Use of hyaluronic acid-based hydrogels for the preparation of fluoride ion release agents

Abstract

The application relates to application of a hyaluronic acid-based hydrogel in preparation of a fluorine ion slow-release agent, the fluorine ion slow-release agent being prepared by mixing a precursor solution of the hyaluronic acid-based hydrogel with a fluorine source on site, the precursor solution of the hyaluronic acid-based hydrogel comprising an A component and a B component, a preparation method of the A component comprising the following steps: S1. mixing and reacting methyl methacrylate group modified hyaluronic acid and furfurylamine to obtain furan-methyl methacrylate modified hyaluronic acid; S2. mixing and reacting the furan-methyl methacrylate modified hyaluronic acid and maleimide to obtain double-modified group hyaluronic acid; S3. mixing the double-modified group hyaluronic acid, a photoinitiator and a solvent to obtain the A component; and the B component is a mercapto polyethylene glycol solution. The fluorine ion slow-release agent not only has a fast gelation speed, but also can be self-healed to form a gel in different ways, and can release fluorine ions stably and long-acting after gelation, and can be used for preventing and treating enamel demineralization or enamel defects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of hydrogel, more particularly, to application of hyaluronic acid-based hydrogel in preparation of fluoride ion sustained-release agent. BACKGROUND

[0002] Fluoride ion has important protective effect on teeth, which can inhibit the demineralization of teeth, promote the repair of teeth with demineralization, and prevent the generation of white spot around brackets after orthodontic treatment.

[0003] There are various fluoride ion adsorption and release materials commonly used for preventing and treating related diseases of teeth. Traditional fluoride ion adsorption and release materials are active aluminum hydroxide and other metal hydroxides and salt fluorides, which are sensitive to pH value, easily degraded and precipitated, and have low biological safety.

[0004] Hydrogel material is a kind of three-dimensional grid structure polymer capable of rapidly absorbing, retaining and stabilizing water. In recent years, there have been related researches on the application of hydrogel material in fluoride ion release agent, such as Chinese patent entitled "Polyphenol / PEG-based hydrogel system for dental lacquer".

[0005] In the prevention and treatment of enamel demineralization and enamel defect around orthodontic brackets, the fluoride ion release agent is required to have long-acting and stable fluoride ion release effect because the application scene is a long period. However, many existing hydrogel fluoride ion release agents have fluoride ion burst release phenomenon, and the release time is relatively short. The hydrogel and fluoride ion cannot stably and long-term act on the target area, so they cannot be applied to the above-mentioned scene or need to be frequently changed when applied.

[0006] In addition, in order to better play the effect of hydrogel fluoride ion release agent, the corresponding part is usually first medicated (hydrogel precursor liquid), and then gelled, but this use mode has high requirements for hydrogel: on the one hand, due to the use in complex oral environment, saliva at the medicated site is easy to contaminate the hydrogel precursor liquid, so the hydrogel precursor liquid needs to be quickly gelled; on the other hand, for irregular enamel defects, the effective gelling of the hydrogel precursor liquid needs to be ensured to realize the effective filling or contact of the hydrogel to the enamel defect position. At present, the hydrogel fluoride ion release agent is difficult to meet these requirements at the same time.

[0007] Therefore, it is of great significance to develop a hydrogel fluoride ion release agent which has fast gelling speed, can effectively gel, and can long-acting and stably release fluoride ion. SUMMARY

[0008] The primary object of the present application is to overcome the problem of poor long-term stable release effect of the hydrogel fluoride ion release agent in the prior art, which cannot simultaneously meet the requirements of rapid gelation and effective gelation, and to provide an application of a hyaluronic acid-based hydrogel in preparation of a fluoride ion sustained-release agent.

[0009] A further object of the present application is to provide a fluoride ion sustained-release agent.

[0010] The above object of the present application is achieved by the following technical solutions.

[0011] The present application provides an application of a hyaluronic acid-based hydrogel in preparation of a fluoride ion sustained-release agent, wherein the fluoride ion sustained-release agent is prepared by mixing a precursor solution of the hyaluronic acid-based hydrogel with a fluoride source on site, the precursor solution of the hyaluronic acid-based hydrogel comprises component A and component B, and the preparation method of the component A comprises the following steps:

[0012] S1. mixing methyl acrylate group modified hyaluronic acid and furfuryl amine, and reacting to obtain furan-methyl acrylate modified hyaluronic acid;

[0013] S2. mixing the furan-methyl acrylate modified hyaluronic acid and maleimide, and reacting to obtain double-modified group hyaluronic acid;

[0014] S3. mixing the double-modified group hyaluronic acid, a photoinitiator and a solvent to obtain the component A;

[0015] The component B is a mercapto polyethylene glycol solution, and the mass ratio of the double-modified group hyaluronic acid in the component A to the mercapto polyethylene glycol in the component B is 1: (2.5-5.0);

[0016] The fluoride source of the fluoride ion sustained-release agent is a magnetic mesoporous nanoparticle containing fluoride ions.

[0017] The main component of the hyaluronic acid-based hydrogel of the fluoride ion sustained-release agent of the present application is a double-modified group hyaluronic acid. The hyaluronic acid is first modified with methacrylate groups, then grafted with furan groups, and then reacted with maleimide through the furan groups to form norbornene-like groups, thereby obtaining hyaluronic acid with both methacrylate groups and norbornene-like groups. The precursor solution (after mixing component A and component B) of the hyaluronic acid-based hydrogel can be cured to form a gel under direct ultraviolet light irradiation within 0.5 s, which is faster than existing hydrogel materials and meets the requirement of rapid gelation after being prepared into a fluoride ion sustained-release agent. In addition, the precursor solution of the hydrogel can be cured to form a gel under the condition of no ultraviolet light, which provides a basis for effective gelation at positions that cannot be irradiated by ultraviolet light after being prepared into a fluoride ion sustained-release agent. In addition, the hyaluronic acid-based hydrogel can effectively improve the sustained-release effect of fluoride ions, and when combined with fluoride ion-containing magnetic mesoporous nanoparticles, the fluoride ion sustained-release agent prepared therefrom can release 30-34% of fluoride ions within 3 h, release 60% of fluoride ions within about 12 h, maintain an effective fluoride ion concentration for more than 168 h, and realize long-term and stable release of fluoride ions for at least one week.

[0018] That is, the present application is based on a specific hyaluronic acid-based hydrogel, and is combined with fluoride ion-containing magnetic mesoporous nanoparticles to prepare a fluoride ion sustained-release agent. The fluoride ion sustained-release agent not only has a fast gelation speed, but also can be cured to form a gel in different ways (ultraviolet light irradiation or no light irradiation), and can release fluoride ions in a long-term and stable manner after gelation. The fluoride ion sustained-release agent can be used for preventing and / or treating enamel demineralization or enamel defects, especially irregular enamel defects.

[0019] It should be understood that the methacrylate group-modified hyaluronic acid in step S1 refers to hyaluronic acid grafted with methacrylate groups after modification.

[0020] Preferably, the degree of substitution of the methacrylate groups in the methacrylate group-modified hyaluronic acid in step S1 is 25-35%.

[0021] It should be understood that the degree of substitution of the methacrylate groups refers to the ratio of the peak area of the methyl hydrogen in the methacrylate groups to the peak area of the methyl hydrogen on the six-carbon ring side chain of the hyaluronic acid in the nuclear magnetic resonance spectrum of the methacrylate group-modified hyaluronic acid.

[0022] Preferably, the methacrylate group-modified hyaluronic acid in step S1 is prepared by the following method: dissolving hyaluronic acid or a salt thereof in water, first adding methacrylic anhydride, then adjusting the pH to 8.45-8.55, reacting for 20-24 hours, and dialyzing to obtain the methacrylate group-modified hyaluronic acid.

[0023] More preferably, the ratio of the hyaluronic acid to the methacrylic anhydride is 1g: (2.8~3.2 mL).

[0024] More preferably, the relative molecular weight of the hyaluronic acid is 90~100 kDa.

[0025] More preferably, the reaction is carried out at 35~40℃.

[0026] More preferably, the dialysis bag used in the dialysis has a specification of 3000~5000 Da.

[0027] Preferably, the specific process of step S1 is as follows: the methacrylate group-modified hyaluronic acid is first dissolved in a morpholine ethanesulfonic acid buffer, then 4-(4,6-dimethoxytriazine)-4-methyl morpholine hydrochloride is added, followed by the addition of furfurylamine, mixing, reaction for 20~24 hours, dialysis, and the furan-methacrylate group-modified hyaluronic acid is obtained.

[0028] More preferably, the pH of the morpholine ethanesulfonic acid buffer is 4.4~4.6.

[0029] More preferably, the ratio of the methacrylate group-modified hyaluronic acid to the furfurylamine is 1g: (0.35~0.40 mL).

[0030] More preferably, the mass ratio of the methacrylate group-modified hyaluronic acid to the 4-(4,6-dimethoxytriazine)-4-methyl morpholine hydrochloride is 1: (2.8~3.0).

[0031] More preferably, the dialysis bag used in the dialysis has a specification of 3000~5000 Da.

[0032] Preferably, the temperature of the reaction in step S1 is 25~30℃.

[0033] Preferably, the degree of substitution of the furan group in the furan-methacrylate group-modified hyaluronic acid in step S1 is 55~65%.

[0034] It should be understood that the degree of substitution of the furan group refers to the ratio of the sum of the peak areas of the three hydrogen atoms of the five-carbon ring of the furan group to the peak area of the methyl hydrogen on the six-carbon ring side chain of the hyaluronic acid in the nuclear magnetic resonance spectrum of the furan-methacrylate group-modified hyaluronic acid.

[0035] Preferably, the specific process of step S2 is as follows: the furan-methacrylate group-modified hyaluronic acid is first dissolved in water, then maleimide is added, mixing, reaction for 20~24 hours, dialysis, and the double-modified group hyaluronic acid is obtained.

[0036] More preferably, the mass ratio of the furan-methyl acrylate modified hyaluronic acid to the maleimide is 1: (1.0~1.2).

[0037] More preferably, the size of the dialysis bag for dialysis is 3000~5000 Da.

[0038] Preferably, the temperature of the reaction in step S2 is 25~30℃.

[0039] Preferably, the concentration of the double-modified group hyaluronic acid in the A component in step S3 is 20~25 mg / mL.

[0040] Preferably, the mass ratio of the double-modified group hyaluronic acid to the photoinitiator in the A component in step S3 is 1: (0.1~0.25).

[0041] Preferably, the photoinitiator in step S3 is lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate.

[0042] Preferably, the solvent in step S3 is water or physiological saline.

[0043] Preferably, the mercapto-polyethylene glycol solution is a four-arm mercapto-polyethylene glycol solution.

[0044] Preferably, the concentration of the mercapto-polyethylene glycol solution is 500~1500 mg / mL.

[0045] Preferably, the mass ratio of the double-modified group hyaluronic acid to the fluorine source is 1: (0.025~0.030).

[0046] Preferably, the volume ratio of the A component to the B component is 10: (1~2).

[0047] The fluorine ion-containing magnetic mesoporous nanoparticles commonly used in the art can be used in the present application. In the present application, the preparation method of the fluorine ion-containing magnetic mesoporous nanoparticles can refer to the preparation method of the fluorine ion-containing magnetic mesoporous nanoparticles in the prior application patent CN109490261A of the inventor of the present application (FSMN in CN109490261A).

[0048] Preferably, the preparation method of the fluorine ion-containing magnetic mesoporous nanoparticles comprises the following steps:

[0049] S4. Mixing the hydrophilic Fe3O4 particles, the organosilane functionalized carbon dots and cetyltrimethylammonium bromide, then adding ammonia water and tetraethyl orthosilicate, reacting, magnetically separating, removing the cetyltrimethylammonium bromide, and obtaining the magnetic nanoparticles;

[0050] S5. Preparing the magnetic nanoparticles into silica-coated magnetic nanoparticles;

[0051] S6. The silica-coated magnetic nanoparticles are mixed with a fluoride salt solution, filtered, and magnetic mesoporous nanoparticles containing fluoride ions are obtained.

[0052] The addition or mixing order of the reagents in step S4 is optimized compared to CN109490261A, which can ensure that the silica-coated magnetic nanoparticles / magnetic mesoporous nanoparticles (FSMN in CN109490261A) are successfully prepared, and the preparation process is more simple.

[0053] More preferably, the organosilane-functionalized carbon dots in step S4 are added in the form of an organosilane-functionalized carbon dot-ethanol solution.

[0054] More preferably, the tetraethyl orthosilicate in step S4 is added in the form of a tetraethyl orthosilicate-ethanol solution.

[0055] More preferably, the ammonium nitrate-ethanol solution is used to reflux to remove the cetyltrimethylammonium bromide in step S4.

[0056] Preferably, the hyaluronic acid-based hydrogel is used in the preparation of a fluoride ion sustained-release agent for preventing and / or treating enamel demineralization or enamel defects.

[0057] A fluoride ion sustained-release agent includes a precursor solution of the above-mentioned hyaluronic acid-based hydrogel and the above-mentioned fluoride source.

[0058] Compared with the prior art, the present application has the following advantages:

[0059] The present application is based on a specific hyaluronic acid-based hydrogel, and is combined with magnetic mesoporous nanoparticles containing fluoride ions to prepare a fluoride ion sustained-release agent. The fluoride ion sustained-release agent not only has a fast gelation speed, but also can be self-healed to form a gel through different ways (ultraviolet light or without light), and can release fluoride ions stably and long-acting after gelation. The fluoride ion sustained-release agent can be used for preventing and / or treating enamel demineralization or enamel defects, especially irregular enamel defects. BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1 NMR spectra of each intermediate product (HA, HM, HFM) and the final product (HFMM) of the double-modified group hyaluronic acid of the hyaluronic acid-based hydrogel of the fluoride ion sustained-release agent of Example 1.

[0061] Figure 2 Process diagram of the ultraviolet light direct irradiation rapid gelation test of the hyaluronic acid-based hydrogel of the fluoride ion sustained-release agent of Example 1.

[0062] Figure 3Schematic diagram of slow self-healing and fast self-healing process of hyaluronic acid-based hydrogel precursor solution of fluoride ion sustained-release agent of Example 1.

[0063] Figure 4 TEM image of silica-coated magnetic nanoparticles as fluoride source of fluoride ion sustained-release agent of Example 1.

[0064] Figure 5 SEM image of silica-coated magnetic nanoparticles (FSMN) as fluoride source of fluoride ion sustained-release agent of Example 1.

[0065] Figure 6 Nitrogen adsorption / desorption isotherm plot of silica-coated magnetic nanoparticles (FSMN) as fluoride source of fluoride ion sustained-release agent of Example 1; Figure 6 b isotherm pore size distribution plot of silica-coated magnetic nanoparticles (FSMN) as fluoride source of fluoride ion sustained-release agent of Example 1.

[0066] Figure 7 Scanning electron microscope image of Example 1 after gelation of fluoride ion sustained-release agent.

[0067] Figure 8 Fluoride ion release curve of each group of fluoride ion sustained-release agents of Example 5. DETAILED DESCRIPTION

[0068] In order to more clearly, completely describe the technical solutions of the present application, the following will further illustrate the present application through specific examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application. Various changes can be made within the scope of the present application.

[0069] Example 1

[0070] The present embodiment provides a fluoride ion sustained-release agent, which comprises a hyaluronic acid-based hydrogel and fluoride ion-containing magnetic mesoporous nanoparticles as a fluoride source. Wherein:

[0071] 1) The precursor solution of the hyaluronic acid-based hydrogel comprises A component and B component; the B component is a four-arm thiol polyethylene glycol (PEG, 2 kDa, AR, Sigma-Aldrich) aqueous solution with a concentration of 1000 mg / mL; the preparation method of the A component comprises the following steps:

[0072] 1.1) Preparation of methyl methacrylate group modified hyaluronic acid

[0073] Take 5 g of solid sodium hyaluronate (HA, 100 kDa, Shanghai Yuan Ye) and add it to 500 mL of deionized water. Stir at room temperature (25°C) for 2 h to completely dissolve into a colorless transparent liquid. Add 15 mL of methacrylic anhydride (MA, Sigma-Aldrich) drop by drop, and drop it in 3 h. Then use 5 M sodium hydroxide solution to adjust the pH of the system to 8.5, and stir overnight in a 37°C water bath. Put the reaction completed mixed liquid into a 3000 Da dialysis bag, and change the peripheral deionized water every 8 h for 3 days. Collect and freeze-dry into a white flocculent solid, which is the methacrylate-modified hyaluronic acid, denoted as HM.

[0074] 1.2) Preparation of furan-methacrylate-modified hyaluronic acid

[0075] First, prepare 100 mM, pH=4.5 morpholine ethanesulfonic acid buffer (MES); take 1.2 L MES and put it into a 2 L volume brown light-proof beaker, and dissolve thoroughly; then weigh 4 g HM and slowly add it to the MES under 1000 rpm / min magnetic stirring; seal with tin foil paper and continue stirring at room temperature for 2 h until the HM is completely dissolved to form a clear transparent solution.

[0076] Weigh 11.2 g 4-(4,6-dimethoxytriazine)-4-methyl morpholine hydrochloride (DMTMM) and slowly add it to the above solution, stir at room temperature for 1 h, and use DMTMM to activate the carboxyl functional groups on the hyaluronic acid backbone to obtain a mixed solution. Take 1.5 mL furan (Furan, AR, Sigma-Aldrich) and add it drop by drop to the above mixed solution within 5 min, seal with tin foil paper, and continue stirring at room temperature for 24 h.

[0077] Put the reaction completed mixed liquid into a 3000 Da dialysis bag, change the peripheral deionized water every 8 h for 3 days, collect, and freeze-dry into a white flocculent solid, which is the furan-methacrylate-modified hyaluronic acid, denoted as HFM.

[0078] 1.3) Preparation of double-modified group hyaluronic acid

[0079] Take 3 g of freeze-dried HFM and add it to 1.2 L of deionized water, stir at room temperature for 2 h to completely dissolve into a colorless transparent liquid; then add 3 g of maleimide (Mal, AR, Sigma-Aldrich) and stir at room temperature for 1 day. Put the reaction completed mixed liquid into a 3000 Da dialysis bag, change the peripheral deionized water every 8 h for 3 days. Collect and freeze-dry into a white flocculent solid, which is the double-modified group hyaluronic acid, denoted as HFMM.

[0080] 1.4) Preparation of Component A

[0081] HFMM 0.4 g and photoinitiator lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (LAP) 0.1 g were weighed and added into 20 mL deionized water, mixed well and uniformly, and prepared into a 2% (wt / v) HFMM solution, which was Component A.

[0082] 2) Preparation of magnetic mesoporous nanoparticles containing fluoride ions

[0083] 2.1) Preparation of magnetic nanoparticles

[0084] 0.325 g of anhydrous FeCl3and 0.45 g of sodium citrate dihydrate were dissolved in 40 mL of ethylene glycol until an orange-yellow solution was formed, followed by the addition of 3.0 g of anhydrous sodium acetate until a uniform yellow-brown solution was obtained. The solution was then transferred to a 50 mL volume Teflon-lined hydrothermal kettle, 200 °C, 10 h. After cooling to room temperature, deionized water was washed 5 times, and vacuum dried to obtain hydrophilic Fe3O4particles.

[0085] 0.1 g of hydrophilic Fe3O4particles, 2.5 mL of deionized water and 10 mL of anhydrous ethanol were mixed and uniformly dispersed by ultrasonic; 2.5 mL of SiCDs dilution solution (silicone-silane functionalized carbon dots), 0.1 g of CTAB (cetyltrimethylammonium bromide), 6 mL of deionized water and 15 mL of anhydrous ethanol were added and mixed uniformly to form a homogeneous solution. Then, 0.4 mL of ammonia water and 2.5 mL of TEOS (tetraethyl orthosilicate) were injected into the previous homogeneous solution, and stirring was continued for 2 hours. The product was collected with a magnet and washed with ethanol and deionized water for about 4-5 times. In order to remove CTAB, the washed product was mixed with an ammonium nitrate-anhydrous ethanol solution (0.6 wt %) and refluxed at 75 °C for 12 hours to obtain Fe3O4@mSiO2-SiCDs nanoparticles.

[0086] 2.2) Preparation of silica-coated magnetic nanoparticles

[0087] Fe3O4@mSiO2-SiCDs nanoparticles were added to 30 mL of isopropanol, followed by the addition of 0.7 mL of APTES, and the reaction was allowed to proceed for 24 h. The product was collected by vacuum drying. The product was then added to 36 mL of 50% v / v acetic acid-ethanol solution. Next, pre-prepared DTPA anhydride was added to the mixture and the reaction was allowed to proceed further by refluxing at 80 °C for 16 h. Finally, Fe3O4@mSiO2-SiCDs@DTPA nanoparticles were recovered by vacuum suction filtration, washed with deionized water and dispersed in 10 mL of deionized water. The above solution was stirred with a slight excess of Ni(NO3)2salt for 24 h and the pH of the solution was set to 6.0. Fe3O4@mSiO2-SiCDs@DTPA-Ni 2+ nanoparticles (silica-coated magnetic nanoparticles / FSMN).

[0088] Steps 2.1)~2.2) can also be carried out by referring to Example 1 and Example 2 of CN109490261A, both of which can obtain FSMN, and the addition or mixing order of reagents of steps 2.1)~2.2) of the present example is optimized, and the process is more simple.

[0089] 2.3) Preparation of magnetic mesoporous nanoparticles containing fluoride ions

[0090] 10 mg of FSMN powder was mixed with 3 mL of deionized water under a mild shaking environment, and after sufficient stirring, a NaF aqueous solution with a concentration of 10 mg / L (30 mL) was added, and shaken for 1 h. Then the lower particles were collected, washed, centrifuged, and freeze-dried to obtain magnetic mesoporous nanoparticles containing fluoride ions (FSMN-F - ).

[0091] 3) Preparation before testing / using

[0092] The fluoride ion slow-release agent needs to be prepared and used immediately, and the specific process is as follows: take the A component prepared in 1.4) (all), add 0.01 g of magnetic mesoporous nanoparticles containing fluoride ions prepared in 2.3), and then add 2 mL of B component to prepare a fluoride ion slow-release agent precursor solution (HFMM / PEG / FSMN-F - ), and the gel after forming is the fluoride ion slow-release agent.

[0093] Example 2

[0094] The fluoride ion slow-release agent of the present example is different from that of Example 1 in that the B component is a four-arm thiol polyethylene glycol aqueous solution with a concentration (wt / v) of 500 mg / mL.

[0095] Example 3

[0096] This example provides a fluoride ion sustained-release agent which differs from Example 1 in that the B component is a four-arm mercapto polyethylene glycol aqueous solution with a concentration (wt / v) of 1500 mg / mL.

[0097] Example 4 Sample characterization

[0098] The following characterization was performed based on Example 1.

[0099] 1) Characterization of each component of the double-modified group hyaluronic acid in the A component and its preparation process

[0100] 0.01 g of HA, HM, HFM, and HFMM, respectively, was weighed and dissolved in 0.5 mL of D2O until completely dissolved. Then, the mixed solution was transferred to a clean NMR tube for NMR hydrogen spectrum test, and the results are shown in Figure 1 . Figure 1 is the NMR spectrum (hydrogen spectrum, 600 MHz) of each component, wherein i is HA, ii is HM, iii is HFM, and iv is HFMM; wherein, Figure 1 A and Figure 1 C are local amplification diagrams of different chemical shift ranges of B, respectively. Figure 1 B.

[0101] It can be seen that: Figure 1

[0102] The peak of HA at a chemical shift of 2.00 ppm is the characteristic peak of the methyl hydrogen atom on the side chain of HA. The appearance of the characteristic peaks of the methyl methacrylate group double bond at 6.22 ppm and 5.75 ppm and the methyl hydrogen (from the methyl methacrylate group) characteristic peak at 1.93 ppm in HM proves that the hydroxyl of the hydroxymethyl on the six-carbon sugar ring side chain of HA reacts with methyl methacrylate to form HM, and the methyl methacrylate group is successfully grafted onto the hyaluronic acid. The hydroxyl in the surface hydroxymethyl of hyaluronic acid is the farthest from the main chain of hyaluronic acid and has the smallest steric hindrance, so it is the easiest to undergo esterification with the methyl methacrylate group. The degree of substitution of the methyl methacrylate group in HM refers to the ratio of the peak area of the methyl hydrogen in the methyl methacrylate group (Area (1.93 ppm)) to the peak area of the methyl hydrogen on the six-carbon ring side chain of hyaluronic acid (Area (2.00 ppm)) in the NMR spectrum of the methyl methacrylate-modified hyaluronic acid, and the calculation formula is as follows:

[0103] .

[0104] Through calculation, the DS of the methyl methacrylate group in HM is about 25 %. ​

[0105] HFM retains the characteristic peak of methyl hydrogen atom at 2.00 ppm, and the peaks of methyl methacrylate at 6.22 ppm, 5.75 ppm and 1.93 ppm, indicating that the methyl methacrylate group remains stable during the furan modification process. New characteristic peaks appear at chemical shifts of 6.35 ppm, 6.48 ppm and 7.42 ppm, which correspond to the three hydrogen atoms on the five-carbon ring of the furan group, proving that the amidation reaction between the carboxyl group on the hyaluronic acid molecule side chain and the amino group on the furan amine molecule has occurred, and the furan group has been successfully grafted onto HM. The degree of substitution of the furan group in HFM refers to the ratio of the sum of the peak areas of the three hydrogen atoms on the five-carbon ring of the furan group (Area (6.35 ppm) + Area (6.48 ppm) + Area (7.42 ppm)) to the peak area of the methyl hydrogen on the six-carbon ring side chain of hyaluronic acid (Area (2.00 ppm)) in the nuclear magnetic resonance spectrum of furan-methyl methacrylate modified hyaluronic acid, and the calculation formula is as follows:

[0106]

[0107] Through calculation, the DS of the furan group in HFM is about 55%.

[0108] HFMM also retains the characteristic peak of methyl hydrogen atom at 2.00 ppm, the characteristic peaks of the furan group at 6.35 ppm, 6.48 ppm and 7.42 ppm, and the peaks of methyl methacrylate at 6.22 ppm, 5.75 ppm and 1.93 ppm, proving that the connection of the maleimide group does not affect the groups already modified on the aforementioned product. By comparing the spectrum of HFM, it can be found that the chemical shift intensity of the characteristic hydrogen atoms on the furan group is significantly weakened, and a new peak appears at 5.29 ppm, which is the characteristic peak of the norbornene-like double bond generated between the maleimide and the furan group through DA click chemistry reaction, indicating that the final norbornene-like group is successfully modified to the hyaluronic acid side chain. The degree of substitution of the norbornene-like group is mainly affected by the degree of substitution of the furan group. In the gel molecule, whether to graft active groups, the type and position of the grafted active groups, all affect the properties and functional activities of the gel. The above nuclear magnetic data show that the double modified group hyaluronic acid of the present application has both norbornene-like groups and methyl methacrylate groups on the hyaluronic acid side chain.

[0109] Compared with hyaluronic acid alone, two kinds of photo-crosslinking groups are grafted on the side chain of HFMM; the steric hindrance effect of different photo-crosslinking groups is different, and in this case, the difference in grafting sequence may affect the success and degree of substitution of the two groups, and ultimately may affect the properties and functional activity of the hydrogel. In this application, due to the fact that the steric hindrance of norbornene-like group is obviously larger than that of methacrylate group, if norbornene-like group is grafted first in the MES environment, although the degree of substitution of norbornene-like group is ensured, the steric hindrance of norbornene-like group will affect the contact between methacrylic anhydride and the surface hydroxyl group of hyaluronic acid, and then affect the degree of substitution of methacrylate group.

[0110] In addition, since methacrylated hyaluronic acid is grafted through the hydroxyl group of the side chain, the reaction itself is more difficult than the amidation of carboxyl group, so the DS of methacrylate group in HM is lower than that of pure methacrylate group grafted on the surface of gelatin. When furan is grafted on HM, due to the existence of methacrylate group, there is steric hindrance, so the DS of furan group in HFM is lower than that of pure furan group grafted on the surface of hyaluronic acid, but in the HFMM of the present application, it is still higher than 50%.

[0111] In addition, the HFMM solution was also tested by ultraviolet absorption spectrum, and there was an absorption peak at 217 nm, which came from the conjugated diene structure of the furan group in the norbornene-like group; there was an absorption peak at 213 nm, which came from the conjugated structure of the maleimide carbonyl group in the norbornene-like group.

[0112] The precursor solution of the hyaluronic acid-based hydrogel of Example 1 was mixed (volume ratio 10:1) and subjected to ultraviolet light direct irradiation rapid gelation test, and the results are shown in Figure 2 . Figure 2 From 2a~2b, the ultraviolet light irradiation time was controlled by an electronic timer, and the hyaluronic acid-based hydrogel precursor solution was subjected to ultraviolet light direct irradiation for 0.5 s, from 2b~2c~2d, the rapid photo-crosslinking Thiol-ene reaction between the norbornene-like group and the methacrylate group of the double modified group hyaluronic acid and the thiol group of the four-arm thiol polyethylene glycol occurred, and it can be seen that the gelation and the hydrogel piece were completely lifted. Figure 2 It can be seen that the shortest time of ultraviolet light direct irradiation of only 0.5 s can realize the gelation of the precursor solution of the hyaluronic acid-based hydrogel, which is much faster than the currently reported hydrogels (generally more than 10 s). At the same time, the hydrogel formed by rapid photo-crosslinking after 0.5 s ultraviolet irradiation has the characteristics of colorless and transparent.

[0113] The precursor solution of hyaluronic acid-based hydrogel of Example 1 was mixed (volume ratio 10:1) with A component and B component to conduct different ways of gelation test: it can be achieved by Thiol-ene rapid photo-crosslinking reaction under UV light to realize the rapid self-healing of hydrogel, see Figure 3 a~f; it can also be achieved by Michael chemical crosslinking without UV irradiation to realize the slow self-healing of hydrogel, see Figure 3 g~h, the time of slow self-healing is about 20 min. Figure 3 i is the effect picture after self-healing, wherein, Figure 3 i, the left side is the rapid self-healing hydrogel, and the right side is the slow self-healing hydrogel.

[0114] 2) Characterization of FSMN

[0115] Figure 4 is the TEM picture of FSMN (silica-coated magnetic nanoparticles of 2.2 of Example 1), wherein, Figure 4 b is a local enlarged view of a. As shown in Figure 4 , FSMN is a rough spherical surface with core-shell structure; the total diameter is about 250 nm, the diameter of core Fe3O4 is about 100 nm, and the thickness of outer mesoporous silica shell is about 75 nm. Figure 4 is the scanning electron microscope picture of FSMN, Figure 5 which shows that FSMN is a rough spherical surface and has good dispersibility. Figure 5

[0116] Figure 6 a is the nitrogen adsorption / desorption isotherm curve of FSMN, which shows the characteristics of IV isotherm curve with hysteresis, which is the characteristic of mesoporous surface of FSMN, according to Figure 6 a, the specific surface area and total pore volume of particles are calculated by BET method and BJH method to be as high as 282.34 m 2 / g and 0.63 cm 3 / g. Figure 6 The isotherm pore size distribution curve of b confirms the formation of mesoporous SiO2 on the surface of FSMN, and the average diameter of mesopore is 3.1 nm. Compared with pure Fe3O4 particles, FSMN has a larger specific surface area.

[0117] 3) Characterization of fluoride ion sustained-release agent (HFMM / PEG / FSMN-F - hydrogel)

[0118] Figure 7 is the scanning electron microscope picture of HFMM / PEG / FSMN-F - hydrogel (after UV light gelation) of Example 1, wherein, Figure 7 b is​Figure 7 a is a close-up view of a. Figure 7 Fluoride ion-containing magnetic mesoporous nanoparticles (FSMN-F - ) were successfully dispersed and immobilized in HFMM / PEG hydrogels (hyaluronic acid-based hydrogels).

[0119] The characterization results of the fluoride ion sustained-release agents of Examples 2 and 3 were similar to those of Example 1.

[0120] Example 5 Determination of fluoride ion release curve of fluoride ion sustained-release agent

[0121] The following tests were based on Example 1.

[0122] First, according to the national standard of “HJ 488-2009 Water Quality-Determination of Fluoride-Fluoride Reagent Spectrophotometric Method”, the fluoride ion mixed color reagent kit was used to mix the fluoride reagent solution, sodium acetate buffer, acetone and lanthanum nitrate solution in a volume ratio of 3:1:3:3 to prepare the mixed color reagent required for fluoride ion detection. It was prepared and used immediately.

[0123] Subsequently, the standard curve of fluoride ion concentration was drawn. In 6 2 mL volume EP tubes, 0.625 μL, 1.25 μL, 2.5 μL, 5 μL, 10 μL and 18 μL of sodium fluoride standard solution (fluoride ion concentration: 200 μg / mL) were added, respectively, followed by adding deionized water to 1 mL, adding 1 mL of mixed color reagent, shaking well, and standing for 30 min. 100 μL of liquid was transferred to a 96-well plate, with deionized water (blank well) as a reference, and the absorbance was measured at 630 nm. The fluoride content was plotted against the absorbance to obtain the standard curve of fluoride ion concentration.

[0124] Finally, 500 μL of HFMM / PEG / FSMN-F - precursor solution, 500 μL of HFMM / PEG / F - precursor solution were added dropwise into 27 15 mL centrifuge tubes, respectively, and gelled under 395 nm wavelength, 50 mW / cm 2 power ultraviolet light irradiation for 10 s. Then 10 mL of deionized water was added and placed on a shaker at 100 rpm. At different predetermined time points, 1 mL of leaching liquid was drawn out, and the above-mentioned drawn leaching liquid was colored using a fluoride ion mixed color reagent kit, and then the absorbance was measured at 630 nm. According to the standard curve of fluoride ion concentration-absorbance, the fluoride ion release curve was obtained by conversion. The above experiment was repeated 3 times.

[0125] 500 μL of HFMM / PEG / FSMN-F -The precursor solution was added to 27 15 mL centrifuge tubes without UV irradiation; then 10 mL of deionized water was added, and the tubes were placed on a shaker, with a magnetic stir bar attached to the bottom wall of the centrifuge tube, so that the FSMN-F - The nanoparticles were separated from the hydrogel precursor solution, and as a first control group, were used to investigate the release of fluoride ions in FSMN-F; 100 rpm shaking, at different predetermined time points, 1 mL of the leaching solution was carefully sucked out, and the same volume of deionized water was added to maintain the stability of the total volume of the solution in the centrifuge tube. In addition, according to the HFMM / PEG / FSMN-F - The amount of particles (0.01 g) was added to 20 mL of the HFMM / PEG hydrogel precursor solution, and 0.473 mg of NaF powder was added to simulate the FSMN-F - The amount of particles (0.01 g) was added to 20 mL of the HFMM / PEG hydrogel precursor solution, and 0.473 mg of NaF powder was added to simulate the FSMN-F - The amount of particles (0.01 g) was added to 20 mL of the HFMM / PEG hydrogel precursor solution, and 0.473 mg of NaF powder was added to simulate the FSMN-F - The precursor solution was added to 27 15 mL centrifuge tubes without UV irradiation; then 10 mL of deionized water was added, and the tubes were placed on a shaker, with a magnetic stir bar attached to the bottom wall of the centrifuge tube, so that the FSMN-F

[0126] The leaching solution was colored using a fluoride ion mixed color reagent kit, and then the absorbance was measured at 630 nm. According to the standard curve of fluoride ion concentration-absorbance, the fluoride ion release curve was obtained.

[0127] When the standard curve of the fluoride ion concentration was determined, the concentrations of fluoride ions in 6 2 mL volume EP tubes were 0.0625 μg / mL, 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL and 1.8 μg / mL, respectively. With deionized water (blank hole) as a reference, the standard curve of fluoride ion concentration-absorbance was obtained after measuring the absorbance at 630 nm wavelength:

[0128]

[0129] where A represents the absorbance at 630 nm wavelength, and C represents the corresponding fluoride ion concentration (μg / mL). The correlation coefficient R 2 = 0.9987. According to the above standard curve, the fluoride ion release rate at different time points was obtained, and the fluoride ion release curve was plotted.

[0130] Figure 8 is the fluoride ion release curve of each group. Figure 8 In the above, the HFMM / PEG / FSMN-F - (gel) is the fluoride ion release curve of the fluoride ion sustained-release agent of the present application (Example 1); the HFMM / PEG / FSMN-F - (liquid) is the fluoride ion release curve of the first control group, and the HFMM / PEG / F- (gel) is the fluorine ion release curve of the second control group. From Figure 8 It can be seen that:

[0131] Among the three, the HFMM / PEG / F -- (gel) releases fluorine ions the fastest and has the highest cumulative fluorine ion concentration. Within the first 3 h, it shows a burst release of fluorine ions, about 60-63 % of the fluorine ions are released into the solution, and then gradually releases in the subsequent time, the cumulative release rate is less than 100 %, indicating that the hydrogel itself has a certain adsorption and slow release effect on the doped fluorine ions.

[0132] Compared with the HFMM / PEG / F -- (gel), the FSMN-F - nanoparticle (i.e. Figure 8 HFMM / PEG / FSMN-F - (liquid)) releases fluorine ions more slowly and has a lower cumulative drug release amount. Within 3 h, 40-43 % of the fluorine ions are released, and about 8 h is needed to release 60 % of the fluorine ions, and the slow release phase is longer and eventually approaches the release equilibrium of the drug. The cumulative release rate of fluorine ions of the FSMN-F - nanoparticle is less than 100 %, indicating that the FSMN-F - nanoparticle itself is relatively firm to the adsorbed fluorine ions, and part of the fluorine ions cannot be released by simple diffusion.

[0133] The slow release effect of the HFMM / PEG / FSMN-F - hydrogel (i.e. Figure 8 HFMM / PEG / FSMN-F - (gel)) is the most obvious, within 3 h, 30-34 % of the fluorine ions are released, about 12 h is needed to release 60 % of the fluorine ions, and eventually also approaches the release equilibrium of the drug, the cumulative fluorine ion release rate is close to 90 %, and the effective fluorine ion concentration can be maintained for 168 h. This indicates that the fluorine ion slow-release agent of the application can achieve long-acting and stable release of fluorine ions within a week.

[0134] The fluorine ion release effect of the fluorine ion slow-release agent of Example 2 and Example 3 is similar to that of Example 1.

[0135] Since the hyaluronic acid-based hydrogel of the fluorine ion slow-release agent of the application has a fast gelation speed (0.5 s gelation) and can be gelled in different ways (with or without ultraviolet light irradiation), and the fluorine ion slow-release agent can release fluorine ions long-acting and stably, the fluorine ion slow-release agent can become a new type of fluorine ion slow-release agent for the prevention and treatment of enamel demineralization and enamel defects around the orthodontic bracket, especially for the treatment of irregular enamel defects.

[0136] Obviously, the above-mentioned embodiments of the present application are only examples for clearly illustrating the present application, but not limitation on the embodiments of the present application. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, it is not necessary and also impossible to enumerate all the embodiments. Any modification, equivalent replacement and improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. Use of a hyaluronic acid-based hydrogel for the preparation of a fluorine ion slow-release agent, said fluorine ion slow-release agent being extemporaneously prepared from a precursor solution of said hyaluronic acid-based hydrogel and a fluorine source, characterized in that, The precursor solution of the hyaluronic acid-based hydrogel comprises an A component and a B component, and the preparation method of the A component comprises the following steps: S1. mixing and reacting methyl acrylate group modified hyaluronic acid and furfuryl amine to obtain furan-methyl acrylate modified hyaluronic acid; S2. mixing and reacting furan-methyl acrylate modified hyaluronic acid and maleimide to obtain double-modified group hyaluronic acid; S3. mixing double-modified group hyaluronic acid, a photoinitiator and a solvent to obtain the A component; The B component is a mercapto polyethylene glycol solution, and the mass ratio of the double-modified group hyaluronic acid in the A component to the mercapto polyethylene glycol in the B component is 1: (2.5-5.0); The fluorine source of the fluoride ion sustained-release agent is a magnetic mesoporous nanoparticle containing fluoride ions; The degree of substitution of the methyl acrylate group in the methyl acrylate group modified hyaluronic acid in step S1 is 25-35%; The degree of substitution of the furan group in the furan-methyl acrylate modified hyaluronic acid in step S1 is 55-65%; The mass ratio of the double-modified group hyaluronic acid to the fluorine source is 1: (0.025-0.03); The preparation method of the magnetic mesoporous nanoparticle containing fluoride ions comprises the following steps: S4. mixing hydrophilic Fe3O4 particles, organosilane functionalized carbon dots and cetyltrimethylammonium bromide, then adding ammonia water and tetraethyl orthosilicate, reacting, magnetically separating, removing cetyltrimethylammonium bromide, and obtaining magnetic nanoparticles; S5. preparing the magnetic nanoparticles into silica-coated magnetic nanoparticles; S6. mixing the silica-coated magnetic nanoparticles with a fluoride salt solution, filtering, and obtaining the magnetic mesoporous nanoparticle containing fluoride ions.

2. Use according to claim 1, characterized in that, The specific process of step S2 is as follows: first, dissolving the furan-methyl acrylate modified hyaluronic acid in water, adding maleimide, mixing, reacting for 20-24 hours, dialyzing, and obtaining the double-modified group hyaluronic acid.

3. The use according to claim 1, characterized in that, The concentration of the double-modified group hyaluronic acid in the A component in step S3 is 20-25 mg / mL.

4. Use according to claim 1, characterized in that, The concentration of the mercapto polyethylene glycol solution is 500-1500 mg / mL.

5. The use according to claim 1, characterized in that, The hyaluronic acid-based hydrogel is used for preparing a fluoride ion sustained-release agent for preventing and / or treating enamel demineralization or enamel defects.

6. A fluoride ion releasing agent, characterized by, The precursor solution of the hyaluronic acid-based hydrogel and the fluorine source; The precursor solution of the hyaluronic acid-based hydrogel comprises an A component and a B component, and the preparation method of the A component comprises the following steps: S1. mixing and reacting methyl acrylate group modified hyaluronic acid and furfuryl amine to obtain furan-methyl acrylate modified hyaluronic acid; S2. mixing and reacting furan-methyl acrylate modified hyaluronic acid and maleimide to obtain double-modified group hyaluronic acid; S3. mixing double-modified group hyaluronic acid, a photoinitiator and a solvent to obtain the A component; The B component is a mercapto polyethylene glycol solution, and the mass ratio of the double-modified group hyaluronic acid in the A component to the mercapto polyethylene glycol in the B component is 1: (2.5-5.0); The fluorine source of the fluoride ion sustained-release agent is a magnetic mesoporous nanoparticle containing fluoride ions; The degree of substitution of the methacrylate groups in the hyaluronic acid modified with the methacrylate groups in step S1 is 25-35%; The degree of substitution of the furan groups in the hyaluronic acid modified with furan-methacrylate in step S1 is 55-65%; The mass ratio of the double-modified group hyaluronic acid to the fluorine source is 1:(0.025-0.03); The preparation method of the magnetic mesoporous nanoparticles containing fluorine ions comprises the following steps: S4. The hydrophilic Fe3O4 particles, the organic silane functionalized carbon dots and cetyltrimethylammonium bromide are mixed, and then ammonia water and tetraethyl orthosilicate are added, reacted, magnetically separated, and the cetyltrimethylammonium bromide is removed to obtain the magnetic nanoparticles; S5. The magnetic nanoparticles are prepared into silica-coated magnetic nanoparticles; S6. The silica-coated magnetic nanoparticles are mixed with a fluorinated salt solution, filtered, and the magnetic mesoporous nanoparticles containing fluorine ions are obtained.

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