Nasal temperature-sensitive hydrogel with immunoregulation and anti-inflammatory functions as well as preparation method and application of nasal temperature-sensitive hydrogel

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to nasal temperature-sensitive hydrogel with immunoregulation and anti-inflammatory functions as well as a preparation method and application of the nasal temperature-sensitive hydrogel. The preparation method of the nasal temperature-sensitive hydrogel with the immunoregulation and anti-inflammatory functions comprises the following steps: preparation of hydroxybutyl chitosan (HBC), preparation of levodopa-hydroxybutyl chitosan (LHBC), and preparation of CELHBC composite hydrogel. The novel temperature-sensitive hydrogel composite material of the composite hydrogel loaded with the chlorella / exosome can be used for the focus position of allergic rhinitis, and enrichment and retention of active ingredients are achieved; the chlorella is combined with the exosome, so that the inflammatory response of nasal mucosa can be relieved, the damaged nasal mucosa can be repaired, and the immune imbalance of Th1 / Th2 cells can be reversed, thereby improving the treatment effect of allergic rhinitis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nasal thermosensitive hydrogel with immunomodulatory and anti-inflammatory functions, its preparation method, and its application. Background Technology

[0002] Currently, treatments for allergic rhinitis (AR) include medication, desensitization therapy, and intranasal phototherapy. Medication is the primary method used clinically to relieve symptoms and improve the quality of life for patients with allergic rhinitis. Common nasal medications include corticosteroids and antihistamines such as budesonide, mometasone furoate, cetirizine, and ketotifen. However, frequent administration inevitably leads to irritation of the respiratory mucosa and rapid drug diffusion to non-lesion sites. Furthermore, while these treatments can provide temporary relief from allergy symptoms, they cannot reverse the immune imbalance associated with AR.

[0003] In recent years, exosomes derived from mesenchymal stem cells have shown great application potential in tissue engineering, exhibiting unique advantages in immune regulation and tissue repair. However, during the onset of inflammation-related diseases, the continuous accumulation of reactive oxygen species (ROS) and a hypoxic microenvironment often accompany the development of these conditions, further leading to immune imbalance. Chlorella (CV), due to its inherent antioxidants (antioxidant enzymes, chlorophyll, carotenoids, and phenolic compounds), can neutralize excess ROS and alleviate oxidative stress-induced inflammatory damage. Furthermore, under light conditions, CV produces oxygen and secretes the growth factor CGF, which can accelerate tissue repair and regeneration. Therefore, the synergistic regulation of immune balance at inflammatory lesions by CV and exosomes is expected to demonstrate superior therapeutic effects.

[0004] Hydroxybutyl chitosan (HBC) is an important chitosan (CS) derivative, synthesized through the etherification reaction of hydroxybutyl with the hydroxyl and amino groups of the CS backbone. The introduction of hydroxybutyl enhances the solubility of chitosan in water, endowing HBC with excellent thermosensitivity. Its solution can rapidly undergo a reversible phase transition to gel simply by changing the ambient temperature, without the need for additional cross-linking agents. HBC hydrogels, as temperature-sensitive injectable hydrogels, possess good physicochemical properties and biocompatibility, and have been widely used in promoting wound healing, drug delivery / sustained release, stem cell culture, and metal scaffold coatings. However, HBC-formed hydrogels have low tissue adhesion and limited biological efficacy, preventing them from functioning effectively at the lesion site of inflammatory diseases.

[0005] Currently, existing inventions mainly focus on loading drug components onto thermosensitive hydrogels to achieve sustained drug release at the site of rhinitis lesions. Although this can reduce the frequency of drug administration and increase the local concentration of the drug at the lesion site, the drugs often only exert a single effect, resulting in limited therapeutic efficacy. The treatment of allergic rhinitis remains at the level of relieving symptoms, and long-term use of steroid drugs can also produce adverse reactions in the body.

[0006] Biomaterials loaded with Chlorella are currently mostly used in wound healing, tumor treatment and other fields, but their application in allergic rhinitis, an inflammatory disease, has not yet been reported. Summary of the Invention

[0007] To address the shortcomings of the existing technology, this invention provides a nasal thermosensitive hydrogel with immunomodulatory and anti-inflammatory functions, its preparation method, and its application. The novel thermosensitive hydrogel composite material loaded with Chlorella / exosomes can be used at the lesion site of allergic rhinitis to achieve enrichment and retention of active ingredients. The combined use of Chlorella and exosomes can not only reduce the inflammatory response of the nasal mucosa and repair the damaged nasal mucosa, but also reverse the immune imbalance of Th1 / Th2 cells, thereby improving the therapeutic effect of allergic rhinitis.

[0008] The specific technical solution is as follows: The first objective of this invention is to provide a method for preparing a nasal thermosensitive hydrogel with immunomodulatory and anti-inflammatory functions, comprising the following steps: (1) Preparation of hydroxybutyl chitosan (HBC): The crude chitosan product was purified to obtain purified chitosan, which was then subjected to alkalization and microwave reaction to obtain solids. The solids were then subjected to microwave reaction, and finally the pH of the system was adjusted to obtain hydroxybutyl chitosan product. (2) Preparation of L-DOPA-hydroxybutyl chitosan (LHBC): Weigh L-DOPA and dissolve it in water to obtain an L-DOPA solution. Then activate the carboxyl group and add pre-dissolved HBC solution to the activated L-DOPA solution. Perform ice bath, dialyze, freeze dry to obtain LHBC. (3) Preparation of CELHBC composite hydrogel: LHBC-hydroxybutyl chitosan was dissolved in PBS at low temperature until completely dissolved to obtain LHBC solution; Chlorella CV was washed by centrifugation (4000 rpm, 5 min) to obtain Chlorella concentrate; LHBC solution was placed in an ice bath and magnetically stirred, Chlorella concentrate and exosome suspension were added, and CELHBC composite hydrogel, i.e., the thermosensitive hydrogel, was obtained after heating.

[0009] Currently, the main treatment for allergic rhinitis is commercially available drugs. Existing research has used nanocarriers / hydrogels to achieve sustained drug release in the nasal cavity. This invention provides a highly efficient and safe nasal drug delivery strategy that combines two active substances, Chlorella and exosomes, to regulate the immune balance at the site of rhinitis lesions, promote anti-inflammatory repair, restore immune homeostasis, and enhance the existing therapeutic effects of allergic rhinitis.

[0010] Two active components, Chlorella and exosomes, are loaded onto a thermosensitive hydrogel and enriched at the lesion site of allergic rhinitis. The antioxidant and oxygen-producing properties of Chlorella are combined with the immunosuppressive properties of exosomes. The biological effects of both can synergistically regulate immune balance, reduce inflammation and repair damage, and enhance the therapeutic effect of allergic rhinitis.

[0011] Furthermore, in step (2), the mass ratio of HBC to L-DOPA during the reaction is 7~15:1. After grafting L-DOPA, LHBC enhances adhesion and retains temperature sensitivity.

[0012] Furthermore, in step (3), the final concentration (w / v) of LHBC dissolved in PBS under low temperature conditions is 2-5%.

[0013] Furthermore, in step (3), the amount of Chlorella added to the LHBC solution is 1×10⁻⁶. 6 ~1×10 9 cells / mL.

[0014] Furthermore, in step (3), the exosomes are derived from human umbilical cord mesenchymal stem cells, and the content of the exosomes added to the LHBC solution is 50~300 μg / mL.

[0015] Furthermore, in step (3), the LHBC solution is placed in an ice bath and the magnetic stirring speed is 400~800 rpm.

[0016] Furthermore, in step (1), the preparation method of the hydroxybutyl chitosan HBC includes the following steps: 1) Alkalization: Weigh out the chitosan sample, stir at room temperature, add sodium hydroxide solution dropwise, and stir at room temperature; 2) Dispersion: Place the alkalized chitosan solution on a gauze and squeeze out the alkali, then transfer it to a beaker, add isopropanol solution, disperse, and stir at room temperature; 3) Synthesis Transfer the dispersed suspension to a three-necked flask, add 1,2-epoxybutane dropwise, heat and stir in a water bath, and reflux the reaction under condensation. 4) Dialysis Hydrochloric acid was added dropwise to the mixture after the reaction to adjust it to neutral pH. The mixture was then transferred to a dialysis bag and dialyzed with running water for 3 days and with pure water for 3 days. After centrifugation, the supernatant was collected and concentrated. After concentration, it was freeze-dried to obtain sponge-like HBC, i.e., hydroxybutyl chitosan product.

[0017] Further, in step 1), the stirring speed is 300-400 rpm, and the sodium hydroxide solution is 50% w / v; in step 2), V 异丙醇 V 水 = 7 : 3, stirring speed is 500-600 rpm; in step 3), the amount of 1,2-epoxybutane used is 20mL per gram of chitosan, stirring speed is 800-1000 rpm.

[0018] The second objective of this invention is to provide a nasal thermosensitive hydrogel with immunomodulatory and anti-inflammatory functions, prepared by the method described above.

[0019] A third objective of this invention is to provide the application of the nasal thermosensitive hydrogel described above in the preparation of a drug for treating allergic rhinitis, wherein the nasal thermosensitive hydrogel can be sprayed.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention develops a nasal thermosensitive hydrogel with immunomodulatory and anti-inflammatory functions, loaded with Chlorella / exosomes (derived from umbilical cord mesenchymal stem cells). The hydrogel can be sprayed into the nasal cavity to achieve the enrichment and retention of the two active ingredients, Chlorella / exosomes, at the site of rhinitis lesions. The two can synergistically treat allergic rhinitis: 1. Regulate the immune balance of Th1 / Th2 cells: Exosomes can exert immunosuppressive properties by delivering miRNA and anti-inflammatory factors, inhibiting Th2 cell differentiation; Chlorella can produce oxygen under light, and reduce the level of NLRP3 inflammasomes, thereby reducing the expression of pro-inflammatory factors in epithelial cells, thereby reducing Th2 differentiation and B cell IgE production; 2. Anti-inflammatory repair: The miRNA carried by exosomes can downregulate the expression of mast cell MRGPRX2 protein, and the inherent antioxidants of Chlorella can neutralize excessive ROS in the inflammatory microenvironment, thereby blocking the initiation of mast cell degranulation. Furthermore, exosomes can upregulate the expression of repair proteins. In addition to scavenging ROS, Chlorella can also secrete the growth factor CGF, which synergistically repairs the damaged nasal barrier. Therefore, the combined use of Chlorella and exosomes can enhance the therapeutic effect of allergic rhinitis.

[0021] (2) The Chlorella and exosomes used in this invention are both bioactive components with good biosafety and no adverse effects on the body. Furthermore, Chlorella / exosomes work together to regulate immune balance. Compared with drug treatment, the composite thermosensitive hydrogel loaded with Chlorella / exosomes can not only relieve the symptoms of allergic rhinitis, but also restore the homeostasis of the immune system, thereby improving the treatment effect of allergic rhinitis.

[0022] (3) In this invention, L-DOPA is used to modify HBC to give it a certain degree of adhesion. After loading Chlorella / exosomes onto LHBC hydrogel, it has the biological effect of regulating the immune balance at the site of inflammation. Attached Figure Description

[0023] Figure 1 This is a graph showing the gelation temperature and gelation time of the hydrogel in this invention; Figure 2 This is a graph showing the ROS level results of HNEpC nasal mucosal epithelial cells in this invention. Figure 3 This is a graph showing the migration-promoting effect of HNEpC on nasal mucosal epithelial cells for 48 hours in this invention. Figure 4 This is a graph showing the results of inhibiting mast cell degranulation rate in this invention; Figure 5 This is a diagram showing the hypoxia of HNEpC nasal mucosal epithelial cells in this invention. Figure 6 The image shows the results of Western blot quantification of NLRP3 inflammasomes and expression levels of IL-1β and IL-18 in the supernatant during this invention. Detailed Implementation

[0024] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0025] Example 1 A method for preparing a nasal thermosensitive hydrogel with immunomodulatory and anti-inflammatory functions includes the following steps: (1) Preparation of hydroxybutyl chitosan (HBC): 1) Alkalization: Weigh 4.0 g of chitosan sample into a 100 mL beaker, stir at room temperature (400 rpm), add 50 mL of sodium hydroxide solution (50%, w / v), and stir at room temperature for 24 h; 2) Dispersion: Place the alkalized chitosan solution on a gauze and squeeze out the alkali, then transfer it to a beaker and add isopropanol solution (V). 异丙醇 V 水Disperse the mixture at a ratio of 7:3 (600 rpm) and stir at room temperature for 24 h. 3) Synthesis The dispersed suspension was transferred to a three-necked flask, and 80 mL of 1,2-epoxybutane was added dropwise using a separatory funnel. The mixture was heated and stirred in a water bath at 55 °C (1000 rpm), and then refluxed for 24 h. 4) Dialysis Hydrochloric acid was added dropwise to the mixture after the reaction to adjust to neutral pH. The mixture was then transferred to a dialysis bag (8-14 kDa) and dialyzed with running water for 3 days and with pure water for 3 days. After centrifugation, the supernatant was collected and concentrated. After concentration, the mixture was freeze-dried to obtain sponge-like HBC, i.e., hydroxybutyl chitosan product. (2) Preparation of L-DOPA-hydroxybutyl chitosan (LHBC): 57.14 mg of L-DOPA powder was weighed and dissolved in 20 mL of deionized water and stirred until the solution was clear to obtain an L-DOPA solution. 34.28 mg of N-hydroxysuccinimide (NHS) and 57.14 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC hydrochloride) were added to the solution as catalysts to activate the carboxyl groups. After the solution was clear, N2 was introduced for 30 min. 40 mL of pre-dissolved 1% HBC solution was added dropwise to the activated L-DOPA solution and the mixture was magnetically stirred overnight under ice bath conditions. After the reaction was completed, the mixture was dialyzed in deionized water for 3 days and then freeze-dried to obtain L-DOPA-hydroxybutyl chitosan (LHBC). (3) Preparation of CELHBC composite hydrogel: Weigh the lyophilized LHBC and dissolve it in PBS at low temperature (4℃) until completely dissolved to obtain LHBC solution; take the sterile cultured Chlorella (CV) solution, centrifuge (4000 rpm, 5 min), wash it 3 times with PBS and resuspend it to obtain CV concentrate; place the LHBC solution in an ice bath and stir magnetically (800 rpm), add 1×10 9 A concentrated CV solution of cells / mL was prepared. After the CV was uniformly dispersed in the LHBC solution, a 300 μg / mL exosome suspension was added dropwise to make the final concentration of LHBC in the mixed solution 5%, thus obtaining a CELHBC solution. After heating, a CELHBC composite hydrogel can be prepared.

[0026] Example 2 A method for preparing a nasal thermosensitive hydrogel with immunomodulatory and anti-inflammatory functions includes the following steps: (1) Preparation of hydroxybutyl chitosan (HBC): 1) Alkalization: Weigh 4.0 g of chitosan sample into a 100 mL beaker, stir at room temperature (300 rpm), add 50 mL of sodium hydroxide solution (50%, w / v), and stir at room temperature for 24 h; 2) Dispersion: Place the alkalized chitosan solution on a gauze and squeeze out the alkali, then transfer it to a beaker and add isopropanol solution (V). 异丙醇 V 水 Disperse the mixture at a ratio of 7:3 (500 rpm) at room temperature for 24 h. 3) Synthesis The dispersed suspension was transferred to a three-necked flask, and 80 mL of 1,2-epoxybutane was added dropwise using a separatory funnel. The mixture was heated and stirred in a water bath at 55 °C (800 rpm) and refluxed for 24 h. 4) Dialysis Hydrochloric acid was added dropwise to the mixture after the reaction to adjust to neutral pH. The mixture was then transferred to a dialysis bag (8-14 kDa) and dialyzed with running water for 3 days and with pure water for 3 days. After centrifugation, the supernatant was collected and concentrated. After concentration, the mixture was freeze-dried to obtain sponge-like HBC, i.e., hydroxybutyl chitosan product. (2) Preparation of L-DOPA-hydroxybutyl chitosan (LHBC): 28.27 mg of L-DOPA powder was weighed and dissolved in 20 mL of deionized water and stirred until the solution was clear to obtain an L-DOPA solution. 16.96 mg of NHS and 28.27 mg of EDC hydrochloride were added to the solution to activate the carboxyl group. After the solution was clear, N2 was passed through for 30 min. 40 mL of pre-dissolved 1% HBC solution was added dropwise to the activated L-DOPA solution and the mixture was magnetically stirred overnight under ice bath conditions. After the reaction was completed, the mixture was dialyzed in deionized water for 3 days and then freeze-dried to obtain LHBC. (3) Preparation of CELHBC composite hydrogel: Weigh the lyophilized LHBC and dissolve it in PBS at low temperature (4℃) until completely dissolved to obtain LHBC solution; take the sterile cultured Chlorella (CV) solution, centrifuge (4000 rpm, 5 min), wash it 3 times with PBS and resuspend it to obtain CV concentrate; place the LHBC solution in an ice bath and stir magnetically (900 rpm), add 1×10 6 A concentrated CV solution of cells / mL was prepared. After the CV was uniformly dispersed in the LHBC solution, 50 μg / mL of exosome suspension was added dropwise to make the final concentration of LHBC in the mixed solution 2%, thus obtaining a CELHBC solution. After heating, a CELHBC composite hydrogel can be prepared.

[0027] Example 3 A method for preparing a nasal thermosensitive hydrogel with immunomodulatory and anti-inflammatory functions includes the following steps: (1) Preparation of hydroxybutyl chitosan (HBC): Refer to Example 1; (2) Preparation of L-DOPA-hydroxybutyl chitosan (LHBC): 33.50 mg of L-DOPA powder was weighed and dissolved in 20 mL of deionized water and stirred until the solution was clear to obtain L-DOPA solution. 20.10 mg of N-hydroxysuccinimide (NHS) and 33.50 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC hydrochloride) were added to the solution to activate the carboxyl group. After the solution was clear, N2 was introduced for 30 min. 40 mL of pre-dissolved 1% HBC solution was added dropwise to the activated L-DOPA solution and the mixture was magnetically stirred overnight under ice bath conditions. After the reaction was completed, the mixture was dialyzed in deionized water for 3 days and then freeze-dried to obtain LHBC. (3) Preparation of CELHBC composite hydrogel: Weigh the lyophilized LHBC and dissolve it completely in PBS at low temperature (4℃) to obtain an LHBC solution; take the sterile cultured Chlorella (CV) solution, centrifuge (4000 rpm, 5 min), wash three times with PBS and resuspend to obtain a concentrated CV solution; place the LHBC solution in an ice bath and stir magnetically (1000 rpm), add 1×10 8 A concentrated CV solution of cells / mL was prepared. After the CV was uniformly dispersed in the LHBC solution, 200 μg / mL of exosome suspension was added dropwise to make the final concentration of LHBC in the mixed solution 3%, thus obtaining the CELHBC solution. The CELHBC composite hydrogel could be prepared by heating.

[0028] Comparative Example 1 A method for preparing a nasal thermosensitive hydrogel includes the following steps: (1) Preparation of hydroxybutyl chitosan (HBC): Refer to Example 1; (2) Preparation of L-DOPA-hydroxybutyl chitosan (LHBC): Refer to Example 3; (3) Preparation of LHBC hydrogel: Weigh the freeze-dried LHBC, dissolve it in PBS or pure water, and after complete dissolution at low temperature, heat it to transform it from liquid to LHBC hydrogel.

[0029] Comparative Example 2 A method for preparing a nasal thermosensitive hydrogel includes the following steps: (1) Preparation of hydroxybutyl chitosan (HBC): Refer to Example 1; (2) Preparation of L-DOPA-hydroxybutyl chitosan (LHBC): Refer to Example 3; (3) Preparation of ELHBC composite hydrogel: without adding CV concentrate, the rest is the same as in Example 3.

[0030] Comparative Example 3 A method for preparing a nasal thermosensitive hydrogel includes the following steps: (1) Preparation of hydroxybutyl chitosan (HBC): Refer to Example 1; (2) Preparation of L-DOPA-hydroxybutyl chitosan (LHBC): Refer to Example 3; (3) Preparation of CLHBC composite hydrogel: No exosome suspension was added, and the rest was the same as in Example 3.

[0031] test: 1. Rheological property testing of hydrogels The rheological properties, such as gelation temperature and gelation time, of the hydrogels prepared in Examples 1-3 and Comparative Examples 1-3 were measured using a rheometer. The results are as follows: Figure 1 As shown, G' represents the storage modulus, G” represents the loss modulus, and the intersection of G' and G” represents the gelation point of the hydrogel; LHBC, ELHBC, CLHBC, and CELHBC are the hydrogels without Chlorella and exosomes in Comparative Example 1, the hydrogel with exosomes in Comparative Example 2, the hydrogel with Chlorella in Comparative Example 3, and the hydrogels with Chlorella and exosomes in Examples 1-3, respectively.

[0032] Depend on Figure 1 The results show that the gelation time of Example 1 (10s) and Example 3 (15s) are both shorter than that of Example 2 (160s), and the grafting amount is greater than that of Example 2. The hydrogels of Examples 1-3 undergo sol-gel transition at 13.1℃, 22.6℃ and 18.1℃, respectively, proving that the present invention can still maintain the thermosensitive reversibility of the hydrogel after chemical grafting of L-DOPA and physical blending of Chlorella and exosomes.

[0033] Since the addition of Chlorella and exosomes does not affect the temperature sensitivity of the hydrogel and the gelation temperature changes little, there are no significant differences among the groups in Examples 1-3 and Comparative Examples 1-3.

[0034] 2. In vitro ROS scavenging ability test The composite hydrogels prepared in Example 3 and Comparative Examples 1-3 were co-cultured with nasal mucosal epithelial cells and fibroblasts using the Transwell method. Cell culture medium containing ROSup was added, and the ROS scavenging ability was detected by the DCFH-DA probe. The cell scratch assay verified the repair-promoting ability of the composite hydrogel in a microenvironment containing ROS.

[0035] Results of ROS levels in HNEpC nasal mucosal epithelial cells, such as Figure 2 As shown. Results of the ability of HNEpC nasal mucosal epithelial cells to promote migration after 48 hours, as shown. Figure 3 As shown. This experiment used Transwell chambers to simulate the inflammatory microenvironment of the nasal cavity. In the Control group, cells were cultured in basal medium without treatment; in the other groups, cells were cultured in medium containing Rosup to induce oxidative stress. Among them, four hydrogel groups (Example 3, Comparative Examples 1-3) were co-incubated with cells, and the ROS group only induced oxidative stress in cells.

[0036] Depend on Figure 2 and Figure 3 The results showed that the intracellular ROS scavenging and cell scratch assays demonstrated that the composite hydrogel prepared in the examples could remove excess ROS in the microenvironment, and that the combination of Chlorella vulgaris and exosomes in the examples had a better repair-promoting effect than the single component in the comparative examples.

[0037] 3. In vitro test to inhibit mast cell degranulation The composite hydrogels prepared in Example 3 and Comparative Examples 1-3 were pre-incubated with mast cells using the Transwell method. Cell culture medium containing ROSup was added, and after incubation at different time points, drugs were added to stimulate degranulation. Mast cells were stained with toluidine blue, and the degranulation of mast cells was observed under a microscope. The degranulation rate of mast cells was calculated from five fields of view. The results are as follows: Figure 4 As shown, the negative control group consisted of mast cells cultured in 1640 basal medium; the compound 48 / 80 group was a single drug that stimulated mast cell degranulation; the reactive oxygen species (ROS) group had ROSup in its cell culture medium to induce oxidative stress; the compound 48 / 80 + ROSup group was a combination of the ROSup group and the compound 48 / 80 group, which further stimulated mast cell degranulation by adding the drug; LHBC, ELHBC, CLHBC, and CELHBC were all cultured in 1640 medium containing a certain concentration of ROSup before being stimulated for degranulation with compound 48 / 80.

[0038] Depend on Figure 4 The results show that the Chlorella in the CELHBC hydrogel prepared in the examples can reduce the ROS stimulation of mast cell degranulation by clearing ROS in the environment, and the miRNA carried by exosomes can downregulate the expression of MRGPRX2 protein in mast cells. The combination of the two can reduce the initiation of mast cell degranulation.

[0039] 4. In vitro hypoxia relief (dissolved oxygen content; HNEpC cell hypoxia relief) test Hypoxia was induced in nasal mucosal epithelial cells using CoCl2. The composite hydrogels prepared in Example 3 and Comparative Example 3 were co-incubated with nasal mucosal epithelial cells, with Dark and Light groups set up respectively. The hypoxia status of cells was detected using a Ru(dpp)3Cl2 probe. The expression of NLRP3 inflammasome in cells was detected by Western blot, and the content of inflammatory factors such as IL-1β and IL-18 in the supernatant was detected by ELISA kit.

[0040] The hypoxia of HNEpC nasal mucosal epithelial cells, such as Figure 5 As shown in the figure. Results of NLRP3 inflammasome quantification by Western blot, and expression levels of IL-1β and IL-18 in the supernatant, are as follows. Figure 6 As shown in the figure. Normoxia represents the normoxic group and Hypoxia represents the hypoxic group.

[0041] Depend on Figure 5 and Figure 6 The results show that the photosynthetic oxygen production of Chlorella enables the composite hydrogel prepared in the example to generate dissolved oxygen after red light irradiation, which can alleviate the hypoxia of nasal mucosal epithelial cells induced by CoCl2, reduce the expression of NLRP3 inflammasome, and reduce the secretion of inflammatory factors such as IL-1β and IL-18.

[0042] In summary, the above in vitro experimental results confirm that the combined use of Chlorella / exosomes in this invention can restore the Th1 / Th2 cell immune balance at the site of rhinitis lesions and repair the damaged nasal barrier.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a nasal thermosensitive hydrogel with immunomodulatory and anti-inflammatory functions, characterized in that, Includes the following steps: (1) Preparation of hydroxybutyl chitosan (HBC): The chitosan sample was alkalized, dispersed, synthesized and dialyzed to obtain hydroxybutyl chitosan product; (2) Preparation of L-DOPA-hydroxybutyl chitosan (LHBC): Weigh L-DOPA and dissolve it in water to obtain an L-DOPA solution. Then activate the carboxyl group and add pre-dissolved HBC solution to the activated L-DOPA solution. Perform ice bath, dialyze, freeze dry to obtain LHBC. (3) Preparation of CELHBC composite hydrogel: LHBC-hydroxybutyl chitosan was dissolved in PBS until completely dissolved to obtain LHBC solution; Chlorella CV was washed by centrifugation to obtain Chlorella concentrate; LHBC solution was placed in an ice bath and magnetically stirred, Chlorella concentrate and exosome suspension were added, and CELHBC composite hydrogel, i.e., the nasal thermosensitive hydrogel, was obtained after heating.

2. The method for preparing the nasal thermosensitive hydrogel with immunomodulatory and anti-inflammatory functions according to claim 1, characterized in that, In step (2), the mass ratio of HBC to L-DOPA during the reaction is 7~15:

1.

3. The method for preparing the nasal thermosensitive hydrogel with immunomodulatory and anti-inflammatory functions according to claim 1, characterized in that, In step (3), the final concentration (w / v) of LHBC dissolved in PBS is 2-5%.

4. The method for preparing the nasal thermosensitive hydrogel with immunomodulatory and anti-inflammatory functions according to claim 1, characterized in that, In step (3), the amount of Chlorella added to the LHBC solution is 1×10⁻⁶. 6 ~1×10 9 cells / mL.

5. The method for preparing the nasal thermosensitive hydrogel with immunomodulatory and anti-inflammatory functions according to claim 1, characterized in that, In step (3), the exosomes are derived from human umbilical cord mesenchymal stem cells, and the content of the exosomes added to the LHBC solution is 50~300 μg / mL.

6. The method for preparing the nasal thermosensitive hydrogel with immunomodulatory and anti-inflammatory functions according to claim 1, characterized in that, In step (3), the LHBC solution is placed in an ice bath and the magnetic stirring speed is 400~800 rpm.

7. The method for preparing the nasal thermosensitive hydrogel with immunomodulatory and anti-inflammatory functions according to claim 1, characterized in that, In step (1), the preparation method of the hydroxybutyl chitosan HBC includes the following steps: alkalization, dispersion, synthesis, and dialysis.

8. A nasal thermosensitive hydrogel with immunomodulatory and anti-inflammatory functions, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 7.

9. The use of the nasal thermosensitive hydrogel as described in claim 8 in the preparation of a medicament for treating allergic rhinitis.

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