Construction and application of nano-enzyme system for targeted therapy of xerostomia

Abstract

The invention provides construction and application of a nano-enzyme system for targeted therapy of xerostomia, and relates to the technical field of biomedicine, the nano-enzyme system comprises a nano-enzyme carrier and an enzyme active substance, the nano-enzyme carrier adopts a polyethyleneimine-polylactic acid-glycolic acid copolymer and has good biocompatibility and stability, and the nano-enzyme carrier has good biocompatibility and stability. Cerium oxide nanoparticles are adopted as the enzyme active substance, the dry mouth symptom can be effectively improved, the treatment effect is improved, the targeting molecule is a tertiary amine (triethylamine) group existing in polyethyleneimine-polylactic acid-glycolic acid with the unmodified surface, and the targeting molecule can easily recognize the submaxillary gland to achieve targeted treatment. According to the invention, a polyethyleneimine-polylactic acid-glycolic acid copolymer is used as a raw material, and a double-layer film-shaped module with a biocompatible gel polymer is synthesized by a double-emulsion method. And when the module is used as a carrier of the active nano-enzyme, the internal module can be transported to a specific region in a targeted manner to play a role.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the construction and application of a nanozyme system for targeted treatment of xerostomia. Background Art

[0002] Sjögren's syndrome Sjögren's syndrome (SS) is a common autoimmune disease characterized by chronic inflammation of the exocrine glands, leading to symptoms such as dry mouth and dry eyes. Xerostomia is one of the most common symptoms in patients with Sjögren's syndrome, which seriously affects the quality of life of patients.

[0003] Existing treatments mainly include local treatment and systemic treatment. Local treatment includes: 1. Artificial saliva substitutes: Relieve dry mouth symptoms by using artificial saliva substitutes. However, the effect of artificial saliva substitutes is limited, and they need to be used frequently, and patient compliance is poor. 2. Saliva stimulants: such as pilocarpine and cevimeline, relieve dry mouth symptoms by stimulating salivary gland secretion. However, these drugs have side effects, such as sweating, palpitations, etc., and are not effective for some patients. Systemic treatment mainly refers to the use of immunosuppressants and biologics. Existing treatments for dry mouth have problems such as poor efficacy and large side effects. Summary of the invention

[0004] The purpose of the present invention is to solve the technical problems of poor efficacy and large side effects in the prior art treatment of xerostomia.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A nanozyme system comprises a nanozyme carrier, a targeting molecule and an enzyme-active substance, wherein the nanozyme carrier is a polyethyleneimine-polylactic acid-glycolic acid copolymer, the enzyme-active substance is a cerium oxide nanoparticle, and the targeting molecule is a tertiary amine (triethylamine) group present in the surface-unmodified polyethyleneimine-polylactic acid-glycolic acid.

[0007] Preferably, the preparation method of the nanozyme system carrier is as follows:

[0008] S1: Dissolve polyethyleneimine-polylactic acid-glycolic acid completely in dichloromethane with double distilled water.

[0009] S2: dissolving the enzyme active substance-cerium oxide nanoparticles in the inner water phase of the mixed solution of S1;

[0010] S3: ultrasonically treating the mixed solution of S2, and adding a polyvinyl alcohol aqueous solution after the ultrasonic treatment;

[0011] S4: Collect the nanozyme system by centrifugation, wash and resuspend to obtain the nanozyme system.

[0012] Preferably, the preparation method of the cerium oxide nanoparticles is as follows:

[0013] Dissolve cerium nitrate hexahydrate in deionized water to form liquid a, and disodium citrate in deionized water to form liquid b;

[0014] Mix liquid A and liquid B thoroughly, add ammonia water, stir and react.

[0015] The mixed solution was centrifuged to remove aggregated particles in the mixed solution, and the supernatant was taken and dialyzed;

[0016] The dialyzed liquid was centrifuged to obtain the supernatant, which was filtered using a sterile syringe filter and then freeze-dried and stored at 4°C.

[0017] Preferably, S3 is carried out in two parts:

[0018] After ultrasonic treatment at 35 W for 5 min, 2 mL of 2% polyvinyl alcohol aqueous solution was added to the mixture;

[0019] The emulsion was then ultrasonicated for another 5 min and then dispersed into a 0.6% polyvinyl alcohol solution under stirring.

[0020] Preferably, in S3, the aqueous solution is divided into a 0.6% polyvinyl alcohol solution and then the remaining organic solvent is removed by rotary evaporation.

[0021] Preferably, the centrifugation condition in S4 is 13000g, 10 min, and washing is performed twice after the centrifugation is completed.

[0022] Preferably, in S4, a 0.22 μm sterile syringe filter is used to filter the supernatant.

[0023] The present application also provides an application of a nanozyme system in the preparation of a drug for the targeted treatment of xerostomia, wherein the nanozyme system is the nanozyme system described above.

[0024] The present invention uses polyethyleneimine-polylactic acid-glycolic acid copolymer as raw material, and synthesizes a double-layer membrane-shaped module with a biocompatible adhesive polymer by a double emulsion method. And the module is used as a carrier of active nanozymes, and the internal module can be targeted and transported to a specific area to play a role. The present invention can simply, quickly and flexibly produce various active nanozyme coated modules, and actively release them from the mold after targeting specific organs; it is expected to provide a flexible and effective alternative treatment for Sjögren's syndrome, and be applied in tissue engineering and regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The following are representative transmission electron micrographs of the nanozyme carrier and the cerium oxide nanoparticles in one embodiment of the present invention; (a) transmission electron micrograph of the nanozyme carrier; (b) transmission electron micrograph of the cerium oxide nanozyme system;

[0026] Figure 2 This is an in vitro verification of the targeting of the nanozyme carrier to the submandibular gland in one embodiment of the present invention;

[0027] Figure 3 This is an in vitro verification of the treatment of xerostomia by a nanozyme carrier loaded with cerium oxide particles in one embodiment of the present invention. (a, b) The expression results of CD4+CD8-CD25+Foxp3+Treg cell subsets in the spleen of the nanozyme system treatment group with different concentrations and the PBS control group showed that the proportion of CD4+CD8-CD25+Foxp3+Treg in the CD4+ cells in the nanozyme system treatment group was higher than that in the PBS control group. (c, d) The Elisa method was used to measure the Sjögren's syndrome-related antibodies in the peripheral blood of mice. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with specific embodiments.

[0029] A nanozyme system comprises a nanozyme carrier and an enzyme-active substance. The nanozyme carrier adopts a polyethyleneimine-polylactic acid-glycolic acid copolymer, which has good biocompatibility and stability. The enzyme-active substance adopts cerium oxide nanoparticles, which can effectively improve dry mouth symptoms and improve the treatment effect. The targeting molecule is a tertiary amine (triethylamine) group present in the surface-unmodified polyethyleneimine-polylactic acid-glycolic acid. The targeting molecule can easily recognize the submandibular gland and achieve targeted treatment.

[0030] In one embodiment, the preparation method of the nanozyme system is as follows:

[0031] S1: 20 mg of polyethyleneimine-polylactic acid-glycolic acid was completely dissolved in 1 mL of dichloromethane to which 0.2 mL of double distilled water had been added.

[0032] S2: Preparation of enzyme active substances - cerium oxide nanoparticles

[0033] 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.

[0034] 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.

[0035] The dialyzed liquid was centrifuged at 15,000 g for 10 minutes, and the supernatant was collected. The supernatant was filtered through a 0.22 μm sterile syringe filter, and then freeze-dried and stored at 4°C.

[0036] S3: Weigh 20 mg of cerium oxide nanoparticles and dissolve them in the internal aqueous phase of the mixed solution of S1;

[0037] S4: After ultrasonic treatment at 35 W for 5 min, 2 mL of 2% polyvinyl alcohol aqueous solution was added to the mixture;

[0038] S5: The emulsion was ultrasonically treated for another 5 min and then dispersed into a 0.6% polyvinyl alcohol solution under stirring, and the remaining organic solvent was removed by rotary evaporation;

[0039] S6: The nanozyme system particles were collected by centrifugation at 13000 g for 10 min, washed twice, and resuspended in water or saline to obtain the nanozyme system.

[0040] The present application also provides the use of the above-mentioned nanozyme system in the preparation of a drug for the targeted treatment of xerostomia.

[0041] The above contents are described below in conjunction with specific embodiments:

[0042] Example 1: Construction of a targeted nanozyme system ( Figure 1 )

[0043] (1) Nanozyme carrier: The present invention uses polyethyleneimine-polylactic acid-glycolic acid copolymer as raw material and synthesizes a double-layer membrane-like module with biocompatible adhesive polymer by double emulsion method. It has good biocompatibility and stability.

[0044] The experimental method is as follows:

[0045] 20 mg of polyethyleneimine-polylactic acid-glycolic acid was completely dissolved in 1 mL of dichloromethane to which 0.2 mL of double distilled water had been added.

[0046] For the complete nanosystem construction, cerium oxide nanoparticles were dissolved in the internal aqueous phase of the system.

[0047] After ultrasonic treatment at a power of 35 W for 5 min, 2 mL of 2% polyvinyl alcohol aqueous solution was added to the mixture.

[0048] The emulsion was sonicated for another 5 minutes and then dispersed into a 0.6% polyvinyl alcohol solution under stirring. The remaining organic solvent was removed by rotary evaporation.

[0049] Nanoparticles were collected by centrifugation at 13000 g for 10 min, washed twice, and resuspended in water or saline for further use.

[0050] The transmission electron microscopy image of the obtained nanozyme carrier is as follows Figure 1 As shown in a.

[0051] (2) Targeting molecules: The tertiary amine (triethylamine) group present in the surface-unmodified polyethyleneimine-polylactic acid-glycolic acid copolymer can specifically recognize the type 3 muscarinic acetylcholine receptor on the surface of the submandibular gland, thereby achieving targeted therapy.

[0052] (3) Enzyme-active substances: Cerium oxide nanoparticles have peroxidase-like activity and can effectively improve dry mouth symptoms and enhance the therapeutic effect.

[0053] The experimental method is as follows:

[0054] 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.

[0055] 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.

[0056] The dialyzed liquid was centrifuged at 15,000 g for 10 minutes, and the supernatant was collected. The supernatant was filtered through a 0.22 μm sterile syringe filter, and then freeze-dried and stored at 4°C.

[0057] Take 20 mg of cerium oxide nanoparticles and dissolve them in the internal aqueous phase of the mixed solution of the nanozyme carrier; after ultrasonic treatment at 35 W power for 5 minutes, add 2 mL of 2% polyvinyl alcohol aqueous solution to the mixture; ultrasonically treat the emulsion for another 5 minutes, then disperse it into a 0.6% polyvinyl alcohol solution under stirring, and remove the remaining organic solvent by rotary evaporation; centrifuge at 13000g for 10 minutes to collect the nanozyme system particles, wash twice, and resuspend them in water or saline to obtain the nanozyme system. The scanning electron microscopy image of the obtained nanozyme system is shown in the figure. Figure 1 As shown in b.

[0058] Example 2: Targeting verification of nanozyme system ( Figure 2 )

[0059] The fluorescently labeled nanozyme system was injected into the tail vein, and the targeting ability of the system was verified by the small animal in vitro imaging system.

[0060] The experimental method is as follows:

[0061] As in Example 1, water-soluble rhodamine B is dissolved in the aqueous phase inside the nanosystem.

[0062] Free saline, rhodamine B, and rhodamine B-labeled nanoparticles were intravenously injected into different groups of BALB / c mice (n=3).

[0063] All mice were sacrificed 8 hours after injection.

[0064] The submandibular gland and other major organs including liver, kidney, spleen, lung, and heart were dissected and imaged using a fluorescence imaging system.

[0065] Experimental results: Please refer to Figure 2 , the nanozyme system carrier has specific targeting ability to the submandibular gland tissue.

[0066] Example 3: In vitro verification of the function of the nanosystem Figure 3 )

[0067] Female NOD mice (spontaneous drying mice) aged 8 weeks were randomly divided into 5 groups, PBS group and nanozyme system treatment group (treatment groups were divided into 5 mg / kg, 15 mg / kg, 30 mg / kg, 50 mg / kg according to concentration) (n=6), and each mouse was injected with 100 μL of the nanozyme system or PBS through the tail vein every week for 8 consecutive weeks. The mice were killed after 8 weeks, and the proportion of Treg cells in the spleen of the mice was detected by flow cytometry. The venous blood of the mice was taken, and the autoantibodies SSA and SSB in the serum of the mice were determined by Elisa method. C57BL / 6 mice were used as positive controls.

[0068] 3.1 Flow cytometry test of mouse spleen Treg cells

[0069] 3.1.1 Preparation of mouse spleen cell suspension

[0070] (1) Remove the mouse spleen and immediately place it in 1640 serum-free and dual antibody-free culture medium.

[0071] (2) Place a 100 μm filter on the culture dish, place the spleen in the filter, add 2 mL of 1640 serum-free and double-antibody-free culture medium, and use the end of a 1 mL syringe to gently and thoroughly grind in the same direction until no visible tissue is left.

[0072] (3) Use a Pasteur pipette to aspirate the culture medium in the above culture dish onto the cap of a blue-capped flow cytometer and filter it. Then use 1 mL of 1640 serum-free and double-antibody-free culture medium to fully rinse the filter and the remaining cells in the culture dish, aspirate it onto the cap of the flow cytometer and filter it. Temporarily store it on ice.

[0073] (4) Centrifuge the filtered cell suspension at 1500 rpm for 10 min and discard the supernatant.

[0074] (5) Resuspend the cell pellet in 5 mL of 1× RBC Lysis Buffer, transfer the cell pellet to a 50 mL centrifuge tube, and incubate the cells for 5 min at room temperature.

[0075] (6) Add 25 mL of PBS to the above 50 mL centrifuge tube to stop the red cell lysis, centrifuge at 1800 rpm for 15 min, and then discard the supernatant.

[0076] (7) After discarding the supernatant, add 500 μL PBS to the cell pellet and resuspend the cells to obtain a spleen lymphocyte suspension.

[0077] 3.1.2 Flow cytometry staining of mouse spleen Treg cell subsets

[0078] (1) Prepare mouse spleen lymphocyte suspension according to step 3.1.1 above.

[0079] (2) 100 μL of the mouse spleen lymphocyte suspension was taken out and added to a flow cytometry tube, and PBS was added to the tube to make up to 1 mL.

[0080] (3) Add 1 μL of FVD to the flow tube in step (2), place on a shaker, and incubate at 4°C in the dark for 20 min.

[0081] (4) After 20 min, add 2 mL of PBS to the flow tube in step (3), centrifuge at 1800 rpm for 6 min, and discard the supernatant.

[0082] (5) Add 100 μL PBS to the cell pellet to resuspend the cells, add 0.2 μL mouse CD3-APC-CY7, 0.5 μL CD4-FITC, and 0.3 μL CD8-AF700 fluorescent antibodies to the cell suspension, vortex mix promptly, place on a shaker, and incubate at 4°C in the dark for 30 min.

[0083] (6) After 30 min, add 2 mL of PBS to the flow tube in step (5), centrifuge at 1800 rpm for 6 min, and discard the supernatant.

[0084] (7) Add 1 mL of the prepared Foxp3 fixation and permeabilization solution to the cell pellet from step (6), resuspend the cells, and permeabilize at room temperature for 30 min.

[0085] (8) After 30 min, add 2 mL of 1×Foxp3 permeabilization buffer to the flow tube in step (7), centrifuge at 2200 rpm for 6 min, and discard the supernatant.

[0086] (9) Add 100 μL of 1×Foxp3 permeabilization solution to the cell pellet in step (8) to resuspend the cells. After resuspension, add 2 μL of mouse Foxp3-eFluor610 fluorescent antibody, place on a shaker, and incubate at room temperature for 40 min.

[0087] (10) Add 2 mL of 1×Foxp3 permeabilization buffer to the flow tube in step (9), centrifuge at 2200 rpm for 6 min, and discard the supernatant.

[0088] (11) Add 200 μL of 1×Foxp3 permeabilization buffer to the cell pellet in step (10) and resuspend the cells for detection.

[0089] 3.2 ELISA method for determination of Sjögren's syndrome related antibodies SSA and SSB in mouse peripheral blood serum

[0090] (1) Return the mouse serum and reagents in the Elisa kit to room temperature;

[0091] (2) Draw the sample layout;

[0092] (3) Dilute mouse serum: dilute 5 μL of mouse serum with 500 μL of serum diluent;

[0093] (4) Preparing a microplate;

[0094] (5) Take 100 μL of standard, negative and positive control reagents, and diluted mouse serum and place them in the corresponding positions according to the drawn sample layout;

[0095] (6) Incubate at room temperature for 30 min;

[0096] (7) Wash 4 times with lotion;

[0097] (8) Add 100 μL of secondary antibody to each microwell and incubate at room temperature for 30 min;

[0098] (9) Wash 4 times with lotion;

[0099] (10) Add 100 μL of enzyme reaction substrate to each microwell;

[0100] (11) Incubate at room temperature for 30 min;

[0101] (12) Add 100 μL of reaction stop solution to each microwell in the order of adding enzyme reaction substrates, observe the color of the reaction solution, and measure the absorbance within 30 min.

[0102] For experimental results, see Figure 3 The nanozyme system of the present invention can significantly increase the production of regulatory T cells and reduce Sjögren's syndrome-related antibodies in peripheral blood, and is dose-dependent within a certain concentration.

[0103] The experimental materials and sources in the above embodiments of this application are as follows:

[0104] Experimental Materials source Polyethylenimine-polylactic-co-glycolic acid Qiyue Biology Cerium nitrate hexahydrate Merck Life Science Disodium Citrate Merck Life Science

[0105] In summary, the nanozyme system provided in the present application uses polyethyleneimine-polylactic acid-glycolic acid copolymer as raw material, synthesizes a double-layer membrane-shaped module with biocompatible polyol polymer by double emulsion method, and uses the module as a carrier of active nanozymes, which can target and transport the internal modules to specific areas to exert their effects. The nanozyme system provided in the present application can simply, quickly and flexibly produce various hundred-fold modules of active nanozymes, and actively release them from the mold after targeting specific organs. The present application uses the nanozyme system as a flexible and effective alternative treatment for Sjögren's syndrome, and it is used in tissue engineering and regenerative medicine.

Claims

1. A nanozyme system, characterized in that: The invention comprises a nanozyme carrier, a targeting molecule and an enzyme-active substance, wherein the nanozyme carrier is a polyethyleneimine-polylactic acid-glycolic acid copolymer, the enzyme-active substance is a cerium oxide nanoparticle, and the targeting molecule is a tertiary amine (triethylamine) group existing in the polyethyleneimine-polylactic acid-glycolic acid with unmodified surface.

2. A nanozyme system according to claim 1, characterized in that: The preparation method of the nanozyme system carrier is as follows: S1: Dissolve polyethyleneimine-polylactic acid-glycolic acid completely in dichloromethane with double distilled water. S2: dissolving the enzyme active substance-cerium oxide nanoparticles in the inner water phase of the mixed solution of S1; S3: ultrasonically treating the mixed solution of S2, and adding a polyvinyl alcohol aqueous solution after the ultrasonic treatment; S4: Collect the nanozyme system particles by centrifugation, wash and resuspend to obtain the nanozyme system.

3. A nanozyme system according to claim 2, characterized in that: The preparation method of the cerium oxide nanoparticles is as follows: Dissolve cerium nitrate hexahydrate in deionized water to form liquid a, and disodium citrate in deionized water to form liquid b; Mix liquid A and liquid B thoroughly, add ammonia water, stir and react. The mixed solution was centrifuged to remove aggregated particles in the mixed solution, and the supernatant was taken and dialyzed; The dialyzed liquid was centrifuged to obtain the supernatant, which was filtered using a sterile syringe filter and then freeze-dried and stored at 4°C.

4. A nanozyme system according to claim 3, characterized in that: The S3 is divided into two parts: After ultrasonic treatment at 35 W for 5 min, 2 mL of 2% polyvinyl alcohol aqueous solution was added to the mixture; The emulsion was then ultrasonicated for another 5 min and then dispersed into a 0.6% polyvinyl alcohol solution under stirring.

5. A nanozyme system according to claim 4, characterized in that: In the step S3, the aqueous solution is divided into a 0.6% polyvinyl alcohol solution and then the remaining organic solvent is removed by rotary evaporation.

6. A nanozyme system according to claim 5, characterized in that: The centrifugation condition in S4 is 13000 g, 10 min, and washing is performed twice after the centrifugation is completed.

7. A nanozyme system according to claim 6, characterized in that: The supernatant was filtered using a 0.22 μm sterile syringe filter as described in S4.

8. Application of a nanozyme system in the preparation of a drug for targeted treatment of xerostomia, characterized in that: The nanozyme system is the nanozyme system according to any one of claims 1 to 7.

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