Preparation method and application of nanocomposite adhesive hydrogel based on astragalus extract

Abstract

The present invention discloses a preparation method and application of a nanocomposite adhesive hydrogel based on astragalus extract. The preparation method comprises: Step 1: grafting triaminophenylboronic acid monohydrate onto bexarotene via an amidation reaction to obtain bexarotene grafted with phenylboronic acid; Step 2: mixing the bexarotene grafted with phenylboronic acid with astragalus polyphenol and conducting a borate ester reaction to obtain astragalus polyphenol nanoparticles; and Step 3: mixing the astragalus polyphenol nanoparticles with astragalus polysaccharide and subjecting them to hydrogen bonding to obtain a nanocomposite adhesive hydrogel. The nanocomposite adhesive hydrogel prepared by the present invention synergizes the efficacy of astragalus extract and bexarotene and can be used to repair cerebral hematoma injuries.

Description

Technical Field

[0001] The present application belongs to the technical field of biomedical materials, and specifically relates to a preparation method and application of a nanocomposite adhesive hydrogel based on astragalus extract. Background Art

[0002] Polymer nanoparticle hydrogels have attracted widespread attention due to their unique advantages, such as strong adhesion, low cost and safety. This multifunctional, tunable dynamic nanocomposite material is formed by the reversible interaction between polymer chains and nanoparticles, and has attracted widespread attention in tissue adhesion, wound healing, organ repair and other biomedical applications. Scientific researchers have widely reported a variety of nanoparticles and nanosheets combined with polymer chains to prepare nanocomposite hydrogels, which can achieve rapid wound repair in special environments. However, these materials generally lack multifunctionality and excellent biocompatibility, and therefore still face severe challenges in coping with special diseases in complex environments and clinical applications (such as cardiovascular, liver and brain diseases).

[0003] As a traditional Chinese medicine, the root of Astragalus has a long history of medicinal use. Pharmacological studies have shown that Astragalus contains a variety of elements, including folic acid, amino acids, betaine, carbohydrates, and proteins. Generally speaking, Astragalus and its extracts have the effects of replenishing qi and raising yang, strengthening the exterior and stopping sweating, promoting diuresis and reducing swelling, promoting fluid production and nourishing blood, relieving stagnation and relieving numbness, expelling toxins and draining pus, and healing sores and promoting tissue regeneration. Astragalus polyphenols are a natural extract containing multiple catechols with biological effects such as antioxidant, anti-inflammatory, and antibacterial properties. Astragalus polysaccharides are an extract with multiple hydrogen bonding groups that can be prepared into hydrogels with excellent adhesion. Therefore, combining Astragalus polyphenols and Astragalus polysaccharide extracts to prepare a new type of antioxidant and anti-inflammatory nanocomposite hydrogel is a feasible strategy. Summary of the Invention

[0004] The purpose of this application is to provide a preparation method and application of nanocomposite adhesive hydrogel based on astragalus extract.

[0005] On the one hand, the present application provides a method for preparing a nanocomposite adhesive hydrogel based on an astragalus extract, comprising:

[0006] Step 1: grafting triaminophenylboronic acid monohydrate onto bexarotene drug through amidation reaction to obtain bexarotene drug grafted with phenylboronic acid;

[0007] Step 2: mixing the bexarotene drug grafted with phenylboronic acid with astragalus polyphenol to carry out a borate ester reaction to obtain astragalus polyphenol nanoparticles;

[0008] Step 3: Mix the astragalus polyphenol nanoparticles and astragalus polysaccharide and form a nanocomposite adhesive hydrogel through hydrogen bonding.

[0009] The structural formula of bexarotene is shown in the following formula (I):

[0010]

[0011] The structural formula of the bexarotene drug grafted with phenylboronic acid is shown in the following formula (II):

[0012]

[0013] The structural formula of Astragalus polyphenols is shown in the following formula (III):

[0014]

[0015] The structural formula of Astragalus polyphenols is shown in the following formula (IV):

[0016]

[0017] In some specific embodiments, step 1 is specifically:

[0018] 1-2 mole parts of triaminophenylboronic acid monohydrate, 1.5-3 mole parts of amidation catalyst and 1-2 mole parts of bexarotene drug are mixed and dissolved in dimethyl sulfoxide or dimethylformamide, and reacted for 12-24 hours to obtain bexarotene drug grafted with phenylboronic acid.

[0019] Furthermore, the amidation catalyst includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, wherein the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:1.

[0020] In some specific implementations, step 2 is specifically:

[0021] Bexarotene grafted with phenylboronic acid and astragalus polyphenol are taken in a mass ratio of 4 to 8:10 to 30, respectively, a dimethyl sulfoxide or dimethylformamide solution of the bexarotene grafted with phenylboronic acid is prepared, and an astragalus polyphenol aqueous solution is prepared. The dimethyl sulfoxide or dimethylformamide solution of the bexarotene grafted with phenylboronic acid is added dropwise to the astragalus polyphenol aqueous solution, and the astragalus polyphenol nanoparticles are obtained after stirring and reacting for 2 to 24 hours.

[0022] In some specific embodiments, the concentration of the dimethyl sulfoxide or dimethylformamide solution of the bexarotene drug grafted with phenylboronic acid is 10-30 mg / mL, and the concentration of the astragalus polyphenol aqueous solution is 4-8 mg / mL.

[0023] In some specific embodiments, step three is specifically:

[0024] 0.5-5 parts by mass of astragalus polyphenol nanoparticles are dispersed in water, 50-100 parts by mass of astragalus polysaccharide are taken to prepare an astragalus polysaccharide aqueous solution, the astragalus polysaccharide aqueous solution is dissolved in the dispersed solution, and the mixture is stirred for 5-12 hours to obtain a nanocomposite adhesive hydrogel.

[0025] In some specific embodiments, the concentration of astragalus polyphenol nanoparticles in the reaction solution is 0.5-5 mg / mL, and the concentration of the astragalus polysaccharide aqueous solution is 50-100 mg / mL.

[0026] Astragalus extract has excellent antioxidant and adhesive properties. Bexarotene, a receptor-selective agent, can bind to relevant brain receptors, selectively activating retinoid X receptor (RXR) agonists, thereby being used to treat brain diseases. The prepared astragalus extract-based nanocomposite adhesive hydrogel, synergistically combining the effects of astragalus extract and bexarotene, can be used to repair brain hematoma injuries. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a hydrogen nuclear magnetic resonance spectrum of the bexarotene drug grafted with phenylboronic acid in the examples of this application;

[0028] Figure 2 This is the mass spectrum of bexarotene grafted with phenylboronic acid in the examples of this application;

[0029] Figure 3 This is a scanning electron micrograph of the astragalus polyphenol nanoparticles prepared in the examples of this application;

[0030] Figure 4 : is the modulus curve of different samples in the examples of this application;

[0031] Figure 5 Viscosity curves of different samples in the examples of this application;

[0032] Figure 6 The adhesion strength of different samples on different substrates in the examples of this application;

[0033] Figure 7 The antioxidant properties of different samples in the examples of this application;

[0034] Figure 8 The biocompatibility cell test results of the nanoparticles in the examples of this application;

[0035] Figure 9 This is the brain hematoma clearance rate of animals by different samples in the examples of this application. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and beneficial effects of this application more clear, this application is further described in detail below in conjunction with embodiments.

[0037] In order to make the purpose, technical solutions and beneficial effects of this application more clear, this application is further described in detail below in conjunction with embodiments.

[0038] Example

[0039] The specific steps of this embodiment are as follows:

[0040] Step 1: 1 mol part of triaminophenylboronic acid monohydrate, 3 mol parts of an amidation catalyst, and 1.5 mol parts of bexarotene are mixed and dissolved in dimethyl sulfoxide, and the mixture is stirred for 12 hours to obtain bexarotene grafted with phenylboronic acid; wherein the amidation catalyst comprises 1.5 mol parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 1.5 mol parts of N-hydroxysuccinimide.

[0041] The product of this step was subjected to nuclear magnetic resonance detection, and the hydrogen spectrum and mass spectrum were respectively shown in Figures 1-2 ,from Figures 1-2 It can be seen that the grafting of phenylboronic acid was successful.

[0042] Step 2: Prepare a dimethyl sulfoxide solution of bexarotene grafted with phenylboronic acid at a concentration of 20 mg / mL; prepare an astragalus polyphenol aqueous solution at a concentration of 5 mg / mL; take 200 microliters of the dimethyl sulfoxide solution of bexarotene grafted with phenylboronic acid, add it dropwise to 5 milliliters of the astragalus polyphenol aqueous solution, and stir the reaction for 5 hours to obtain astragalus polyphenol nanoparticles.

[0043] The scanning electron photo of the product of this step is shown in Figure 3 , it can be seen that nano-scale astragalus polyphenol extract has been successfully prepared.

[0044] Step 3: Disperse the astragalus polyphenol nanoparticles in an aqueous solution to obtain a dispersion with a concentration of 1 mg / mL; prepare an astragalus polysaccharide aqueous solution with a concentration of 60 mg / mL; take 500 microliters of the dispersion, and then take 500 microliters of the astragalus polysaccharide aqueous solution and dissolve it in the dispersion. After 12 hours of hydrogen bonding physical action, a nanocomposite adhesive hydrogel based on astragalus extract is obtained.

[0045] The product of this step was subjected to rheological tests under the following conditions: 1% strain, 25°C, 10 rad / s. Figures 4-5 , Figure 4 and Figure 5 The modulus curve and viscosity curve are respectively, where "nanogel" is the nanocomposite adhesive hydrogel of this embodiment. Figures 4-5 It can be seen that the nanocomposite adhesive hydrogel based on Astragalus extract has been successfully prepared.

[0046] To verify the performance advantages of the nanocomposite adhesive hydrogel based on Astragalus extract in this application, the performance of different samples will be evaluated separately below. Among them, the "nanogel" sample is the product of step three of the embodiment, that is, the nanocomposite adhesive hydrogel based on Astragalus extract; the "nanoparticle" sample is the product of step two of the embodiment, that is, Astragalus polyphenol nanoparticles; and the "gel" sample is an Astragalus polysaccharide gel without Astragalus polyphenol nanoparticles, and its preparation method is as follows:

[0047] Astragalus polyphenols were dispersed in an aqueous solution to obtain a dispersion with a concentration of 5 mg / mL; an astragalus polysaccharide aqueous solution was prepared with a concentration of 60 mg / mL; 1000 microliters of the dispersion and the astragalus polysaccharide aqueous solution were taken respectively, and the astragalus polysaccharide aqueous solution was dissolved in the dispersion, and a gel based on the astragalus extract was obtained through the physical action of hydrogen bonds.

[0048] The performance evaluation of the above three samples is carried out as follows.

[0049] 1. Evaluation of Adhesion

[0050] Evaluate the adhesion of gels and nanogels to different substrates. The gel sample was evenly applied to the surface of a 25mm*25mm substrate. When the substrate was pig skin, the gel sample was applied to the inner epidermis of the pig skin. A universal testing machine was used to measure the lap shear stress of the PDL gel sample adhering to the substrate. The ends of the tissue sample were fixed to a single clamp and stretched at a tensile rate of 10mm / min using a 500N load cell. The adhesion strength was calculated by dividing the maximum stress by the contact area between the gel sample and the substrate. The adhesion strength data can be found in Figure 6 .from Figure 6 It can be seen that the adhesion of nanogel is significantly better.

[0051] 2. Antioxidant Capacity Assessment

[0052] The antioxidant capacity of nanoparticles, gels and nanogels was evaluated. The antioxidant capacity of different samples in aqueous solution was evaluated by scavenging 2,2'-azido-bis(3-ethylbenzothiazoline-6-sulfonic acid) radicals (ABTS). ABTS solution was reacted with 2.45 mM potassium persulfate at room temperature in the dark for 12 h to generate ABTS cation radicals. Sample solution and 7 mM ABTS aqueous solution were prepared. Sample solution and 100 μL of ABTS solution were added to meet the test requirement of a total of 3 mL and the absorbance of the test sample at 734 nm was recorded for 30 minutes. The above test was repeated three times for each group of samples. The test results are shown in Figure 7 , used to evaluate the antioxidant capacity of nanoparticles and nanocomposite hydrogels. Figure 7 It can be seen that both nanoparticles and nanogels have relatively excellent antioxidant properties.

[0053] 3. Biocompatibility Cell Testing

[0054] Test the biocompatibility of nanoparticles Cell viability. NIH 3T3 cells (5% CO2 and 37°C) were cultured for cellular evaluation of nanoparticles, and the in vitro cell viability of each sample was assessed using the Alamar blue method. NIH 3T3 cells were plated on 96-well plates (2000 cells per well) and cultured for 24 hours. The cells were then treated with different concentrations of nanoparticle solutions and incubated for 24 hours. The corresponding cell viability of each sample was then assessed using the standard Alamar blue method. The test results are shown in Figure 8 .

[0055] 4. Animal Experiment on Repair of Brain Hematoma Trauma in Rats

[0056] (1) Establishment of brain hematoma injury model:

[0057] Mice weighing 25-30 g were used. Anesthetized preoperatively with intraperitoneal injection of tribromoethanol. After anesthesia was achieved and respiratory stability was confirmed by loss of the righting reflex, the mice were placed in a prone position and fixed on a stereotaxic apparatus. The head was prepared, and after disinfection, a midline incision was made. Blunt dissection was performed to expose the bregma. Positioning was performed 0.2 mm anterior to the bregma and 2.25 mm to the right of the sagittal suture. A bone hole approximately 1 mm in diameter was drilled with a burr drill and set aside. The right femoral artery and vein were dissected and exposed. The proximal end was temporarily occluded, and the distal end was ligated and blocked. The femoral artery and vein were straightened and tensioned. A small incision was made near the distal end of the femoral artery with microscissors. The femoral artery was cannulated with a plastic catheter. After cannulation, approximately 40 μL of femoral arterial blood was collected into a 1 mL syringe. Then, 30 μl of autologous blood was injected into the right basal ganglia area (3.7 mm deep) of the mouse through a microinjection pump. The injection speed was set to 3 μL / min. After the injection, the needle was kept for 10 minutes before being withdrawn to see if there was any blood reflux. If no reflux was observed, the skull hole was sealed with bone wax and the skin of the head and thigh was sutured.

[0058] (2) Experimental grouping and sampling method:

[0059] Eighteen rats with a brain hematoma injury model were randomly divided into three groups according to body weight, including a control group of 6 rats (no treatment), a Gel group, and a Gel-NPs group of 6 rats (bioadhesive hydrogel loaded with nanoparticles). The drugs were injected through the tail vein at a dose of 1.0 mg / Kg / Day and were continuously administered for 1-7 days after modeling.

[0060] (3) The wound healing was observed every day and imaging analysis was performed. The edema and hematoma of the mice were observed by MRI T2 and T2* sequences on the day of modeling and on the 3rd, 7th, and 14th days respectively. The degree of edema and hematoma volume were calculated by ImageJ and MRI analysis was performed.

[0061] The results of the rat brain hematoma clearance test are shown in Figure 9 , shows the hematoma clearance rate calculated based on the wound healing status on the 7th day.

[0062] The above embodiments are merely for the purpose of illustrating the embodiments and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications may be made based on the above descriptions. It is not necessary and impossible to enumerate all implementation methods here. Therefore, any obvious variations or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A method for preparing a nanocomposite adhesive hydrogel based on an astragalus extract, comprising: Step 1: grafting triaminophenylboronic acid monohydrate onto bexarotene drug through amidation reaction to obtain bexarotene drug grafted with phenylboronic acid; Step 2: mixing the bexarotene drug grafted with phenylboronic acid with astragalus polyphenol to carry out a borate ester reaction to obtain astragalus polyphenol nanoparticles; Step 3: Mixing the astragalus polyphenol nanoparticles and astragalus polysaccharide and subjecting them to hydrogen bonding to obtain a nanocomposite adhesive hydrogel; Step three comprises dispersing 0.5 to 5 parts by weight of astragalus polyphenol nanoparticles in water, preparing an astragalus polysaccharide aqueous solution by taking 50 to 100 parts by weight of astragalus polysaccharide, dissolving the astragalus polysaccharide aqueous solution in the dispersed solution, and mixing and stirring for 5 to 12 hours to obtain a nanocomposite adhesive hydrogel; The structural formula of the astragalus polyphenol is The structural formula of the astragalus polysaccharide is:

2. The method for preparing the nanocomposite adhesive hydrogel based on Astragalus extract according to claim 1, wherein: Step 1 is as follows: 1-2 mole parts of triaminophenylboronic acid monohydrate, 1.5-3 mole parts of amidation catalyst and 1-2 mole parts of bexarotene drug are mixed and dissolved in dimethyl sulfoxide or dimethylformamide, and reacted for 12-24 hours to obtain bexarotene drug grafted with phenylboronic acid.

3. The method for preparing the nanocomposite adhesive hydrogel based on Astragalus extract according to claim 2, characterized in that: The amidation catalyst includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, wherein the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:

1.

4. The method for preparing the nanocomposite adhesive hydrogel based on Astragalus extract according to claim 1, wherein: Step 2 is as follows: Bexarotene grafted with phenylboronic acid and astragalus polyphenol are taken in a mass ratio of 4 to 8:10 to 30, respectively, a dimethyl sulfoxide or dimethylformamide solution of the bexarotene grafted with phenylboronic acid is prepared, and an astragalus polyphenol aqueous solution is prepared. The dimethyl sulfoxide or dimethylformamide solution of the bexarotene grafted with phenylboronic acid is added dropwise to the astragalus polyphenol aqueous solution, and the astragalus polyphenol nanoparticles are obtained after stirring and reacting for 2 to 24 hours.

5. Use of the nanocomposite adhesive hydrogel prepared by the preparation method according to any one of claims 1 to 4 in preparing a drug for repairing brain hematoma injury.

6. Use of the nanocomposite adhesive hydrogel prepared by the preparation method according to any one of claims 1 to 4 in preparing drugs for treating brain diseases.

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