A nanoenzyme adhesive hydrogel microsphere and preparation method thereof
By using nanoenzyme sticky hydrogel microspheres and using materials such as ceria sodium alginate microspheres and gelatin, the problem that traditional drugs may have side effects in the treatment of dry mouth disease is solved, and long-term sustained release and antioxidant and anti-inflammatory effects are achieved, providing a new and effective way to treat dry mouth disease.
Patent Information
- Application Number
- CN202510001302.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-02
- Publication Date
- 2025-04-04
AI Technical Summary
Traditional medicines for dry mouth may have side effects, and existing treatments are difficult to relieve symptoms for a long time.
The nanoenzyme viscous hydrogel microspheres are made of sodium alginate microspheres in the core of ceria sodium alginate microspheres, and the outer layer is a viscous hydrogel made of a combination of gelatin and N-hydroxysuccinimide ester grafted crosslinked polyacrylic acid. The hydrogel forms microspheres through ultrasonic shock, which can provide long-term sustained release of ceria with antioxidant and anti-inflammatory effects.
This nanoenzyme sticky hydrogel microsphere can firmly adhere to the oral mucosa, long-term sustained release of ceria, providing antioxidant and anti-inflammatory effects, thereby effectively treating dry mouth and reducing the occurrence of side effects.
Smart Images
Figure CN119745827A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of medical materials, and in particular to a nanoenzyme viscous hydrogel microsphere and a preparation method thereof. Background Art
[0002] Xerostomia is a disease that affects the quality of life of many patients and can be caused by a variety of factors, such as certain diseases (such as Sjögren's syndrome), drug side effects, radiotherapy, etc. Current methods for treating xerostomia have limitations, such as artificial saliva can only temporarily relieve symptoms and requires frequent use; traditional drug treatments may be accompanied by side effects, etc. Therefore, the development of new and effective therapeutic materials is urgent. Summary of the invention
[0003] The purpose of the invention is to solve the technical problem that traditional medicines for treating xerostomia may be accompanied by side effects.
[0004] In order to achieve the above object, the present invention adopts the following technical solutions:
[0005] A nanoenzyme viscous hydrogel microsphere, wherein the inner core of the microsphere is a cerium dioxide sodium alginate microsphere, and the outer layer is a viscous hydrogel made of a combination of gelatin and cross-linked polyacrylic acid grafted with N-hydroxysuccinimide ester.
[0006] Preferably, the cerium dioxide sodium alginate microspheres include cerium dioxide with a particle size of 1-50 nm and sodium alginate microspheres with a particle size of 100-300 μm, and the concentration of cerium dioxide in the sodium alginate microspheres is 60 μg / mL.
[0007] Preferably, in the viscous hydrogel made of the combination of gelatin and cross-linked polyacrylic acid grafted with N-hydroxysuccinimide ester, the mass ratio of gelatin, acrylic acid, gelatin methacrylate (gelMA), α-ketoglutaric acid, and acrylic acid N-hydroxysuccinimide ester (AAc-NHS ester) is 100:300:1:2:10, and the thickness of the viscous hydrogel is 10-100 μm.
[0008] The present application also provides a method for preparing nanozyme sticky hydrogel microspheres, which is used to prepare the above-mentioned nanozyme sticky hydrogel microspheres, and the steps are as follows:
[0009] S1: Preparation of sodium alginate cerium dioxide microspheres: First, cerium dioxide nanoparticles are prepared, and then a sodium alginate solution is prepared. The cerium dioxide nanoparticles are dispersed in the sodium alginate solution to obtain a mixed solution. The solution is dripped into a calcium chloride solution by microfluidics, and the microspheres are washed with deionized water;
[0010] S2: Preparation of viscous hydrogel: dissolving acrylic acid, gelatin, AAc-NHS ester, gelMA and α-ketoglutaric acid in water, stirring for 12 h, and filtering with a sterile syringe filter to obtain a viscous hydrogel;
[0011] S3: Soak the cerium dioxide sodium alginate microspheres in S1 in the sticky hydrogel of S2, and perform ultrasonic vibration to form nanozyme sticky hydrogel microspheres.
[0012] Preferably, the preparation method of the cerium dioxide nanoparticles in S1 is as follows:
[0013] Preferably, the sodium alginate solution in S1 is a solution with a mass fraction of 2%, and the maximum concentration of cerium dioxide dissolved in the sodium alginate solution is 60 μg / mL.
[0014] Preferably, the added amounts of the materials in S2 are as follows: 30% (w / w) acrylic acid, 10% (w / w) gelatin, 1% (w / w) AAc-NHS ester, 0.1% (w / w) gelMA and 0.2% (w / w) α-ketoglutaric acid.
[0015] The present application also provides the use of the above-mentioned nanozyme sticky hydrogel microspheres in the preparation of drugs for treating dry mouth.
[0016] Based on the above content, the present application also provides a drug for treating dry mouth, which is prepared by adding 1% photocuring agent to the above-mentioned nanozyme adhesive hydrogel microspheres.
[0017] Preferably, the drug is an oral spray.
[0018] The nanozyme sticky hydrogel microspheres described above, wherein cerium dioxide (CeO2) has unique redox properties, can scavenge free radicals, and has potential anti-inflammatory and anti-oxidation effects; sodium alginate is a natural polysaccharide with good biocompatibility and degradability, gelatin is also a material with good biocompatibility and has a certain viscosity; polyacrylic acid is a commonly used polymer material. The prepared nanozyme sticky hydrogel microspheres can be applied to the oral cavity for the treatment of xerostomia. The hydrogel firmly adheres to the oral mucosa, and the long-term sustained release of cerium dioxide with antioxidant and anti-inflammatory effects provides a new and effective way to treat xerostomia. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1The scanning electron microscope images and performance diagrams of the products in each step of the preparation process of a nanozyme sticky hydrogel microsphere in one embodiment of the present invention, wherein: (A) scanning electron microscope image of cerium dioxide sodium alginate microspheres; (B) scanning electron microscope image of sticky hydrogel; (C) scanning electron microscope image of nanozyme sticky hydrogel microspheres: (D) SOD enzyme activity and hydroxyl radical scavenging rate of cerium dioxide; (E) cell viability of RAW264.7 co-cultured with cerium dioxide;
[0020] Figure 2 The figure is a comparison of the survival rates of cells cultured in the culture medium after being immersed in the viscous hydrogel (A) of the cerium dioxide sodium alginate microspheres and the viscous hydrogel (B) in one embodiment of the present invention;
[0021] Figure 3 This is a comparison diagram of salivary gland samples in Example 4 of the present invention. DETAILED DESCRIPTION
[0022] The present invention is further described in detail below in conjunction with specific embodiments.
[0023] The invention discloses a nanoenzyme viscous hydrogel microsphere, the inner core of which is a cerium dioxide sodium alginate microsphere, and the outer layer is a viscous hydrogel made of a combination of gelatin and cross-linked polyacrylic acid grafted with N-hydroxysuccinimide ester.
[0024] Among them, in the cerium dioxide sodium alginate microspheres, the particle size of cerium dioxide is 1-50nm, the concentration in the sodium alginate microspheres is 60μg / mL, and the particle size of the sodium alginate microspheres is 100-300μm. In the viscous hydrogel made of the combination of gelatin and cross-linked polyacrylic acid grafted with N-hydroxysuccinimide ester, the mass ratio of gelatin, acrylic acid, gelatin methacrylate (gelMA), α-ketoglutaric acid, and acrylic acid N-hydroxysuccinimide ester (AAc-HNS ester) is 100:300:1:2:10, and the thickness of the viscous hydrogel is 10-100μm.
[0025] Based on the above content, the present application also provides a method for preparing nanozyme sticky hydrogel microspheres, which comprises the following steps:
[0026] S1: Preparation of cerium dioxide sodium alginate microspheres:
[0027] ① Preparation of cerium dioxide nanoparticles:
[0028] Dissolve 108.5 mg of cerium nitrate hexahydrate in 2 mL of deionized water to prepare solution A, and dissolve 100 mg of disodium citrate in 1 mL of deionized water to prepare solution B. Mix solution A and solution B thoroughly, add 50 mL of 0.4 mol / L ammonia water, and stir for 24 hours. Centrifuge the mixture at 2600 g for 30 minutes to remove aggregated particles, take the supernatant, and dialyze it for 24 hours using a 3 kDa dialysis bag. Centrifuge the dialyzed liquid at 15,000 g for 10 minutes, take the supernatant, filter the supernatant with a 0.22 μm sterile syringe filter, and freeze-dry it and store it at 4°C.
[0029] ② Preparation of sodium alginate microspheres:
[0030] Sodium alginate was dissolved in deionized water to prepare a solution with a mass fraction of 2%, and the prepared cerium dioxide nanoparticles were dispersed in the sodium alginate solution to make the concentration of cerium dioxide reach 60 μg / mL. The mixed solution was dripped into the calcium chloride solution by microfluidic dripping. The reaction time was 10-30 minutes, and then the microspheres were washed with deionized water for 3-5 times.
[0031] S2: Preparation of a viscous hydrogel made of gelatin and N-hydroxysuccinimide ester grafted cross-linked polyacrylic acid:
[0032] 30% (w / w) acrylic acid, 10% (w / w) gelatin, 1% (w / w) AAc-NHS ester, 0.1% (w / w) gelMA and 0.2% (w / w) α-ketoglutaric acid were dissolved in deionized water, stirred for 12 hours and filtered through a 0.45 μm sterile syringe filter.
[0033] S3: Preparation of Nanozyme Adhesive Hydrogel Microspheres:
[0034] The cerium dioxide sodium alginate microspheres prepared in S1 are immersed in the viscous hydrogel solution in S2, and the hydrogel is evenly wrapped on the surface of the microspheres by ultrasonic vibration to form nanozyme viscous hydrogel microspheres.
[0035] The present application also provides the use of the above-mentioned nanozyme sticky hydrogel microspheres in the preparation of drugs for treating dry mouth.
[0036] In addition, based on the above content, the present application provides a drug for treating dry mouth, which is an oral smear, which is prepared by adding 1% photocuring agent to the above-mentioned nanoenzyme sticky hydrogel microspheres. When used, it is cured for 5-10 seconds in ultraviolet light at 284nm and 10W power to firmly adhere to the oral mucosa.
[0037] The above contents are described below in conjunction with specific embodiments:
[0038] Example 1: Preparation of nanozyme adhesive hydrogel microspheres:
[0039] (1) Preparation of cerium dioxide sodium alginate microspheres:
[0040] ① Preparation of cerium dioxide nanoparticles:
[0041] 108.5 mg of cerium nitrate hexahydrate was dissolved in 2 mL of deionized water to prepare solution A, and 100 mg of disodium citrate was dissolved in 1 mL of deionized water to prepare solution B. Solution A and solution B were fully mixed, 50 mL of 0.4 mol / L ammonia water was added, and stirred for 24 hours.
[0042] The mixed solution was centrifuged at 2600g for 30 minutes to remove aggregated particles, and the supernatant was dialyzed for 24 hours using a 3 kDa dialysis bag. The dialyzed liquid was centrifuged at 15000g for 10 minutes, and the supernatant was filtered through a 0.22 μm sterile syringe filter, freeze-dried, and stored at 4°C.
[0043] ② Preparation of sodium alginate microspheres:
[0044] Sodium alginate was dissolved in deionized water to prepare a solution with a mass fraction of 2%, and the prepared cerium dioxide nanoparticles were dispersed in the sodium alginate solution to make the concentration of cerium dioxide reach 60 μg / mL. The mixed solution was dripped into the calcium chloride solution by microfluidic dripping. The reaction time was 10-30 minutes, and then the microspheres were washed with deionized water for 3-5 times.
[0045] The scanning electron microscope image of the prepared cerium dioxide sodium alginate microspheres is as follows Figure 1 As shown in A.
[0046] (2) Preparation of viscous hydrogels made of gelatin and cross-linked polyacrylic acid grafted with N-hydroxysuccinimide ester:
[0047] 30% (w / w) acrylic acid, 10% (w / w) gelatin, 1% (w / w) AAc-NHS ester, 0.1% (w / w) gelMA and 0.2% (w / w) α-ketoglutaric acid were dissolved in deionized water, stirred for 12 hours and then filtered with a 0.45 μm sterile syringe filter to obtain a viscous hydrogel. The scanning electron microscopy image is shown in FIG. Figure 1 As shown in B.
[0048] (3) Preparation of nanozyme sticky hydrogel microspheres:
[0049] The prepared cerium dioxide sodium alginate microspheres were immersed in the above-mentioned viscous hydrogel solution, and the hydrogel was evenly wrapped on the surface of the microspheres by ultrasonic vibration to form nanozyme viscous hydrogel microspheres. The scanning electron microscope image of the nanozyme viscous hydrogel microspheres is as follows: Figure 1 As shown in C.
[0050] Example 2: Antioxidant Capacity of Cerium Oxide Nanoparticles
[0051] 1. Experimental methods:
[0052] (1) Total superoxide dismutase (T-SOD) assay: according to the kit operation table, add the sample to be tested (cerium dioxide solution of different concentrations), distilled water, enzyme working solution, enzyme diluent, and substrate application solution, gently shake the well plate to mix, incubate at 37°C for 20 minutes, take out and read the value of the microplate reader at 450 nm, and calculate according to the formula described in the instruction manual.
[0053] (2) Hydroxyl free radical determination: Add double distilled water, 0.03% H2O2 standard application solution, substrate application solution, sample (cerium dioxide solution of different concentrations), and reagent 3 application solution to a 37°C water bath according to the kit operation table. Start the timer when reagent 3 is added, vortex and mix quickly, and react at 37°C for 1 minute. Add the color developer and mix well. After leaving at room temperature for 20 minutes, adjust the light path to 1 cm, 550 nm, and double distilled water to zero. Measure the absorbance value of each tube and calculate according to the formula described in the instructions.
[0054] 2. Experimental results: Please refer to Figure 1 D. Cerium dioxide nanoparticles have good SOD enzyme activity and hydroxyl radical scavenging rate, strong antioxidant capacity, and gradually increase with increasing concentration.
[0055] Example 3: Cerium dioxide nanoparticles can promote cell proliferation
[0056] 1. Experimental method: Cultivate mouse RAW267.4 cells, prepare complete culture medium of different concentrations with cerium dioxide nanoparticles and high-glucose basal culture medium, plant the cells in a 96-well plate, add 100 μL of cell suspension to each well, place in an incubator and culture for 24 hours and 48 hours, then add 10 μL of CCK8 solution to each well and continue to culture for 0.5-4 hours. Use an ELISA reader to measure the absorbance value of each well at a wavelength of 450 nm. Calculate the cell proliferation rate based on the absorbance value.
[0057] 2. Experimental results: Please refer to Figure 1 E, CeO2 nanoparticles have no inhibitory effect on cell proliferation, high proliferation rate and good biocompatibility.
[0058] Example 4: Biocompatibility of Nanozyme Adhesive Hydrogel Microspheres
[0059] 1. Experimental methods:
[0060] 200 μL of viscous hydrogel containing sodium alginate microspheres of ceria and 200 μL of viscous hydrogel without microspheres were cured in 284 nm, 10 W ultraviolet light for 5 minutes, and then soaked in 30 mL of high-glucose complete medium containing 10% serum for 24 hours and taken out to obtain liquid A and liquid B. The culture medium containing 25%, 50%, 75%, and 100% of liquid A and liquid B were prepared with high-glucose complete medium containing 10% serum, and mouse RAW264.7 cells were cultured with this culture medium and culture medium without liquid A and liquid B. After 24 hours, the cell death and viability were fluorescently stained, and the survival of each group of cells was counted.
[0061] 2. Experimental results:
[0062] See also Figure 2 Compared with the control culture medium, culturing cells with nanozyme sticky hydrogel microspheres and culture medium soaked with sticky hydrogel has little effect on cell survival rate and good biocompatibility.
[0063] Example 5: Application of Nanozyme Adhesive Hydrogel Microspheres in Xerostomia Mice
[0064] 1. Experimental methods:
[0065] 24 NOD mice were randomly divided into three groups: a group smeared with viscous hydrogel (n=8), a group smeared with viscous hydrogel containing CeO2 sodium alginate microspheres (n=8), and a blank control group smeared with saline (n=8). At the same time, 8 B6 mice smeared with saline were used as a normal control group. From the thirteenth week, 15 μL of hydrogel, hydrogel containing microspheres, and saline were smeared on the sublingual oral mucosa of each group of mice, and cured under 284nm, 10W ultraviolet light for 5-10 seconds. The drug was administered for four weeks, twice a week. In the eighteenth week, salivary gland specimens were collected for HE staining.
[0066] 2. Experimental results:
[0067] See also Figure 3 Compared with the blank control group, the salivary gland lymphocyte aggregates of mice in the group treated with CeO2 sodium alginate microspheres adhesive hydrogel were significantly reduced, and the therapeutic effect on xerostomia model mice was good.
[0068] The experimental materials and sources of the above embodiments are as follows:
[0069] Experimental Materials source Polyethylenimine-polylactic-co-glycolic acid Qiyue Biology Cerium nitrate hexahydrate Merck Life Science Disodium Citrate Merck Life Science Total Superoxide Dismutase (SOD) Assay Kit Nanjing Jiancheng Bioengineering Institute Hydroxyl Radical Test Kit Nanjing Jiancheng Bioengineering Institute Reactive oxygen species detection kit Solebao CCK8 New Saimei
[0070] In summary, the nanozyme sticky hydrogel microspheres prepared in this application, wherein cerium dioxide (CeO2) has unique redox properties, can scavenge free radicals, and has potential effects in anti-inflammatory and anti-oxidation aspects; sodium alginate is a natural polysaccharide substance with good biocompatibility and degradability, gelatin is also a material with good biocompatibility and has a certain viscosity; polyacrylic acid is a commonly used polymer material. The prepared nanozyme sticky hydrogel microspheres can be applied to the oral cavity for the treatment of xerostomia. The hydrogel firmly adheres to the oral mucosa, and the long-term sustained release of cerium dioxide with antioxidant and anti-inflammatory effects provides a new and effective way to treat xerostomia.
Claims
1. A nanozyme viscous hydrogel microsphere, characterized in that: The inner core of the microsphere is a cerium dioxide sodium alginate microsphere, and the outer layer is a viscous hydrogel made of a combination of gelatin and cross-linked polyacrylic acid grafted with N-hydroxysuccinimide ester.
2. The nanozyme adhesive hydrogel microsphere according to claim 1, characterized in that: The cerium dioxide sodium alginate microspheres include cerium dioxide with a particle size of 1-50 nm and sodium alginate microspheres with a particle size of 100-300 μm, and the concentration of cerium dioxide in the sodium alginate microspheres is 60 μg / mL.
3. The nanozyme adhesive hydrogel microsphere according to claim 1, characterized in that: In the viscous hydrogel prepared by combining gelatin and cross-linked polyacrylic acid grafted with N-hydroxysuccinimide ester, the mass ratio of gelatin, acrylic acid, gelatin methacrylate (gelMA), α-ketoglutaric acid, and acrylic acid N-hydroxysuccinimide ester (AAc-NHS ester) is 100:300:1:2:10, and the thickness of the viscous hydrogel is 10-100 μm.
4. A method for preparing nanozyme viscous hydrogel microspheres, characterized in that: The steps for preparing the nanozyme sticky hydrogel microspheres according to any one of claims 1 to 3 are as follows: S1: Preparation of sodium alginate cerium dioxide microspheres: First, cerium dioxide nanoparticles are prepared, and then a sodium alginate solution is prepared. The cerium dioxide nanoparticles are dispersed in the sodium alginate solution to obtain a mixed solution. The solution is dripped into a calcium chloride solution by microfluidics, and the microspheres are washed with deionized water; S2: Preparation of viscous hydrogel: dissolving acrylic acid, gelatin, AAc-NHS ester, gelMA and α-ketoglutaric acid in water, stirring for 12 h, and filtering with a sterile syringe filter to obtain a viscous hydrogel; S3: Soak the cerium dioxide sodium alginate microspheres in S1 in the sticky hydrogel of S2, and perform ultrasonic vibration to form nanozyme sticky hydrogel microspheres.
5. The method for preparing nanozyme sticky hydrogel microspheres according to claim 4, characterized in that: The sodium alginate solution in S1 is a solution with a mass fraction of 2%, and the maximum concentration of cerium dioxide dissolved in the sodium alginate solution is 60 µg / mL.
6. The method for preparing nanozyme sticky hydrogel microspheres according to claim 4, characterized in that: The amounts of the materials added in S2 are as follows: 30% (w / w) acrylic acid, 10% (w / w) gelatin, 1% (w / w) AAc-NHS ester, 0.1% (w / w) gelMA and 0.2% (w / w) α-ketoglutaric acid.
7. Use of the nanozyme adhesive hydrogel microspheres as described in any one of claims 1 to 3 in the preparation of a drug for treating xerostomia.
8. A drug for treating dry mouth, characterized in that: The drug is prepared by adding 1% photocuring agent to the nanozyme adhesive hydrogel microspheres described in any one of claims 1 to 3.
9. A drug for treating xerostomia according to claim 8, characterized in that: The medicine is an oral spray.