Hydrogel for adjuvant therapy of periodontitis and preparation method thereof

Abstract

The invention discloses hydrogel for adjuvant therapy of periodontitis and a preparation method, and relates to the technical field of medical preparations, the hydrogel comprises astaxanthin, cationic antibacterial peptide and a hydrogel matrix, and the preparation method comprises the following steps: preparing astaxanthin PLGA micro-nanospheres by an ultrasonic emulsion solvent evaporation method; preparing oxidized hyaluronic acid by adopting a sodium periodate oxidation method; the preparation method comprises the following steps: mixing a carboxymethyl chitosan aqueous solution and an oxidized hyaluronic acid aqueous solvent which are equal in volume to form a hydrogel matrix; and fully mixing the astaxanthin PLGA micro-nanospheres, the cationic antibacterial peptide and the hydrogel matrix. According to the invention, the astaxanthin is prepared into the PLGA micro-nanospheres, so that the problems that the astaxanthin is easy to oxidize and the solubility is poor are solved; the cationic antibacterial peptide is used for bacteriostatic supplement; the hydrogel is locally delivered through the hydrogel matrix astaxanthin and the cationic antibacterial peptide, has good biocompatibility and antibacterial property, and can improve the internal environment of periodontal tissues and accelerate recovery of periodontal steady state.

Description

Technical Field

[0001] The present invention relates to the technical field of medical preparations, and in particular to a hydrogel for assisting in the treatment of periodontitis and a preparation method thereof. Background Art

[0002] Periodontitis is an inflammatory disease caused by the complex interaction between plaque microorganisms and the host immune system. Its main manifestations are red, swollen and bleeding gums, the formation of periodontal pockets, and alveolar bone resorption. In the late stage, teeth may become loose and displaced, or even fall out. It is the primary cause of tooth loss in adults. Periodontitis not only affects oral health, but is also closely related to systemic diseases such as diabetes, arthritis, cardiovascular and respiratory diseases. It seriously affects physical and mental health and quality of life. Plaque microorganisms are the initiating factors that destroy periodontal tissues. They can cause the host's immune response through their own antigenic components and toxic products, leading to the recruitment of too many neutrophils in the inflammatory area. Neutrophils can produce excessive reactive oxygen species (ROS) composed of superoxide anions, hydroxyl free radicals, hydrogen peroxide, etc. through respiratory bursts. This large amount of reactive oxygen species (ROS) that cannot be balanced by oxidants in the body can activate inflammatory mediators, activate the NF-kβ / JNK pathway, induce cell apoptosis, inhibit osteoblast differentiation, promote osteoclast maturation, accelerate attachment loss, and cause oxidative stress in tissue cells, thereby destroying cell membranes and damaging lipids, proteins, and DNA. The oxidative damage products generated can lead to telomere shortening and loss, induce normal cell apoptosis, and reactive oxygen species (ROS) can also inhibit osteoblast differentiation and promote osteoclast maturation, causing alveolar bone destruction and accelerating attachment loss.

[0003] At present, the traditional clinical plaque control and treatment of periodontitis are mainly surgical and non-surgical treatments, and their main purpose is to control inflammation, eliminate plaque, and promote the repair and regeneration of periodontal tissues. However, traditional treatment methods still have certain limitations. Some patients need to use antibiotics for plaque control after treatment. Currently, the commonly used routes of administration in clinical practice are systemic and local delivery. Although this method is effective, it still has many shortcomings. For example, systemic delivery of drugs can easily lead to problems such as dysbiosis and drug resistance. Commonly used local delivery dosage forms are mostly in the form of ointments, such as Palio ointment. This method of administration has problems such as being unable to penetrate deep into the periodontal pocket and having a short retention time in the affected area.

[0004] Therefore, it is of great research value to construct a new biomaterial with antibacterial and anti-inflammatory properties and regulate the level of reactive oxygen species (ROS) for local application in the treatment of periodontitis. Summary of the invention

[0005] The present invention aims to provide a hydrogel for the auxiliary treatment of periodontitis and a preparation method thereof, so as to solve the problems that systemic delivery of drugs easily leads to bacterial flora imbalance and drug resistance, and that ointment-type drug administration methods have the problems of being unable to penetrate into the periodontal pocket and having a short retention time in the affected area.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A hydrogel for assisting the treatment of periodontitis, the hydrogel comprising the following components and contents: 6 μM astaxanthin (ASX), 100 μg / ml cationic antimicrobial peptide (AMP) and a hydrogel matrix.

[0008] Furthermore, the astaxanthin (ASX) is water-soluble astaxanthin (ASX) PLGA micro-nanospheres.

[0009] Furthermore, the hydrogel matrix includes oxidized hyaluronic acid (OHA) and carboxymethyl chitosan (CMCS).

[0010] Furthermore, the oxidized hyaluronic acid (OHA) and carboxymethyl chitosan (CMCS) in the hydrogel matrix are both 4% aqueous solutions.

[0011] A method for preparing a hydrogel for assisting the treatment of periodontitis, the drug preparation steps comprising:

[0012] S1, preparation of astaxanthin (ASX) PLGA micro-nanospheres by ultrasonic emulsion solvent evaporation method;

[0013] S2, preparing oxidized hyaluronic acid (OHA) by sodium periodate oxidation method;

[0014] S3, fully mixing equal volumes of carboxymethyl chitosan (CMCS) aqueous solution and oxidized hyaluronic acid (OHA) aqueous solvent to form an OHA-CMCS (OC) hydrogel matrix;

[0015] S4. Thoroughly mix astaxanthin (ASX) PLGA micro-nanospheres, cationic antimicrobial peptide (AMP) and OHA-CMCS (OC) hydrogel matrix.

[0016] Furthermore, in the step S1, 10 mg of PLGA and 1 ml of astaxanthin are dissolved in a mixed solution of 1 ml of dichloromethane and acetone, and the mixture is thoroughly mixed by ultrasonication for 1 min using a probe cell ultrasonic disruptor as the organic phase. The organic phase is added dropwise to a 1% PVA solution for ultrasonic emulsification. The final solution is evaporated of the organic solvent by a rotary evaporator, and then centrifuged at 14,000 rpm for 40 minutes using a high-speed refrigerated centrifuge to obtain a precipitate of astaxanthin-loaded PLGA micro-nanoparticles, which is then washed three times with deionized water and freeze-dried to obtain astaxanthin PLGA micro-nanospheres.

[0017] Furthermore, in the step S2, the specific method for preparing oxidized hyaluronic acid (OHA) by sodium periodate oxidation method is as follows: 0.05 mol / L sodium periodate is dropped into a 20 mg / ml hyaluronic acid (HA) aqueous solution, the molar ratio of sodium periodate to the hydroxyl group of hyaluronic acid (HA) is 1:1, and after stirring in a water bath at 25°C in the dark for 12 hours, 1 mL of ethylene glycol is added to quench the unreacted sodium periodate, and stirring is continued for 1 hour. The product is then loaded into a dialysis bag (molecular weight 3500), dialyzed with distilled water for 72 hours, and then freeze-dried to obtain cotton-like oxidized hyaluronic acid (OHA).

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. Water-soluble astaxanthin (ASX) PLGA micro-nanospheres solve the problems of easy oxidation and solubility of astaxanthin (ASX). The micro-nanospheres are spherical particles with smooth surface and basically uniform particle size. They have good stability in water and good water solubility.

[0020] 2. Antibacterial supplementation through cationic antimicrobial peptides (AMPs). Cationic antimicrobial peptides (AMPs) are a natural spectrum bactericidal small molecule polypeptide. Its antibacterial mechanism is that the positively charged lysine residues carried by it and the negative charges on the bacterial cell membrane produce electrostatic adsorption and aggregate on the bacterial lipid membrane. The hydrophobic residues can be inserted into the cell membrane, increasing the permeability of the cell membrane, and the cell cannot maintain normal osmotic pressure and die. Since the bactericidal mechanism of cationic antimicrobial peptides (AMPs) is completely different from traditional antibiotics, it is difficult for bacteria to develop drug resistance, and there are almost no toxic side effects.

[0021] 3. The delivery of astaxanthin (ASX) and cationic antimicrobial peptides (AMP) through a hydrogel matrix can not only improve the utilization of astaxanthin (ASX) and cationic antimicrobial peptides (AMP), but also effectively deliver the drug according to the complex morphology of the periodontium. It also has good biocompatibility and degradability. The hydrogel matrix also has the advantages of good drug sustained release, imitation of natural extracellular matrix, mesh scaffold structure, adjustable mechanical properties, adaptive changes according to the environment, and absorbable tissue exudates. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a scanning electron micrograph of a freeze-dried sample of a hydrogel used for the adjuvant treatment of periodontitis;

[0023] Figure 2 is the standard curve of astaxanthin;

[0024] Figure 3 The particle size diagram of astaxanthin PLGA micro-nanospheres;

[0025] Figure 4This is the zeta potential diagram of astaxanthin PLGA micro-nanospheres;

[0026] Figure 5 This is a transmission electron microscopy image of astaxanthin PLGA micro-nanospheres;

[0027] Figure 6 This is the antibacterial experiment diagram of hydrogel;

[0028] Figure 7 This is a diagram of the hydrogel antioxidant test. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments:

[0030] A hydrogel for assisting the treatment of periodontitis, comprising the following components and contents: 6 μM astaxanthin (ASX), 100 μg / ml cationic antimicrobial peptide (AMP) and a hydrogel matrix.

[0031] Astaxanthin is water-soluble astaxanthin PLGA micro-nanospheres. Water-soluble astaxanthin (ASX) PLGA micro-nanospheres solve the problems of easy oxidation and solubility of astaxanthin (ASX), and the micro-nanospheres are spherical particles with smooth surface and basically uniform particle size, good stability in water, and good water solubility.

[0032] like Figure 1 As shown, the microscopic morphology of the freeze-dried sample of the present invention was observed by scanning electron microscopy, and it was found that the hydrogel matrix had an interconnected three-dimensional porous network structure with a pore size of about 20-40 μm, and the presence of astaxanthin (ASX) PLGA micro-nanospheres could be observed.

[0033] like Figure 2 As shown, the encapsulation efficiency (EE) and drug loading (DL) of astaxanthin (ASX) PLGA micro-nanospheres were measured. The encapsulation efficiency was 82.65%, and the drug loading rate was 8.27%.

[0034] like Figure 3 and Figure 4 As shown, astaxanthin (ASX) PLGA micro-nanospheres were diluted with ultrapure water to a solution of 500 μg / ml to measure the particle size distribution and zeta potential. The average particle size was measured to be 694±3.57 nm, the polydispersity index (PDI) was 0.16±0.14, and the zeta potential was -22.06±0.955 mV. The absolute value was greater than 20, indicating that the prepared PLGA micro-nanospheres had uniform particle size distribution and good stability in water.

[0035] like Figure 5 As shown, TEM morphology analysis revealed that astaxanthin (ASX) PLGA micro-nanospheres were spherical particles with smooth surfaces and basically uniform particle sizes.

[0036] Astaxanthin and PLGA micro-nanospheres with the same concentration of astaxanthin were dissolved in deionized water respectively. After 5 minutes, the astaxanthin solution became turbid with many particles attached to the bottle wall. As time went by, astaxanthin began to slowly precipitate, while the micro-nanospheres maintained good water solubility.

[0037] The matrix of the hydrogel matrix is ​​oxidized hyaluronic acid (OHA) and carboxymethyl chitosan (CMCS). The aldehyde group of oxidized hyaluronic acid (OHA) and the amino group of carboxymethyl chitosan (CMCS) will form a dynamically self-assembled Schiff base bond, and the negative charge in oxidized hyaluronic acid (OHA) and the positive charge in carboxymethyl chitosan (CMCS) will also have electrostatic interaction. This makes it have the characteristics of self-healing and pH response.

[0038] Oxidized hyaluronic acid (OHA) and carboxymethyl chitosan (CMCS) in the hydrogel matrix are both 4% aqueous solutions. The hydrogel matrix sample is placed in a 3ml syringe, and the hydrogel matrix can be easily squeezed out through the needle, indicating that the hydrogel matrix has good injectability; the hydrogel matrix is ​​cut in the middle, and after 2.5 minutes, the hydrogel matrix self-heals and the fracture disappears.

[0039] like Figure 6 As shown, after the hydrogels of different components were co-cultured with Staphylococcus aureus and Escherichia coli for 12 hours, 20 μL of the bacterial solutions of different groups were spread on the agar plate and incubated at 37°C overnight. The experiment was repeated three times.

[0040] Group A: Blank group

[0041] Group B: ASX+OHA / CMCS

[0042] Group C: AMP+OHA / CMCS group

[0043] Group D: The antibacterial experiment of ASX+AMP+OHA / CMCS group proved that the hydrogel had good antibacterial properties due to the addition of AMP.

[0044] like Figure 7 As shown in the figure, through the DPPH free radical scavenging experiment, it was found that with the addition of astaxanthin micro-nanospheres, the scavenging effect of the hydrogel on free radicals became obvious, and the free radical scavenging rate increased with the extension of time in the first 60 minutes, which was related to the continuous release of astaxanthin from the micro-nanospheres.

[0045] A preparation step of a hydrogel for assisting the treatment of periodontitis is as follows:

[0046] S1. Prepare micro-nano particles according to the emulsion solvent volatilization technology. Dissolve 10 mg of PLGA and 1 ml of astaxanthin in a mixed solution of 1 ml of dichloromethane and acetone, use a probe cell ultrasonic disruptor to ultrasonically mix it for 1 min to make it fully mixed as the organic phase, add the organic phase dropwise to the 1% PVA solution for ultrasonic emulsification, and volatilize the organic solvent from the final solution by a rotary evaporator. Then, use a high-speed refrigerated centrifuge to centrifuge at 14000 rpm for 40 minutes to obtain a precipitate of PLGA micro-nanoparticles loaded with astaxanthin, and wash it three times with deionized water. After freeze drying, astaxanthin PLGA micro-nanospheres are obtained, which are slightly dark red powders.

[0047] S2. Prepare oxidized hyaluronic acid (OHA) by sodium periodate oxidation method. Drop 0.05 mol / L sodium periodate into 20 mg / ml hyaluronic acid (HA) aqueous solution, and the molar ratio of sodium periodate to hydroxyl group of hyaluronic acid (HA) is 1:1. After stirring in a 25°C water bath in the dark for 12 hours, add 1 mL of ethylene glycol to quench the unreacted sodium periodate, continue stirring for 1 hour, then put the product into a dialysis bag (molecular weight 3500), dialyze with distilled water for 72 hours, and then freeze-dry to obtain cotton-like oxidized hyaluronic acid (OHA);

[0048] S3, fully mixing equal volumes of carboxymethyl chitosan (CMCS) aqueous solution and oxidized hyaluronic acid (OHA) aqueous solvent to form an OHA-CMCS (OC) hydrogel matrix;

[0049] S4. Thoroughly mix astaxanthin (ASX) PLGA micro-nanospheres, cationic antimicrobial peptide (AMP) and OHA-CMCS (OC) hydrogel matrix.

[0050] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A hydrogel for adjuvant treatment of periodontitis, characterized in that: The hydrogel comprises the following components and contents: 6 μM astaxanthin (ASX), 100 μg / ml cationic antimicrobial peptide (AMP) and a hydrogel matrix.

2. The hydrogel for adjuvant treatment of periodontitis according to claim 1, characterized in that: The astaxanthin (ASX) is water-soluble astaxanthin (ASX) PLGA micro-nanosphere.

3. The hydrogel for adjuvant treatment of periodontitis according to claim 1, characterized in that: The hydrogel matrix includes oxidized hyaluronic acid (OHA) and carboxymethyl chitosan (CMCS).

4. The hydrogel for adjuvant treatment of periodontitis according to claim 3, characterized in that: The oxidized hyaluronic acid (OHA) and carboxymethyl chitosan (CMCS) in the hydrogel matrix are both 4% aqueous solutions.

5. A method for preparing a hydrogel for adjuvant treatment of periodontitis, characterized in that: The hydrogel preparation steps are: S1, preparation of astaxanthin (ASX) PLGA micro-nanospheres by ultrasonic emulsion solvent evaporation method; S2, preparing oxidized hyaluronic acid (OHA) by sodium periodate oxidation method; S3, fully mixing equal volumes of carboxymethyl chitosan (CMCS) aqueous solution and oxidized hyaluronic acid (OHA) aqueous solvent to form an OHA-CMCS (OC) hydrogel matrix; S4. Thoroughly mix astaxanthin (AMP) PLGA micro-nanospheres, cationic antimicrobial peptide (AMP) and OHA-CMCS (OC) hydrogel matrix.

6. The method for preparing a hydrogel for assisting the treatment of periodontitis according to claim 5, characterized in that: In the step S1, 10 mg of PLGA and 1 ml of astaxanthin are dissolved in a mixed solution of 1 ml of dichloromethane and acetone, and the mixture is thoroughly mixed by ultrasonication for 1 min using a probe cell ultrasonic disruptor as an organic phase. The organic phase is added dropwise into a 1% PVA solution for ultrasonic emulsification, and the final solution is evaporated of the organic solvent by a rotary evaporator. Then, the solution is centrifuged at 14,000 rpm for 40 minutes using a high-speed refrigerated centrifuge to obtain a precipitate of astaxanthin-loaded PLGA micro-nanoparticles, which is then washed three times with deionized water and freeze-dried to obtain astaxanthin PLGA micro-nanospheres.

7. The method for preparing a hydrogel for assisting the treatment of periodontitis according to claim 5, characterized in that: In the step S2, 0.05 mol / L sodium periodate is dropped into a 20 mg / ml hyaluronic acid (HA) aqueous solution, the molar ratio of sodium periodate to the hydroxyl group of hyaluronic acid (HA) is 1:1, and after stirring in a water bath at 25° C. in the dark for 12 h, 1 mL of ethylene glycol is added to quench the unreacted sodium periodate, and stirring is continued for 1 h. The product is then loaded into a dialysis bag (molecular weight 3500), dialyzed with distilled water for 72 h, and then freeze-dried to obtain cotton-like oxidized hyaluronic acid (OHA).

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