regenerated GFAP-positive cells were detected at the edge of the brain tissue cavity of the rats in the model group, indicating the significant proliferation of glial scars at the site of brain tissue damage after cerebral hemorrhage; while fewer corresponding cells were expressed in the hydrogel group, especially in the hydrogel-loaded exosome group, basically no GFAP-positive cells were observed around the cavity. This shows that hydrogel-loaded exosomes have a significant effect in inhibiting the proliferation of glial scars. In addition, compared with the model group, more regenerated NeuN-positive cells were detected in the cavity and around the cavity of the brain tissue of the rats in the hydrogel treatment group, and the hydrogel-loaded exosome group had more NeuN expression than the simple hydrogel group, indicating that hydrogel-loaded exosomes can provide a more favorable microenvironment for neuronal repair and regeneration.

[0065] MAP-2 is an indicator of mature neurons, and GAP-43 is a marker of nerve axons. The expression of MAP-2 and GAP-43 in the samples was detected, and the regeneration of blood vessels (CD 31) was also evaluated. The results are as follows Figure 6 As shown: the model group basically did not express MAP-2 and GAP-43. The present invention observed that in the hydrogel group, MAP-2 and GAP-43 were concentrated in the front side of the center of the damaged cavity, and there was also a small amount distributed at its edge; while MAP-2 and GAP-43 in the hydrogel-loaded exosome group were more evenly distributed throughout the cavity, and the expression level was significantly higher than that in the model group and the hydrogel group, indicating that the hydrogel-loaded exosomes are beneficial to the differentiation of neurons, and further promote the regeneration of nerve fibers, myelin sheaths and axons, which provides conditions for subsequent nerve repair and tissue regeneration. In addition, the positive expression rate of CD 31 in the hydrogel-loaded exosome group was higher than that in the model group and the hydrogel group, indicating that the hydrogel-loaded exosome group is more conducive to vascular reconstruction.

[0066] TNF-α, IL-1β, and IL-10 have been shown to play a key role in the inflammatory process. Therefore, Western Blot was used to measure the protein levels of TNF-α, IL-1β, and IL-10 in brain tissue. Figure 7 shown.

[0067] Compared with the model group, the expression levels of TNF-α and IL-1β in the hydrogel group and the hydrogel-loaded exosome group were significantly reduced; and the expression levels of TNF-α and IL-1β in the hydrogel-loaded exosome group were significantly lower than those in the hydrogel group. At the same time, the expression of IL-10 in the hydrogel group and the hydrogel-loaded exosome group was significantly higher than that in the model group; and the expression of IL-10 in the hydrogel-loaded exosome group was more significant than that in the hydrogel group. The above results show that the hydrogel provided by the present invention has an anti-inflammatory effect, and the anti-inflammatory effect is significantly enhanced after encapsulating exosomes.

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A multifunctional hydrogel, characterized in that: Made from raw materials including the following ingredients: Gelatin, transglutaminase, and thiolated hyaluronic acid; In the multifunctional hydrogel, the final concentration of gelatin is 5-15%; And / or, in the multifunctional hydrogel, the final concentration of the transglutaminase is 10-40 μg / mL; And / or, in the multifunctional hydrogel, the final concentration of the thiolated hyaluronic acid is 0.5-1.5%; The preparation method of the multifunctional hydrogel comprises the following steps: The gelatin, transglutaminase and thiolated hyaluronic acid are mixed and stirred, and placed under the condition of 35-39° C. to obtain the multifunctional hydrogel.

2. The method for preparing the multifunctional hydrogel according to claim 1, characterized in that: The following steps are involved: The gelatin, transglutaminase and thiolated hyaluronic acid are mixed and stirred, and placed under the condition of 35-39° C. to obtain the multifunctional hydrogel.

3. The method for preparing the multifunctional hydrogel according to claim 2, characterized in that: The gelatin participates in the reaction in the form of a gelatin solution; The transglutaminase participates in the reaction in the form of a transglutaminase solution; The thiolated hyaluronic acid participates in the reaction in the form of a thiolated hyaluronic acid solution.

4. The method for preparing the multifunctional hydrogel according to claim 3, characterized in that: The solvent used in the gelatin solution, transglutaminase solution or thiolated hyaluronic acid solution is a buffer solution; The buffer is PBS buffer.

5. A drug-loaded hydrogel, characterized in that: The invention comprises the multifunctional hydrogel according to claim 1 and an active ingredient dispersed in the multifunctional hydrogel.

6. The drug-loaded hydrogel according to claim 5, characterized in that: The active ingredient is exosomes.

7. The drug-loaded hydrogel according to claim 6, characterized in that: The concentration of the exosomes in the multifunctional hydrogel is 10 7 ~10 8 Pieces / mL.

8. Use of the multifunctional hydrogel according to claim 1, the multifunctional hydrogel prepared by the preparation method according to any one of claims 2 to 4, or the drug-loaded hydrogel according to any one of claims 5 to 7 in the preparation of a drug for treating cerebral hemorrhage.

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