A dentin hypersensitivity desensitizer based on chitosan self-assembly and enzymatic mineralization technology and a preparation method thereof

Abstract

The present application belongs to the technical field of medical material preparation, and particularly relates to a dentin sensitivity desensitizer based on chitosan self-assembly and enzymatic mineralization technology and a preparation method thereof. The amorphous chitosan core-based desensitizer of the present application can not only deeply penetrate into dentin tubules, but also realize rapid mineralization through the loaded phosphatase. Compared with traditional dentin sensitivity desensitizers, the amorphous chitosan desensitizer of the present application has rapid effect and good blocking effect. The results of the examples show that the dentin sensitivity desensitizer provided by the present application can fully and effectively penetrate into the dentin tubules, can form a dense mineralized layer on the surface and inside of the dentin tubules, and the blocking depth exceeds 200 mu m.

Description

Technical Field

[0001] This invention belongs to the field of medical material preparation technology, specifically relating to a dentin-sensitive desensitizing agent based on chitosan self-assembly and enzymatic mineralization technology and its preparation method. Background Technology

[0002] Dentin hypersensitivity is a common oral disease characterized by brief but intense pain when exposed dentin is exposed to non-noxious stimuli such as temperature, mechanical, osmotic, or chemical stimuli. The pain disappears once the stimulus is removed, and it is not accompanied by other tooth defects or diseases. Epidemiological studies indicate that the global prevalence of this condition is approximately 10%-30%, often leading to a decline in patients' quality of life. Therefore, effectively relieving dentin hypersensitivity pain is an urgent need in the field of oral medicine.

[0003] Currently, the desensitization mechanisms for dentin hypersensitivity mainly fall into two categories: one is to temporarily suppress pain signals by blocking the transmission of nerve signals in the dental pulp through substances such as potassium ions; the other is to seal exposed dentinal tubules through physical or chemical means, isolating them from external stimuli. Commonly used products, such as desensitizing toothpaste containing potassium nitrate, can relieve symptoms in the short term, but cannot achieve long-term sealing of dentinal tubules; fluoride products can form a deposition layer on the tooth surface, but their sealing structure is not stable enough in the acidic environment of the oral cavity and is prone to dissolution; laser treatment can seal tubules by melting dentin, but there is a risk of local thermal damage, and the long-term efficacy needs further verification. In addition, the sealing layer formed by commercially available desensitizing toothpastes is relatively shallow (usually only 2–10 μm), and is easily worn away by daily mechanical friction such as brushing and chewing, making it difficult to maintain the desensitization effect for a long time.

[0004] In summary, in order to overcome the shortcomings of existing desensitization treatment products and obtain better treatment results for dentin hypersensitivity, there is an urgent need to develop a dentin hypersensitivity desensitizing agent. Summary of the Invention

[0005] The purpose of this invention is to provide a dentin-sensitive desensitizing agent. The desensitizing agent provided by this invention can form a sealing layer in situ deep within the dentinal tubules through mineralization, and is a fast, safe and long-lasting dentinal tubule sealing material.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a dentin-sensitive desensitizing agent comprising amorphous chitosan bound to phosphatase and calcium phosphate mineralization solution.

[0007] The present invention also provides a method for preparing a dentin-sensitive desensitizing agent, the method comprising preparing amorphous chitosan bound to phosphatase and preparing a calcium phosphate mineralization solution; The method for preparing amorphous chitosan bound to phosphatase includes the following steps: a) Chitosan nanocrystal dispersion was prepared by dissolution, regeneration, and physical crushing; b) Amorphous chitosan is obtained by adjusting the pH value, wherein the pH adjustment range is 3.5-9.5; c) Mix amorphous chitosan with a phosphatase solution to obtain amorphous chitosan bound to phosphatase; The method for preparing calcium phosphate mineralization solution includes: dissolving soluble organic phosphate calcium salt to obtain calcium phosphate mineralization solution.

[0008] Preferably, in step a), the physical breaking includes ultrasonic dispersion, high-pressure homogenization, or high-speed shearing.

[0009] Preferably, in step a), the degree of deacetylation of the chitosan is 50-100%. Preferably, in step a), the concentration of the chitosan nanocrystal dispersion is 1 wt%.

[0010] Preferably, in step b), the acid used for pH adjustment includes phosphoric acid, hydrochloric acid, lactic acid, acetic acid, or citric acid; the base used includes sodium hydroxide, sodium bicarbonate, or sodium carbonate.

[0011] Preferably, in step b), the pH adjustment method is as follows: 1% acetic acid is added dropwise to the chitosan nanocrystal dispersion until its pH value is 3.5-6.5, and ultrasonic dispersion is performed using an ultrasonic disruptor to prepare amorphous chitosan.

[0012] Preferably, in step c), the concentration of the amorphous chitosan is 0.01-5 wt%.

[0013] Preferably, in step c), the phosphatase includes alkaline phosphatase, acid phosphatase, phosphodiesterase, or lipid phosphatase.

[0014] Preferably, in step c), the concentration of the phosphatase is 0.001-0.5 wt%.

[0015] Preferably, in step c), the method of mixing amorphous chitosan with phosphatase solution is as follows: 1% alkaline phosphatase solution is added dropwise to the amorphous chitosan solution until the mass ratio of alkaline phosphatase to chitosan is 1:10, thereby obtaining amorphous chitosan bound to phosphatase.

[0016] Preferably, the soluble organic phosphate calcium salt includes glucose-6-phosphate calcium, glycerophosphate calcium, phytate calcium, or 5-ribonucleotide calcium.

[0017] Preferably, the concentration of the calcium phosphate mineralization solution is 0.005-5 wt%.

[0018] This invention also provides the following applications: The application of the dentin hypersensitivity desensitizer in the preparation of anti-dentin hypersensitivity drugs. Amorphous chitosan bound to phosphatase is used to treat dentin hypersensitivity. It possesses excellent flow properties and self-assembly characteristics, enabling it to deeply penetrate dentinal tubules and, in synergy with calcium-phosphorus mineralization solutions, mineralize and seal dentinal tubules to treat dentin hypersensitivity.

[0019] The dentin-sensitive desensitizing agent is used in the preparation of anti-caries drugs. Amorphous chitosan bound to phosphatase is used to prevent tooth decay and has a significant antibacterial effect. In synergy with calcium-phosphomineralizing solution, it forms an antibacterial mineralization layer on the tooth surface, preventing tooth decay.

[0020] The beneficial effects of this invention are: The desensitizing agent of this invention, with amorphous chitosan as its core, can not only penetrate deeply into dentinal tubules, but also achieve rapid mineralization through loaded phosphatase; combined with the antibacterial effect of chitosan itself, the desensitizing agent provided by this invention can achieve rapid and long-lasting treatment of dentin hypersensitivity and relieve patient pain.

[0021] The desensitizing agent of this invention is prepared by utilizing the acid-base dependent self-assembly characteristics of amorphous chitosan. First, chitosan nanocrystals are prepared by dissolution and regeneration technology and physical crushing. Then, by adjusting the acid-base, the chitosan nanocrystals are dispersed into amorphous chitosan. Chitosan is a positively charged polysaccharide that can stably bind to negatively charged phosphatases.

[0022] The amorphous chitosan in the desensitizing agent of this invention has a small size and strong fluidity. After being coated on the dentin surface, it can effectively penetrate into the interior of the dentinal tubules. The amorphous chitosan that has penetrated into the dentinal tubules reassembles into a three-dimensional network at physiological pH. When calcium and phosphate ions are replenished using calcium phosphate mineralization solution, the surface of the chitosan three-dimensional network rapidly produces biocompatible minerals—hydroxyapatite—due to the catalytic action of phosphatases, which can seal exposed dentinal tubules. The positively charged amino groups of chitosan electrostatically bind to the bacterial surface, destroying the bacterial structure, thereby achieving the purpose of inhibiting bacteria and preventing caries.

[0023] Compared to traditional dentin-sensitive desensitizing agents, the amorphous chitosan desensitizing agent of this invention has a rapid onset of action, excellent sealing effect, and produces a mineralized layer similar to natural dentin, exhibiting good biocompatibility while also possessing antibacterial and anti-caries effects. Furthermore, the preparation method of this desensitizing agent is simple and suitable for production and practical application. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a photograph of 1 wt% amorphous chitosan desensitizing agent in Example 1 of this invention; Figure 2 This is a transmission electron microscope image of the amorphous chitosan desensitizing agent in Example 1 of the present invention; Figure 3 These are confocal micrographs of the amorphous chitosan desensitizing agent penetrating dentinal tubules in Example 2 of this invention; Figure 4 These are scanning electron microscope images of the in vitro model of dentin hypersensitivity treated with the amorphous chitosan desensitizing agent from Example 1 of the present invention in Embodiment 3 of the present invention; Figure 5 This is a scanning electron microscope image showing the results of treating an SD rat dentin hypersensitivity model with the amorphous chitosan desensitizing agent from Example 1 of this invention in Example 4 of this invention. Detailed Implementation

[0026] This invention provides a two-component desensitizing agent for dentin hypersensitivity, the desensitizing agent comprising a first component: amorphous chitosan that binds to phosphatase and a second component: calcium phosphate mineralization solution.

[0027] Chitosan, a natural linear polysaccharide derived from the partial deacetylation of chitin, is widely found in shrimp and crab shells, insect exoskeletons, and fungal cell walls. It possesses advantages such as good biocompatibility, biosafety, biodegradability, low immunogenicity, and antibacterial properties. Its molecular chain is rich in amino and hydroxyl groups, which can adsorb mineral ions such as calcium and phosphorus through electrostatic interactions and guide their orderly arrangement, thereby inducing biomimetic mineralization on the surface of dental hard tissues and promoting the deposition of an enamel-like hydroxyapatite layer. Furthermore, chitosan exhibits good biocompatibility, biodegradability, low immunogenicity, and certain antibacterial properties.

[0028] The method for preparing the dentin hypersensitivity desensitizer of the present invention includes preparing amorphous chitosan bound to phosphatase and calcium phosphate mineralization solution; The steps for preparing amorphous chitosan bound to phosphatase are as follows: a) Chitosan nanocrystal dispersion was prepared by dissolution, regeneration, and physical crushing; b) Obtain amorphous chitosan by adjusting the pH; c) Mix amorphous chitosan with phosphatase solution to prepare amorphous chitosan bound to phosphatase; d) Prepare calcium phosphate mineralization solution using soluble organic phosphate calcium salt.

[0029] In step a), the degree of deacetylation of the chitosan is 50-100%, preferably 70-100%.

[0030] In step a), the physical fragmentation methods include, but are not limited to, ultrasonic dispersion, high-pressure homogenization, and high-speed shearing.

[0031] In step b), the pH value is adjusted within the range of 3.5-9.5, preferably 5.5-7.5 or 3.5-6.5.

[0032] In step b), the acids used include, but are not limited to, phosphoric acid, hydrochloric acid, lactic acid, acetic acid, and citric acid. The bases used include, but are not limited to, sodium hydroxide, sodium bicarbonate, and sodium carbonate.

[0033] In step c), the concentration of the amorphous chitosan is 0.01-5 wt%, preferably 0.5-2 wt%.

[0034] In step c), the phosphatases used include, but are not limited to, alkaline phosphatase, acid phosphatase, phosphodiesterase, and lipid phosphatase.

[0035] In step c), the concentration of the phosphatase is 0.001-0.5 wt%, preferably 0.01-0.5 wt%.

[0036] In step d), the soluble organic phosphate calcium salts used include, but are not limited to, calcium glucose-6-phosphate, calcium glycerophosphate, calcium phytate, and calcium 5-ribonucleotide.

[0037] In step d), the concentration of the mineralizing solution is 0.005-5 wt%, preferably 0.1-2 wt%.

[0038] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0039] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0040] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0041] Example 1

[0042] The specific method for preparing amorphous chitosan is as follows: 1. Dissolve chitosan powder in a 2 wt% acetic acid solution and stir until completely dissolved. While stirring at high speed, add 5 wt% sodium hydroxide solution dropwise until the pH reaches 7-8, resulting in a white precipitate. Collect the precipitate by centrifugation and wash with deionized water until neutral. Redisperse the precipitate in water and homogenize it three times using a high-pressure homogenizer at 800 bar to obtain a 1 wt% chitosan nanofiber dispersion. 2. Slowly add 1% acetic acid dropwise to the chitosan nanocrystal dispersion until the pH value reaches 6.2, and then use an ultrasonic disruptor to ultrasonically disperse the mixture to prepare an amorphous chitosan solution (e.g., Figure 1 (as shown) 3. Slowly add 1% alkaline phosphatase solution to the amorphous chitosan solution until the mass ratio of alkaline phosphatase to chitosan solute is 1:10, thus completing the preparation of the amorphous chitosan desensitizing agent.

[0043] The amorphous chitosan desensitizing agent was observed using a transmission electron microscope (e.g., Figure 2 (As shown).

[0044] Example 2

[0045] The specific method for using amorphous chitosan to penetrate dentinal tubules is as follows: 1. A 1 wt% chitosan nanocrystal dispersion was prepared by acid-base regeneration and physical crushing; 2. A 1 wt% chitosan nanocrystal dispersion was stained with fluorescein isothiocyanate; 3. An amorphous chitosan desensitizing agent was prepared using a 1 wt% chitosan nanocrystal dispersion after dyeing; 4. Using extracted human teeth, dentin sections were prepared under water-cooled conditions using a diamond abrasive cutter. The dentin sections were then etched with 37% phosphoric acid to open the dentinal tubules. 5. Add 100 μL of amorphous chitosan desensitizing agent to the surface of the dentin section and rub it with a disposable dental swab for 10 minutes.

[0046] Dentin sections can be observed using a confocal microscope, and fluorescence images can be obtained. Figure 3 The penetration of the amorphous chitosan desensitizing agent into the dentinal tubules was observed. The results showed that the desensitizing agent could fully and effectively penetrate into the dentinal tubules.

[0047] Example 3

[0048] The specific method for treating an in vitro model of dentin hypersensitivity using amorphous chitosan desensitizing agents is as follows: 1. An amorphous chitosan desensitizing agent was prepared according to the method of Example 1; 2. Prepare a 0.5% glycerol calcium phosphate solution using deionized water; 3. Using extracted human teeth, dentin sections were prepared under water-cooled conditions using a diamond abrasive cutter. The dentin sections were then etched with 37% phosphoric acid to open the dentinal tubules. 4. Add 100 μL of amorphous chitosan desensitizing agent to the surface of the dentin section and rub it with a disposable dental swab for 10 minutes; 5. Add 500 μL of 0.5% calcium glycerophosphate solution to the surface of the dentin section and let it stand for 12 hours; Scanning microscopy was used to observe the sealing of dentinal tubules after treatment with amorphous chitosan desensitizing agent. A dense mineralized layer was observed on the surface and inside of the dentinal tubules, with a sealing depth exceeding 200 μm. Figure 4 ).

[0049] Example 4

[0050] The following is a method for treating a rat model of dentin hypersensitivity using amorphous chitosan desensitizing agent: Amorphous chitosan desensitizing agents were evaluated for their sealing effect in a rat model of dentin hypersensitivity. Male SD rats, weighing 100-150 g, were used as experimental animals, sourced from the Experimental Animal Center of East China Normal University and housed under standard conditions. All animal experiments were conducted in accordance with the regulations of the Animal Experimentation Ethics Committee. Rats were randomly assigned to groups, and after complete anesthesia, 3-layer desensitizing agents were prepared on the surface of the rat incisors using a handheld dental instrument under water-cooling conditions. 2 A 1mm defect was etched with 37% phosphoric acid for 30 seconds, followed by thorough rinsing with deionized water. Then, the rats were treated sequentially with the amorphous chitosan desensitizing agent from Example 1 of this invention for 10 minutes, and then with 0.5% glycerol calcium phosphate solution for 30 minutes. After recovery from anesthesia, the rats were fed normally, and euthanized 24 hours later, their incisors were removed. Scanning microscopy was used to observe the rat incisors, revealing the closure of the dentinal tubules in SD rats after treatment with the amorphous chitosan desensitizing agent. A mineralized layer was observed on the surface and inside the dentinal tubules of the SD rats. Figure 5 ).

[0051] As can be seen from the above embodiments, the dentin-sensitive desensitizing agent provided by the present invention can fully and effectively penetrate into the dentinal tubules, forming a dense mineralized layer on the surface and inside of the dentinal tubules, with a sealing depth exceeding 200 μm. Simultaneously, animal experimental results show that after treatment with the amorphous chitosan desensitizing agent of the present invention, a mineralized layer is formed on the surface and inside of the dentinal tubules in experimental animals, achieving a sealing effect.

[0052] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A dentin hypersensitivity desensitizing agent, characterized in that, The desensitizing agent comprises amorphous chitosan that binds to phosphatase and calcium phosphate mineralization solution.

2. A method for preparing a dentin-sensitive desensitizing agent, characterized in that, The method includes preparing amorphous chitosan bound to phosphatase and preparing calcium phosphate mineralization solution; The method for preparing amorphous chitosan bound to phosphatase includes the following steps: a) Chitosan nanocrystal dispersion was prepared by dissolution, regeneration, and physical crushing; b) Amorphous chitosan is obtained by adjusting the pH value, wherein the pH adjustment range is 3.5-9.5; c) Mix amorphous chitosan with a phosphatase solution to obtain amorphous chitosan bound to phosphatase; The method for preparing calcium phosphate mineralization solution includes: dissolving soluble organic phosphate calcium salt to obtain calcium phosphate mineralization solution.

3. The method according to claim 2, characterized in that, In step a), the physical breaking includes ultrasonic dispersion, high-pressure homogenization, or high-speed shearing.

4. The method according to claim 2, characterized in that, In step b), the acids used for pH adjustment include phosphoric acid, hydrochloric acid, lactic acid, acetic acid, or citric acid; the bases used include sodium hydroxide, sodium bicarbonate, or sodium carbonate.

5. The method according to claim 2, characterized in that, In step b), the pH adjustment method is as follows: 1% acetic acid is added dropwise to the chitosan nanocrystal dispersion until its pH value is 3.5-6.5, and ultrasonic dispersion is performed using an ultrasonic disruptor to prepare amorphous chitosan.

6. The method according to claim 2, characterized in that, In step c), the phosphatase includes alkaline phosphatase, acid phosphatase, phosphodiesterase, or lipid phosphatase.

7. The method according to claim 2, characterized in that, In step c), the method of mixing amorphous chitosan with phosphatase solution is as follows: 1% alkaline phosphatase solution is added dropwise to amorphous chitosan solution until the mass ratio of alkaline phosphatase to chitosan is 1:10, thereby obtaining amorphous chitosan bound to phosphatase.

8. The method according to claim 2, characterized in that, The soluble organic phosphate calcium salts include calcium glucose-6-phosphate, calcium glycerophosphate, calcium phytate, or calcium 5-ribonucleotide.

9. The method according to claim 2, characterized in that, The concentration of the calcium phosphate mineralization solution is 0.005-5 wt%.

10. The use of the desensitizing agent according to claim 1, or the desensitizing agent prepared by the method according to any one of claims 2-9, in the preparation of anti-dentin hypersensitivity drugs or in the preparation of anti-caries drugs.

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