Temperature-sensitive hydrogel drug delivery system for periodontitis treatment and preparation method thereof

Abstract

The invention discloses a temperature-sensitive hydrogel drug delivery system for periodontitis treatment and a preparation method of the temperature-sensitive hydrogel drug delivery system. The hydrogel is prepared from a temperature-sensitive and biocompatible material, the material comprises a natural biomacromolecule modifying a polyphenol component, a triblock polymer and cationic natural polysaccharide, the temperature-sensitive hydrogel is formed through a cross-linking reaction, and drugs can be carried in a grid structure of the hydrogel for treating periodontitis. The hydrogel can be rapidly converted into a gel state at the body temperature, can stay in a periodontal pocket for a long time and slowly release drugs, remarkably improves the treatment effect, reduces side effects and overcomes the limitation of a traditional drug delivery system in periodontitis treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and particularly to a temperature-sensitive hydrogel for the treatment of periodontitis and a preparation method thereof. Background Art

[0002] Periodontitis is an infectious disease initiated by dental plaque, mainly manifested as gingival inflammation, alveolar bone resorption, and destruction of periodontal tissues. As the disease progresses, periodontitis can lead to tooth loosening or even tooth loss, and cause loss of alveolar bone, seriously affecting the oral function and quality of life of patients. More importantly, periodontitis is not limited to local tooth damage, but also exacerbates the occurrence and progression of various systemic diseases, such as cardiovascular diseases, diabetes, etc. Periodontitis has become a widely prevalent disease globally. Existing studies show that approximately 743 million people worldwide are affected by periodontitis.

[0003] Currently, the main clinical treatment methods for periodontitis include mechanical treatments such as supra-gingival scaling, sub-gingival scaling, and root planing, aiming to remove dental plaque in the gingiva and periodontal pockets and control inflammation. However, due to the influence of anatomical features such as periodontal pocket depth and root bifurcation, simple mechanical treatment often cannot completely remove all pathogenic bacteria, and the treatment effect is limited. Therefore, drug treatment, as an important auxiliary means for the treatment of periodontitis, has become a research hotspot.

[0004] Traditional drug treatment usually adopts the method of systemic administration, but systemic drug use may lead to adverse reactions and drug resistance problems, especially in the case of long-term use of antibiotics. To avoid these problems, local drug delivery has gradually become an important direction in the treatment of periodontitis. Local drugs can directly act on the lesion site, ensuring that a high drug concentration continuously acts in the periodontal tissue, thereby improving the curative effect and reducing systemic side effects. However, existing local drug delivery systems are mostly in the form of ointments, gels, etc. These drug carriers usually cannot effectively penetrate the periodontal pocket, and the local retention time is short, resulting in limited curative effect. Therefore, developing a new drug carrier system that can maintain drug release in the periodontal tissue for a long time has become a key problem to be solved urgently.

[0005] As a novel drug delivery system, hydrogel has shown great potential in the field of drug delivery due to its unique physicochemical properties. Hydrogel is a three-dimensional polymer network system formed by monomers through physical or chemical cross-linking reactions, which can effectively load various drugs and has a simple and easy preparation process. In the treatment of periodontitis, the hydrogel drug delivery system needs to have certain viscosity, appropriate degradation period, thermosensitivity and other characteristics. However, under the pathological conditions of periodontitis, the increase of matrix metalloproteinase-8 (MMP-8) will accelerate the degradation of hydrogel and shorten its degradation period. Since drug release is closely related to the degradation process of hydrogel, a too short degradation period cannot meet the requirement of long-term drug action in the treatment of periodontitis, and too fast degradation rate may lead to explosive release of drugs, resulting in too high local drug concentration and thus causing toxic reactions. Therefore, it is necessary to design an appropriate degradation period to achieve stable and slow drug release. To solve the problem of too short degradation period, the degradation rate of hydrogel can be adjusted by optimizing the cross-linking method and adjusting the matrix components. Compared with physical cross-linking, the hydrogel network formed by chemical cross-linking is more compact and not easily decomposed, so the degradation period can be prolonged. At the same time, using a hydrogel matrix with sustained release effect and endowing it with anti-inflammatory, antioxidant, antibacterial and other properties can act synergistically with the therapeutic drug to effectively treat periodontitis. In addition, by endowing the hydrogel with thermosensitivity, that is, the hydrogel drug delivery system undergoes a liquid-gel state transition at a temperature higher than a certain temperature, it can be injected into the periodontal pocket in the form of a solution and then transformed into a gel state to fit the irregular notch, further optimizing its application performance. Therefore, in the treatment of periodontitis, the thermosensitive hydrogel prepared by selecting appropriate hydrogel materials can maintain the drug concentration locally, regulate the drug release rate, reduce systemic side effects and improve the treatment effect. Summary of the Invention

[0006] In view of the above deficiencies of the prior art, the purpose of the present invention is to provide a temperature-sensitive hydrogel drug delivery system for the treatment of periodontitis and its preparation method, aiming to provide a new local drug delivery method for the treatment of periodontitis. The present invention uses a hydrogel material with good biocompatibility, thermosensitivity and sustained release ability, and through reasonable proportioning and mixing, a thermosensitive hydrogel is prepared for loading drugs for the treatment of periodontitis.

[0007] The technical solution of the present invention is as follows.

[0008] A temperature-sensitive hydrogel drug delivery system for the treatment of periodontitis, the drug delivery system includes a temperature-sensitive hydrogel and a therapeutic drug, and the temperature-sensitive hydrogel is composed of a natural biopolymer modified by a polyphenol component, a triblock polymer and a cationic natural polysaccharide, and can be transformed from a liquid state to a gel state at body temperature and can load drugs for sustained release.

[0009] Preferably, the body temperature range is 20-40 °C.

[0010] Preferably, in the temperature-sensitive hydrogel drug delivery system, the natural biopolymers modified by polyphenol components are beneficial to enhancing the crosslinking property of the hydrogel and providing reactive sites, including hyaluronic acid modified with dopamine (HA-DA); the triblock polymer is beneficial to controlling the temperature to achieve precise drug release and can crosslink with HA-DA, including Pluronic capped with thiol (Plu-SH); the cationic natural polysaccharide is chitosan with good biocompatibility and antibacterial ability.

[0011] Preferably, the therapeutic drug is selected from antibiotics, miRNA or anti-inflammatory drugs.

[0012] Preferably, in the temperature-sensitive hydrogel drug delivery system, the mesh structure of the temperature-sensitive hydrogel can load therapeutic drugs.

[0013] The preparation method of the above temperature-sensitive hydrogel drug delivery system includes the following steps:

[0014] S1. First, prepare hyaluronic acid modified with dopamine (HA-DA) and Pluronic capped with thiol (Plu-SH);

[0015] S2. Dissolve hyaluronic acid modified with dopamine (HA-DA), Pluronic capped with thiol (Plu-SH) and chitosan in a certain amount of PBS solution respectively to form corresponding solutions, and mix and stir them in an ice bath according to a certain ratio to form a temperature-sensitive hydrogel;

[0016] S3. Mix and stir the hyaluronic acid modified with dopamine (HA-DA) solution, Pluronic capped with thiol (Plu-SH) solution, chitosan solution and therapeutic drug in an ice bath according to a certain ratio to form a drug-loaded hydrogel solution.

[0017] In the above method, in S1, in the preparation method of the temperature-sensitive hydrogel drug delivery system, hyaluronic acid modified with dopamine (HA-DA) is prepared by dissolving hyaluronic acid in PBS solution and stirring to completely dissolve HA; then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) are added successively, and stirred in a water bath. After the reaction is completed, dopamine is added and then N 2 is used to block the reaction, and then the HA-DA solid is obtained after dialysis and freeze-drying. The addition amounts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and dopamine satisfy: the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, dopamine is 2-3:1-2:3-6.

[0018] In the above method, in S2, in the preparation method of the temperature-sensitive hydrogel drug delivery system, Pluronic capped with mercapto (Plu-SH) is prepared by dissolving Pluronic in dichloromethane, adding triethylamine. The obtained solution is slowly added dropwise to p-NPC. After stirring, the activated Pluronic is extracted with saturated sodium chloride aqueous solution. Dichloromethane is dried with sodium sulfate and then precipitated with cold ether. The activated Pluronic is dissolved in dichloromethane, cysteamine is added, and a reaction occurs. The product is extracted with saturated sodium chloride aqueous solution, dichloromethane is dried with sodium sulfate, and then cold ether is added for precipitation to obtain Plu-SH. The addition amounts of Pluronic, triethylamine, and p-NPC satisfy the following: the ratio among Pluronic, triethylamine, and p-NPC is 10 - 20 g: 200 - 300 μL: 0.5 - 1 g.

[0019] In the preparation method of the temperature-sensitive hydrogel drug delivery system, the volume ratio of the HA-DA solution, Plu-SH solution, chitosan solution, and therapeutic drug solution is 4:4:1:1 - 5:5:1:1, preferably 4:4:1:1; the concentration of HA-DA in the PBS solution is 100 - 200 mg / mL, w / v, preferably 125 mg / mL, w / v; the concentration of Plu-SH in the PBS solution is 200 - 300 mg / mL, w / v, preferably 282.5 mg / mL, w / v; the concentration of chitosan in the temperature-sensitive hydrogel system is (0.1% - 10%, w / w), and the concentration of the therapeutic drug in the PBS solution is (10 - 200 μg / mL, w / v).

[0020] In the preparation method of the temperature-sensitive hydrogel drug delivery system, the therapeutic drug can be an antibiotic, miRNA, anti-inflammatory drug, or other drug combinations.

[0021] In the preparation method of the temperature-sensitive hydrogel drug delivery system, the temperature-sensitive hydrogel exhibits a liquid-gel state transition within the body temperature range (about 20 - 40 °C).

[0022] In the temperature-sensitive hydrogel drug delivery system, the hydrogel is in an injectable form and can be directly injected into the periodontal pocket for local drug delivery. It can maintain a local drug concentration in the periodontal pocket for a long time, achieve slow release, effectively reduce systemic side effects, and improve the local drug efficacy, making it suitable for local treatment of periodontitis. The hydrogel material has excellent biocompatibility, degradability, and antibacterial ability and will not cause adverse reactions in the periodontal tissue.

[0023] Compared with the prior art, the advantages of the present invention are as follows:

[0024] (1) The temperature-sensitive hydrogel drug delivery system of the present invention can change from a liquid state to a gel state after being injected into the periodontal pocket, closely adhere to the irregular periodontal pocket structure, and simultaneously release the drug in the hydrogel network structure loaded with the drug, thereby maintaining a stable drug concentration locally and regulating the drug release rate, providing a favorable environment for the regeneration of periodontal tissue, and thus significantly improving the treatment effect.

[0025] (2) The thermosensitive hydrogel drug delivery system provided by the present invention not only effectively solves the limitations of traditional treatment methods, but also better meets the needs of clinical treatment of periodontitis, providing a new drug delivery method for local drug treatment of periodontitis.

[0026] (3) In the temperature-sensitive hydrogel drug delivery system of the present invention, HA-DA is cross-linked with Plu-SH through dynamic covalent bonds to form a reversible thiol-catechol bond network, endowing the hydrogel with self-healing properties and pH responsiveness, and can adapt to the changes in the inflammatory microenvironment in the periodontal pocket to achieve intelligent drug release. The cationic property of chitosan interacts with the negative charge of the bacterial cell membrane, and synergistically with the antioxidant effect of the polyphenol component, forming a dual antibacterial mechanism, effectively inhibiting the formation of biofilms of periodontal pathogenic bacteria. The catechol group in HA-DA specifically binds to the collagen of periodontal tissue, enhancing the adhesion and retention of the hydrogel in the moist oral environment and extending the local action time to more than 48 hours. Description of the Drawings

[0027] Figure 1 is the NMR spectrum of HA-DA;

[0028] Figure 2 is the NMR spectrum of Plu-SH;

[0029] Figure 3 are the morphological diagrams of the drug-free hydrogel at 4°C and 37°C respectively;

[0030] Figure 4 is the rheological property of the drug-free hydrogel;

[0031] Figure 5 is the safety of the drug-free hydrogel. Detailed Embodiments

[0032] The present invention provides a temperature-sensitive hydrogel drug delivery system and its preparation method. To make the purpose, technical solution and effect of the present invention clearer and more definite, the present invention will be further described in detail below. It should be noted here that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation to the present invention.

[0033] In the experimental methods of the following examples, unless otherwise specified, they are all conventional methods. Unless otherwise specified, the test materials used in the following examples can all be obtained through conventional commercial channels.

[0034] A preparation method of a temperature-sensitive hydrogel drug delivery system includes the following steps:

[0035] The temperature-sensitive hydrogel drug delivery system includes a temperature-sensitive hydrogel and a therapeutic drug. The temperature-sensitive hydrogel is composed of a natural biopolymer modified by a polyphenol component, a triblock polymer, and a cationic natural polysaccharide, and can be transformed from a liquid state to a gel state at body temperature and can load drugs for sustained release. The natural biopolymer modified by the polyphenol component is beneficial to enhancing the crosslinking of the hydrogel and providing reactive sites, including hyaluronic acid modified by dopamine (HA-DA); the triblock polymer is beneficial to controlling the temperature to achieve precise drug release and can crosslink with HA-DA, including Pluronic with a thiol end group (Plu-SH); the cationic natural polysaccharide is chitosan with good biocompatibility and antibacterial ability.

[0036] Example 1: Preparation of hyaluronic acid modified by dopamine (HA-DA):

[0037] Dissolve 500 mg of hyaluronic acid in 20 mL of PBS with pH = 5, and stir to completely dissolve HA; then add 242.6 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 145.7 mg of N-hydroxysuccinimide (NHS), stir and activate in a water bath at 37 °C overnight, then add 250 mg of dopamine and 10 mL of PBS with pH = 5, 2 Seal the reaction and react for 12 h; put the reacted solution into an activated dialysis bag and dialyze for 72 h with stirring; place the mixed solution in the dialysis bag at -80 °C for 24 h; after freeze-drying, obtain the HA-DA solid; verify the prepared HA-DA by nuclear magnetic resonance spectrum, and the results are as Figure 1 shown. Its characteristic peaks in the nuclear magnetic resonance spectrum are consistent with the structure, indicating that the synthesis of HA-DA is successful.

[0038] The reaction formula of this example is as follows:

[0039]

[0040] Example 2: Preparation of Pluronic with a thiol end group (Plu-SH):

[0041] Dissolve 10 g of Pluronic ( CAS#: 9003-11-6) Dissolve it in 70 mL of dichloromethane, and add 221.2 μL of triethylamine. Slowly drop the resulting solution into p-nitrophenyl chloroformate (p-NPC). After stirring for 48 h, extract the activated Pluronic with saturated sodium chloride aqueous solution. Dry the dichloromethane with sodium sulfate for 30 min. Then, precipitate with cold diethyl ether. Dissolve 5 g of the activated Pluronic in 50 mL of dichloromethane, add 423.4 mg of cysteamine, and react for 24 h. Extract the product with saturated sodium chloride aqueous solution, and dry the dichloromethane with sodium sulfate for 30 min. Add cold diethyl ether to precipitate Plu-SH. Verify the prepared Plu-SH by NMR spectrum. The results are as Figure 2 shown. The characteristic peaks of its NMR spectrum are consistent with the structure, indicating the successful synthesis of Plu-SH.

[0042] The reaction formula of this example is as follows:

[0043]

[0044] Example 3: Preparation of temperature-sensitive hydrogel:

[0045] Mix and stir the HA-DA solution, Plu-SH solution and chitosan solution in an ice bath according to a volume ratio of (4:4:1). The concentration of HA-DA in PBS solution is (125 mg / mL, w / v), the concentration of Plu-SH in PBS solution is (282.5 mg / mL, w / v), and the concentration of chitosan in the temperature-sensitive hydrogel system is (2%, w / w) to form a drug-loaded hydrogel solution.

[0046] Place the prepared hydrogel solution without drug at 4 °C and 37 °C successively, observe and record the morphology of the hydrogel. As Figure 3 shown, the hydrogel is in a liquid state at 4 °C and transforms into a gel state at 37 °C, indicating that the prepared hydrogel has thermosensitivity and can achieve the liquid-gel state transition at body temperature.

[0047] Measure the rheological properties (elastic modulus G' and viscous modulus G”) of the hydrogel using a rotational rheometer. To verify the thermosensitive properties of the hydrogel, the changes of G' and G” from 0 to 50 °C were measured at a shear strain of 1%. As Figure 4 shown, as the temperature increases, the temperature corresponding to the intersection of the two parameters is 20 °C, indicating that the gelation temperature of the hydrogel is 20 °C.

[0048] Seed HUVEC cells in a 96-well plate, set the gradient concentrations (0, 20, 50, 80%) of the temperature-sensitive hydrogel respectively, and then add hydrogels with different concentrations to each well. Use a CCK-8 kit to detect cell viability and evaluate the effect of the hydrogel on cells. As Figure 5As shown, there was no obvious change in cell viability, indicating that the prepared thermosensitive hydrogel has good biosafety.

[0049] Example 4: Preparation of a drug-loaded temperature-sensitive hydrogel:

[0050] Mix and stir the HA-DA solution, Plu-SH solution, chitosan solution, and the therapeutic drug doxycycline (DOX) in a volume ratio of (4:4:1:1) under ice bath. The concentration of HA-DA in the PBS solution is (125 mg / mL, w / v), the concentration of Plu-SH in the PBS solution is (282.5 mg / mL, w / v), the concentration of chitosan in the temperature-sensitive hydrogel system is (2%, w / w), and the concentration of DOX in the PBS solution is (20 μg / mL, w / v) to form a drug-loaded hydrogel solution.

[0051] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A temperature-sensitive hydrogel drug delivery system for the treatment of periodontitis, characterized in that: It includes temperature-sensitive hydrogel and therapeutic drugs; the temperature-sensitive hydrogel is composed of natural biomacromolecules modified by polyphenol components, triblock polymers and cationic natural polysaccharides, can be transformed from liquid to gel within the body temperature range, and can load drugs for sustained release.

2. A temperature-sensitive hydrogel drug delivery system for the treatment of periodontitis according to claim 1, characterized in that: The therapeutic drug is selected from antibiotics, miRNA or anti-inflammatory drugs.

3. The temperature-sensitive hydrogel drug delivery system for the treatment of periodontitis according to claim 1, characterized in that: The natural biomacromolecule modified by the polyphenol component includes dopamine-modified hyaluronic acid.

4. The temperature-sensitive hydrogel drug delivery system for the treatment of periodontitis according to claim 1, characterized in that: The triblock polymer includes mercapto-terminated pluronic.

5. The temperature-sensitive hydrogel drug delivery system for the treatment of periodontitis according to claim 1, characterized in that: The cationic natural polysaccharide is chitosan.

6. The temperature-sensitive hydrogel drug delivery system for treating periodontitis according to claim 1, characterized in that: The body temperature range is 20-40°C.

7. The method for preparing the temperature-sensitive hydrogel drug delivery system for treating periodontitis according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1. preparing dopamine-modified hyaluronic acid and thiol-terminated pluronic; S2, dissolving dopamine-modified hyaluronic acid, thiol-terminated pluronic and chitosan in PBS solution to form corresponding solutions, and mixing and stirring under ice bath to form temperature-sensitive hydrogel; S3. Mix and stir the dopamine-modified hyaluronic acid solution, the thiol-terminated Pluronic solution, the chitosan solution and the therapeutic drug in an ice bath to form a drug-loaded hydrogel solution.

8. The method for preparing the temperature-sensitive hydrogel drug delivery system for treating periodontitis according to claim 7, characterized in that: In S1, the dopamine-modified hyaluronic acid is prepared by dissolving hyaluronic acid in a PBS solution and stirring to completely dissolve HA; then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide are added in sequence, and stirred in a water bath at 20 to 40°C. After the reaction is completed, dopamine is added to conduct a N2-blocking reaction, and then HA-DA solid is obtained after dialysis and freeze-drying; the added amounts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and dopamine satisfy the following conditions: the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and dopamine is 2 to 3:1 to 2:3 to 6.

9. The method for preparing the temperature-sensitive hydrogel drug delivery system for treating periodontitis according to claim 7, characterized in that: In S2, the thiol-terminated Pluronic is prepared by dissolving Pluronic in dichloromethane, adding triethylamine, and dropping the resulting solution into p-nitrobenzene chloroformate (p-NPC); after stirring, extracting the activated Pluronic with a saturated sodium chloride aqueous solution; drying the dichloromethane with sodium sulfate, and then precipitating with cold ether; dissolving the activated Pluronic in dichloromethane, adding cysteamine, and reacting; extracting the product with a saturated sodium chloride aqueous solution, drying the dichloromethane with sodium sulfate, and then adding cold ether to precipitate to obtain Plu-SH; the added amounts of the Pluronic, triethylamine, and p-NPC satisfy the following ratios: 10-20 g: 200-300 μL: 0.5-1 g.

10. The method for preparing the temperature-sensitive hydrogel drug delivery system for treating periodontitis according to claim 7, characterized in that: The volume ratio of the HA-DA solution, Plu-SH solution, chitosan solution and therapeutic drug solution is 4:4:1:1-5:5:1:1, the concentration of HA-DA in the PBS solution is 100-200 mg / mL; the concentration of Plu-SH in the PBS solution is 200-300 mg / mL; the mass percentage concentration of chitosan in the temperature-sensitive hydrogel system is 0.1%-10%; and the mass volume ratio concentration of the therapeutic drug in the PBS solution is 10-200 μg / mL.

Images