Orally disintegrating film for promoting remineralization of enamel and preparation method thereof

Abstract

The invention relates to the technical field of medical supplies, and discloses an orally disintegrating film for promoting remineralization of enamel and a preparation method thereof.The orally disintegrating film takes hydroxypropyl methyl cellulose, microcrystalline cellulose, starch and the like as film base materials and has the advantages of being thin, light, good in flexibility, free of toxicity and stimulation, good in biocompatibility and biodegradability and the like; and the composition can be quickly dissolved in the oral cavity to take effect. The device has the advantages of quick effect, convenience in carrying, easiness in use and the like. The orally disintegrating film serves as a direct carrier of an inducer for inducing remineralization of enamel, so that the orally disintegrating film becomes a more effective transmission system. A bacteriophage display technology is utilized, a peptide fragment with strong adsorption capacity with HAP is integrated into a carrier bacteriophage DNA, and purification is performed after large-scale amplification, so that the peptide fragment is used as an important effective component of the orally disintegrating film.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical supplies, in particular to an oral disintegrating film for promoting enamel remineralization and a preparation method thereof. BACKGROUND

[0002] Enamel is mainly composed of inorganic components such as nanorod-shaped hydroxyapatite, and has a highly ordered multi-level structure. Enamel can be damaged by local cariogenic bacteria in dental plaque, non-bacterial factors in the oral environment, etc. To repair the damaged enamel, various repair materials have been developed, including resin, metal and ceramic fillings, etc.

[0003] Biomineralization technology is an effective strategy for repairing acid-etched enamel. Biomineralization is a mineralization process regulated by organic matrix, for example, the formation of enamel is regulated by amelogenin. In order to simulate the process of biomineralization and enamel regeneration, scientists have developed a variety of natural or synthetic materials to induce enamel regeneration. For example, directly using amelogenin and recombinant amelogenin, a mineralized layer containing ordered needle-shaped fluorine-doped hydroxyapatite crystals is induced on the surface of acid-etched enamel.

[0004] There is a rich casein protein in the oral cavity, which is a small peptide containing 39 amino acid residues with a molecular weight of about 5.4 kDa. The N-terminal (amino-terminal) is rich in tyrosine and glutamine, which is the core region of functional activity and can form an α-helix structure and be adsorbed in hydroxyapatite. Patent No. CN111000747B discloses a mineralized material for preventing enamel demineralization and its application. The composition prepared from amelogenin, amelogenin C-terminal peptide, and enamel maturation protein can achieve the biomimetic remineralization of micrometer-scale demineralized enamel and form new hydroxyapatite with a structure similar to enamel and no obvious interface. However, the composition of the patent is not easy to carry and is difficult to use in practice.

[0005] In this context, finding a method that can directly prevent dental caries or promote enamel remineralization has become the focus of dental research. One of the most promising strategies is to develop a delivery system that can efficiently carry active ingredients, thereby enhancing its clinical effect against enamel damage. Currently, there are many delivery systems under study, among which gel, coating agent, toothpaste and mouthwash are the most commonly studied carriers due to their clinical effectiveness and ease of use. Each carrier has its own characteristics: gel can provide good surface coverage, but has limited retention and usually requires professional operation; coating agent can achieve sustained release of active ingredients, but requires precise application technique; toothpaste and mouthwash are usually well accepted by patients, but may not be suitable for people with swallowing difficulties. SUMMARY

[0006] (I) Technical problems solved: The present application provides a kind of oral disintegrating film for promoting enamel remineralization and preparation method thereof, solve the existing product for promoting enamel remineralization repair effect is not good, not easy to carry, inconvenient to use and other problems.

[0007] (II) Technical scheme: a kind of preparation method of oral disintegrating film for promoting enamel remineralization: (1) 40ng HAP adsorption peptide gene, 5 μL of PCR buffer, 0.6 μL of 5U / μL TaqPlus DNA polymerase, 1 μL of deoxyribonucleotide triphosphate, 1 μL of 10 μM E-VHback, 1 μL of 10 μM E-VLfor are added to PCR tube, sterilized distilled water is added, and constant volume is 50 μL, and placed in PCR instrument, and denatured in 94 DEG C for 5 min, then cyclic reaction is carried out, the process of reaction is first in 94 DEG C for 1 min, then in 60 DEG C for 1 min, finally in 72 DEG C for 1.5 min;The reaction is cycled 5 times;72 DEG C is kept for 10 min, the product is electrophoresed, the gel is recovered, and the purified PCR product is obtained.

[0008] (2) 0.25-1 μg of PCR product, 2 μg of phagemid vector pCANTAB5E, 2 μL of 12U / μL restriction enzyme Sfi I are added to enzyme cutting buffer, sterilized distilled water is added, and constant volume is 50 μL, and placed in PCR instrument, and enzyme cutting reaction is carried out in 50 DEG C for 4h, and cooled to room temperature, and centrifugal separation is carried out, and DNA recovery kit is recovered, and scFv fragment carrier DNA is obtained.

[0009] (3) 25 μL of scFv fragment carrier DNA, 4 μL of 0.1% bovine serum albumin solution, 4 μL of 0.1% triton X-100 solution, 3 μL of 10U / μL restriction enzyme Not I solution are added to 4 μL enzyme cutting buffer, and placed in PCR instrument, and enzyme cutting reaction is carried out in 37 DEG C for 4h, and the product is electrophoresed, and recovered, to obtain enzyme cutting product.

[0010] (4) 250 ng of phagemid vector pCANTAB5E, 150 ng of enzyme cutting product, 1 μL of 5-7U / μL T4 DNA ligase are added to 2.5 uL ligation enzyme buffer, sterilized distilled water is added, and constant volume is 25 μL, and placed in PCR instrument, and reaction is carried out in 16 DEG C for 12h, and separated, to obtain ligation product.

[0011] (5) In an ice water bath, add competent E. coli to the test tube, add the ligation product, mix well, and then ice bath for 30 min, then heat to 42℃, heat treatment for 90 s, ice water bath cooling for 1-2 min, then add 800 uL of YT medium with a temperature of 37℃, 37℃ in the shaking culture for 1 h, then remove 100 μL of bacteria liquid to be coated on YT-Amp agar plate, 37℃ culture for 12 h, and obtain the ligation product transformed E. coli.

[0012] (6) The ligation product transformed E. coli is inoculated in 3 mL of YT-G-Amp liquid medium containing ampicillin and glucose, the concentration of ampicillin in the medium is 100 μg / mL, and the mass fraction of glucose is 2%; 30℃ shaking culture for 12 h, after culture, the bacteria liquid is diluted in 20 mL of YT-G-Amp liquid medium containing ampicillin and glucose at a ratio of 1:100, the concentration of ampicillin in the medium is 100 μg / mL, and the mass fraction of glucose is 2%; 30℃ shaking culture until the OD value is about 0.5, add helper phage M13KO7, 37℃ culture for 1 h, centrifugal separation, and add the precipitate to 200 mL of YT-Amp-Kan liquid medium containing ampicillin and kanamycin, the concentration of ampicillin in the medium is 100 μg / mL, and the concentration of kanamycin is 50 μg / mL; 30℃ shaking culture for 12 h, add 1 / 5 of the volume of the bacteria liquid to the NaCl solution containing polyethylene glycol 8000, the concentration of NaCl in the solution is 2.5 mol / L, and the mass fraction of polyethylene glycol 8000 is 20%; 4℃ preservation for 30 min, centrifugal separation for 20 min, and resuspend the centrifugal precipitate with 2 mL of PBS buffer to obtain the purified phage suspension. 600

[0013] (7) Add 2 mL of the purified phage suspension and 5 μL of enterokinase to the PCR tube, mix well, and then incubate in the PCR instrument at 25℃ for 12 h, filter with a filter with a molecular weight cut-off of 5 kDa, centrifugal separation, and freeze-dry the centrifugal supernatant to obtain the HAP adsorbed peptide.

[0014] (8) Add hydroxypropyl methylcellulose to deionized water, stir, then add sorbitol, stir, then add starch, microcrystalline cellulose, stir to disperse uniformly, and finally add the HAP adsorbed peptide, stir to dissolve uniformly, vacuum degassing, and spray the solution on the continuously rolling silicone film with a pressurized spray gun, dry, and form the oral disintegration film for promoting enamel remineralization on the surface of the silicone film.

[0015] Preferably, the mass ratio of hydroxypropyl methylcellulose, sorbitol, corn starch, microcrystalline cellulose, and HAP adsorbed peptide in (8) is (10-30):(10-30):(6-18):(4-12):(0.1-10).​

[0016] (III) Beneficial technical effects: The oral disintegrating film of the present application is used as a direct carrier of the inducer for inducing the remineralization of dental enamel, thus becoming a more effective delivery system. By using the phage display technology, the peptide segment with strong adsorption to HAP is integrated into the carrier phage DNA, and after being amplified in large quantities, it is purified and used as an important effective component of the oral disintegrating film.

[0017] The oral disintegrating film of the present application uses hydroxypropyl methylcellulose, microcrystalline cellulose, starch and the like as the film base material, has the advantages of thinness, lightness, good flexibility, non-toxicity and non-irritation, good biocompatibility and biodegradability, and can be quickly dissolved in the oral cavity to take effect. It is convenient for children, the elderly and people with difficulty in swallowing to use, has the advantages of rapid effect, easy to carry and easy to use.

[0018] In actual application, a piece of the oral disintegrating film can be held in the oral cavity, the oral disintegrating film can be quickly disintegrated in the oral cavity, and the HAP adsorption peptide contained therein can quickly combine with the dental enamel on the surface of the teeth to form a protective film. In addition, the oral disintegrating film can be cut into small square pieces and added into toothpaste. During the use of the toothpaste for brushing teeth, the oral disintegrating film is quickly disintegrated, and the HAP adsorption peptide can help the HAP in the toothpaste to deposit on the surface of the dental enamel, thereby promoting the remineralization of the dental enamel. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a confocal scanning electron microscope image of a dental enamel sample.

[0020] Figure 2 is a scanning electron microscope image of the surface of a normal dental enamel sample and the surface of an acid-etched dental enamel sample.

[0021] Figure 3 is a scanning electron microscope image of the surface of a dental enamel sample after remineralization in artificial saliva. DETAILED DESCRIPTION

[0022] The following E-VHback is a forward primer, and the sequence number is 5'-GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAG GTG CAG CTG SWG SAG TCW GG-3'. R=A, G; S=G, C; W=A, T; M=A, C; K=G, T.

[0023] E-VLfor is a reverse primer, and the sequence number is 5'-GAG TCA TTC TCG ACT TGC GGC CGC TTT GAT CTG CAS CTT GGT CC-3'. R=A, G; S=G, C; W=A, T; M=A, C; K=G, T.

[0024] The HAP adsorption peptide gene was synthesized by Bioengineering (Shanghai) Co., Ltd. using Fmoc solid-phase synthesis method, and the amino acid sequence was QPHypQPHypQPHypQPMQPSKRKRKRK, Q represented glutamine, P represented proline, Hyp represented hydroxyproline, and M represented methionine.

[0025] The restriction endonuclease Sfi I and the restriction endonuclease Not I were provided by TAKARA Co., Ltd.

[0026] The PCR buffer was provided by Bioengineering (Shanghai) Co., Ltd. The Taq Plus DNA polymerase was provided by Bioengineering (Shanghai) Co., Ltd.

[0027] Example 1: (1) 40 ng of the HAP adsorption peptide gene, 5 μL of the PCR buffer, 0.6 μL of the Taq Plus DNA polymerase with a concentration of 5 U / μL, 1 μL of deoxyribonucleotide triphosphate, 1 μL of E-VHback with a concentration of 10 μM, and 1 μL of E-VLfor with a concentration of 10 μM were added to a PCR tube, sterilized distilled water was added to make the volume 50 μL, and the PCR tube was placed in a PCR instrument. Denaturation was performed at 94°C for 5 min, and then the cycle reaction was performed. The reaction process was as follows: first, denaturation was performed at 94°C for 1 min, then annealing was performed at 60°C for 1 min, and finally extension was performed at 72°C for 1.5 min. The reaction was cycled 5 times, and then the product was incubated at 72°C for 10 min. The product was loaded for electrophoresis, the target band was recovered by cutting the gel, and the purified PCR product was obtained.

[0028] (2) 0.25 μg of the PCR product, 2 μg of the phagemid vector pCANTAB5E, and 2 μL of the restriction endonuclease Sfi I with a concentration of 12 U / μL were added to the enzyme digestion buffer, sterilized distilled water was added to make the volume 50 μL, and the mixture was placed in a PCR instrument. Enzymatic digestion was performed at 50°C for 4 h, the mixture was cooled to room temperature, centrifugal separation was performed, and the scFv fragment vector DNA was recovered by using a DNA recovery kit.

[0029] (3) 25 μL of the scFv fragment vector DNA, 4 μL of a 0.1% bovine serum albumin solution, 4 μL of a 0.1% triton X-100 solution, and 3 μL of the restriction endonuclease Not I with a concentration of 10 U / μL were added to 4 μL of the enzyme digestion buffer, and the mixture was placed in a PCR instrument. Enzymatic digestion was performed at 37°C for 4 h, the product was loaded for electrophoresis, and the enzyme digestion product was recovered.

[0030] (4) To 2.5 uL of ligation buffer, 250 ng of phagemid vector pCANTAB5E, 150 ng of the enzyme-digested product, 1 uL of T4 DNA ligase with a concentration of 5 U / uL were added, sterilized distilled water was added to make up to 25 uL, and the mixture was placed in a PCR instrument for reaction at 16 °C for 12 h. After separation, a ligation product was obtained.

[0031] (5) In an ice water bath, competent E. coli was added to a test tube, and the ligation product was added. After mixing and ice bath standing for 30 min, the mixture was heated to 42 °C for heat treatment for 90 s, and then cooled in an ice water bath for 2 min. Then, 800 uL of YT medium at a temperature of 37 °C was added, and the mixture was cultured at 37 °C for 1 h. Then, 100 uL of the bacterial solution was taken and spread on a YT-Amp agar plate, which was cultured at 37 °C for 12 h. Thus, the ligation product transformed E. coli was obtained.

[0032] (6) The ligation product transformed E. coli was inoculated in 3 mL of YT-G-Amp liquid medium containing ampicillin and glucose. The concentration of ampicillin in the medium was 100 ug / mL, and the mass fraction of glucose was 2%. The mixture was cultured at 30 °C for 12 h. After the culture, the bacterial solution was diluted in 20 mL of YT-G-Amp liquid medium containing ampicillin and glucose at a ratio of 1:100. The concentration of ampicillin in the medium was 100 ug / mL, and the mass fraction of glucose was 2%. The mixture was cultured at 30 °C until the OD value was about 0.5. Then, helper phage M13KO7 was added, and the mixture was cultured at 37 °C for 1 h. The precipitate was added to 200 mL of YT-Amp-Kan liquid medium containing ampicillin and kanamycin. The concentration of ampicillin in the medium was 100 ug / mL, and the concentration of kanamycin was 50 ug / mL. The mixture was cultured at 30 °C for 12 h. Then, 1 / 5 of the volume of the bacterial solution was added to a NaCl solution containing polyethylene glycol 8000. The concentration of NaCl in the solution was 2.5 mol / L, and the mass fraction of polyethylene glycol 8000 was 20%. The mixture was stored at 4 °C for 30 min. Then, the mixture was centrifuged for 20 min. The precipitate was resuspended in 2 mL of PBS buffer to obtain a purified phage suspension. 600

[0033] (7) 2 mL of the purified phage suspension and 5 uL of enterokinase were added to a PCR tube. After mixing, the mixture was incubated in a PCR instrument at 25 °C for 12 h. The mixture was filtered with a filter with a molecular weight cutoff of 5 kDa. The centrifugal supernatant was freeze-dried to obtain an HAP adsorbed peptide.

[0034] ​(8) 160 g of hydroxypropyl methylcellulose is added to deionized water, stirred, 250 g of sorbitol is added, stirred, 130 g of starch, 100 g of microcrystalline cellulose is added, stirred and dispersed uniformly, finally 1 g of HAP adsorbed peptide is added, stirred and dissolved uniformly, vacuum degassing, and the solution is sprayed on the continuously rolling silicone film with a pressurized spray gun, dried to form an oral disintegration film for promoting enamel remineralization on the surface of the silicone film.

[0035] Example 2: (1) 40 ng of HAP adsorbed peptide gene, 5 μL of PCR buffer, 0.6 μL of TaqPlus DNA polymerase with a concentration of 5 U / μL, 1 μL of deoxyribonucleotide triphosphate, 1 μL of E-VHback with a concentration of 10 μM, 1 μL of E-VLfor with a concentration of 10 μM, sterilized distilled water, constant volume to 50 μL, placed in a PCR instrument, denatured at 94°C for 5 min, then cycled, first at 94°C for 1 min, then at 60°C for 1 min, and finally at 72°C for 1.5 min; cycle the reaction 5 times; 72°C for 10 min, load the product for electrophoresis, cut the gel to recover the target band, and obtain the purified PCR product.

[0036] (2) Add 1 μg of PCR product, 2 μg of phagemid vector pCANTAB5E, and 2 μL of restriction endonuclease Sfi I with a concentration of 12 U / μL to the enzyme digestion buffer, add sterilized distilled water, constant volume to 50 μL, place in a PCR instrument, and perform enzyme digestion at 50°C for 4 h, cool to room temperature, centrifugal separation, recover with a DNA recovery kit, and obtain the scFv fragment vector DNA.

[0037] (3) Add 25 μL of scFv fragment vector DNA, 4 μL of 0.1% bovine serum albumin solution, 4 μL of 0.1% triton X-100 solution, and 3 μL of restriction endonuclease Not I solution with a concentration of 10 U / μL to 4 μL of enzyme digestion buffer, place in a PCR instrument, and perform enzyme digestion at 37°C for 4 h, load the product for electrophoresis, recover, and obtain the enzyme digestion product.

[0038] (4) Add 250 ng of phagemid vector pCANTAB5E, 150 ng of enzyme digestion product, and 1 μL of T4 DNA ligase with a concentration of 7 U / μL to 2.5 uL of ligation enzyme buffer, add sterilized distilled water, constant volume to 25 μL, place in a PCR instrument, and react at 16°C for 12 h, separate, and obtain the ligation product.

[0039] (5) In an ice water bath, add competent E. coli to the test tube, add the ligation product, mix well, and then ice bath for 30 min, then heat to 42℃, heat treatment for 90 s, ice water bath cooling for 1 min, then add 800 uL of YT medium with a temperature of 37℃, 37℃ in the shaking culture for 1 h, then remove 100 μL of bacteria liquid to be coated on YT-Amp agar plate, 37℃ culture for 12 h, and obtain the ligation product transformed E. coli.

[0040] (6) The ligation product transformed E. coli is inoculated in 3 mL of YT-G-Amp liquid medium containing ampicillin and glucose, the concentration of ampicillin in the medium is 100 μg / mL, and the mass fraction of glucose is 2%; 30℃ shaking culture for 12 h, after culture, the bacteria liquid is diluted in 20 mL of YT-G-Amp liquid medium containing ampicillin and glucose at a ratio of 1:100, the concentration of ampicillin in the medium is 100 μg / mL, and the mass fraction of glucose is 2%; 30℃ shaking culture until the OD 600 value is about 0.5, add helper phage M13KO7, 37℃ culture for 1 h, centrifugal separation, and add the precipitate to 200 mL of YT-Amp-Kan liquid medium containing ampicillin and kanamycin, the concentration of ampicillin in the medium is 100 μg / mL, and the concentration of kanamycin is 50 μg / mL; 30℃ shaking culture for 12 h, add 1 / 5 of the volume of the bacteria liquid containing polyethylene glycol 8000 NaCl solution, the concentration of NaCl in the solution is 2.5 mol / L, and the mass fraction of polyethylene glycol 8000 is 20%; 4℃ preservation for 30 min, centrifugal separation for 20 min, and resuspend the precipitate with 2 mL of PBS buffer to obtain a purified phage suspension.

[0041] (7) Add 2 mL of purified phage suspension and 5 μL of enterokinase to the PCR tube, mix well, and then incubate in the PCR instrument at 25℃ for 12 h, filter with a filter with a molecular weight cut-off of 5 kDa, centrifugal separation, and freeze-dry the centrifugal supernatant to obtain HAP adsorbed peptide.

[0042] (8) Add 100 g of hydroxypropyl methylcellulose to deionized water, stir, add 170 g of sorbitol, stir, add 140 g of starch, 120 g of microcrystalline cellulose, stir to disperse uniformly, and finally add 10 g of HAP adsorbed peptide, stir to dissolve uniformly, vacuum degassing, and spray the solution on the continuously rolling silicone film with a pressurized spray gun, dry, and form a oral disintegration film for promoting enamel remineralization on the surface of the silicone film.

[0043] Example 3: (1) Add 40 ng of HAP adsorbed peptide gene, 5 μL of PCR buffer, 0.6 μL of TaqPlus DNA polymerase with a concentration of 5 U / μL, 1 μL of deoxyribonucleotide triphosphate, 1 μL of E-VHback with a concentration of 10 μM, 1 μL of E-VLfor with a concentration of 10 μM, and sterilized distilled water into a PCR tube, and make up to 50 μL. Place the PCR tube in a PCR instrument, and denature at 94°C for 5 min. Then, perform a cycle reaction, in which 1 min is performed at 94°C, 1 min is performed at 60°C, and 1.5 min is performed at 72°C. Repeat the cycle reaction 5 times, and keep the temperature at 72°C for 10 min. Electrophorese the product, recover the target band by cutting the gel, and obtain a purified PCR product.

[0044] (2) Add 0.5 μg of the PCR product, 2 μg of phagemid vector pCANTAB5E, and 2 μL of restriction endonuclease Sfi I with a concentration of 12 U / μL into enzyme digestion buffer, and make up to 50 μL with sterilized distilled water. Place the mixture in a PCR instrument, and perform an enzyme digestion reaction at 50°C for 4 h. Cool to room temperature, separate by centrifugation, recover using a DNA recovery kit, and obtain a scFv fragment vector DNA.

[0045] (3) Add 25 μL of the scFv fragment vector DNA, 4 μL of 0.1% bovine serum albumin solution, 4 μL of 0.1% triton X-100 solution, and 3 μL of restriction endonuclease Not I with a concentration of 10 U / μL into 4 μL of enzyme digestion buffer, and place the mixture in a PCR instrument. Perform an enzyme digestion reaction at 37°C for 4 h. Electrophorese the product, recover, and obtain an enzyme digestion product.

[0046] (4) Add 250 ng of phagemid vector pCANTAB5E, 150 ng of the enzyme digestion product, and 1 μL of T4 DNA ligase with a concentration of 5 U / μL into 2.5 μL of ligation buffer, and make up to 25 μL with sterilized distilled water. Place the mixture in a PCR instrument, and perform a reaction at 16°C for 12 h. Separate, and obtain a ligation product.

[0047] (5) In an ice water bath, add competent E. coli to a test tube, add the ligation product, mix, and then place the test tube in the ice water bath for 30 min. Heat to 42°C, and heat treat for 90 s. Cool in the ice water bath for 2 min, add 800 μL of YT medium with a temperature of 37°C, and cultivate in the shaking incubator at 37°C for 1 h. Then, take 100 μL of the bacterial solution, and spread it on a YT-Amp agar plate. Cultivate at 37°C for 12 h, and obtain ligation product transformed E. coli.

[0048] (6) The ligator is transformed into E. coli and inoculated in 3 mL YT-G-Amp liquid medium containing ampicillin and glucose, the concentration of ampicillin in the medium is 100 μg / mL, and the mass fraction of glucose is 2%; 30℃, shake culture for 12 h, after culture, the bacterial liquid is diluted in 20 mL YT-G-Amp liquid medium containing ampicillin and glucose at a ratio of 1:100, the concentration of ampicillin in the medium is 100 μg / mL, and the mass fraction of glucose is 2%; 30℃, shake culture until the OD 600 value is about 0.5, add helper phage M13KO7, incubate at 37℃ for 1 h, centrifugal separation, add the precipitate to 200 mL YT-Amp-Kan liquid medium containing ampicillin and kanamycin, the concentration of ampicillin in the medium is 100 μg / mL, and the concentration of kanamycin is 50 μg / mL; 30℃, shake culture for 12 h, add 1 / 5 volume of NaCl solution containing polyethylene glycol 8000 to the bacterial liquid, the concentration of NaCl in the solution is 2.5 mol / L, and the mass fraction of polyethylene glycol 8000 is 20%; store at 4℃ for 30 min, centrifugal separation for 20 min, resuspend the precipitate with 2 mL PBS buffer to obtain a purified phage suspension.

[0049] (7) Add 2 mL of the purified phage suspension and 5 μL of enterokinase to a PCR tube, mix well, and incubate in a PCR instrument at 25℃ for 12 h, filter with a filter with a molecular weight cutoff of 5 kDa, centrifugal separation, freeze-dry the centrifugal supernatant to obtain HAP adsorbed peptides.

[0050] (8) Add 300 g of hydroxypropyl methylcellulose to deionized water, stir, add 100 g of sorbitol, stir, add 110 g of starch, 40 g of microcrystalline cellulose, stir and disperse uniformly, and finally add 25 g of HAP adsorbed peptides, stir to dissolve uniformly, vacuum degassing, and spray the solution on a continuously rolling silicone film with a pressurized spray gun, dry, and form a tooth enamel remineralization promoting oral disintegration film on the surface of the silicone film.

[0051] Example 4: (1) Add 130 g of hydroxypropyl methylcellulose to deionized water, stir, add 300 g of sorbitol, stir, add 60 g of starch, 90 g of microcrystalline cellulose, stir and disperse uniformly, and finally add 40 g of HAP adsorbed peptides (prepared in Example 1), stir to dissolve uniformly, vacuum degassing, and spray the solution on a continuously rolling silicone film with a pressurized spray gun, dry, and form a tooth enamel remineralization promoting oral disintegration film on the surface of the silicone film.

[0052] Example 5: (1) 240 g hydroxypropyl methylcellulose was added to deionized water, stirred, then 70 g sorbitol was added, stirred, then 80 g starch, 50 g microcrystalline cellulose was added, stirred and dispersed uniformly, and finally 60 g HAP adsorbed peptide (prepared from Example 1) was added, stirred and dissolved uniformly, vacuum degassed, and the solution was sprayed on a continuously rolling silicone film using a pressurized spray gun, and after drying, an oral disintegrating film for promoting enamel remineralization was formed on the surface of the silicone film.

[0053] Example 6: (1) 140 g hydroxypropyl methylcellulose was added to deionized water, stirred, then 180 g sorbitol was added, stirred, then 90 g starch, 70 g microcrystalline cellulose was added, stirred and dispersed uniformly, and finally 80 g HAP adsorbed peptide (prepared from Example 1) was added, stirred and dissolved uniformly, vacuum degassed, and the solution was sprayed on a continuously rolling silicone film using a pressurized spray gun, and after drying, an oral disintegrating film for promoting enamel remineralization was formed on the surface of the silicone film.

[0054] Example 7: (1) 120 g hydroxypropyl methylcellulose was added to deionized water, stirred, then 170 g sorbitol was added, stirred, then 60 g starch, 120 g microcrystalline cellulose was added, stirred and dispersed uniformly, and finally 100 g HAP adsorbed peptide (prepared from Example 1) was added, stirred and dissolved uniformly, vacuum degassed, and the solution was sprayed on a continuously rolling silicone film using a pressurized spray gun, and after drying, an oral disintegrating film for promoting enamel remineralization was formed on the surface of the silicone film.

[0055] Using bovine teeth as the original material, the crowns and roots were separated, the crowns were longitudinally sectioned, and rectangular sample pieces of 6 mm x 8 mm were prepared. Except for the enamel surface, the other surfaces were covered and protected with polymethyl methacrylate resin. The enamel surface was polished flat and polished with a sand table to obtain normal enamel samples, which were stored in a phosphate buffer solution.

[0056] The normal enamel samples were placed in an ultrasonic instrument and ultrasonically cleaned for 5 min, then the enamel samples were immersed in a 10% citric acid solution for 1 min. The acid-etched samples were rinsed with a phosphate buffer solution and ultrasonically cleaned for 5 min to obtain acid-etched enamel samples, which were stored in a phosphate buffer solution.

[0057] 1.37 g of fluorescent dye tetramethyl rhodamine isothiocyanate TRITC was added to 1 L of dimethyl sulfoxide to prepare a fluorescent dye solution. The fluorescent solution was added dropwise to the surface of the normal enamel samples and the acid-etched enamel samples, respectively, and vacuum dried for 2 h. The samples were rinsed with dimethyl sulfoxide 3 times and vacuum dried for 2 hours. The sample enamel surface was observed using a confocal scanning electron microscope.

[0058] To 1 L dimethyl sulfoxide, add 1.37 g fluorescent dye tetramethyl rhodamine isothiocyanate TRITC, add 0.44 g 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride, 0.33 g HAP adsorbed peptide (prepared by Example 1, same below), stir for 72 h, dialyze with deionized water, freeze-dry the product to obtain fluorescent dye labeled HAP adsorbed peptide.

[0059] The fluorescent dye labeled HAP adsorbed peptide is added to deionized water to prepare a 0.1% HAP adsorbed peptide fluorescent solution by stirring. The solution is added dropwise to the surface of the normal enamel sample and the acid-etched enamel sample, respectively, and vacuum dried for 2 h. The sample is washed with dimethyl sulfoxide for 3 times, and vacuum dried for 2 hours. The sample enamel surface is observed by confocal scanning electron microscopy, and the test results are shown in Figure 1 .

[0060] Figure 1 A is the surface of the normal enamel sample; B is the surface of the acid-etched enamel sample; C is the surface of the normal enamel sample treated with fluorescent dye; D is the surface of the acid-etched enamel sample treated with fluorescent dye; E is the surface of the normal enamel sample treated with fluorescent dye labeled HAP adsorbed peptide; F is the surface of the acid-etched enamel sample treated with fluorescent dye labeled HAP adsorbed peptide.

[0061] From Figure 1 It can be seen from the results that the fluorescent dye labeled HAP adsorbed peptide is distributed significantly and uniformly on the acid-etched enamel surface, while it is sporadic and unevenly distributed on the normal enamel surface. This is because the specific surface area of the acid-etched sample is larger, providing more adsorption sites for the HAP adsorbed peptide.

[0062] To 20 mL deionized water, add 0.075 g sorbic acid, 3 g xylitol, 0.015 g calcium chloride, 0.12 g potassium chloride, 0.085 g sodium chloride, 0.005 g magnesium chloride, 0.034 g dipotassium hydrogen phosphate, 0.1 g sodium benzoate, heat to 80℃, stir, then add 0.25 g hydroxypropyl methyl cellulose, stir, then add sodium hydroxide solution to adjust the pH to 5.5, add deionized water to 100 mL, heat to 100℃, stir for 45 min, cool, and filter. The filtrate is artificial saliva.

[0063] The tea polyphenol is added to deionized water, and the tea polyphenol solution is stirred. The solution is added dropwise to the surface of the acid-etched enamel sample, respectively, and vacuum dried for 2 h. The sample is washed with water for 3 times, and vacuum dried for 2 hours.

[0064] Each enamel sample is immersed in 5 mL artificial saliva at 37℃ water bath for 1 day, 7 days, and 14 days, respectively, and the artificial saliva is replaced every 24 h during the period. The enamel surface is observed by scanning electron microscopy, and the results are shown inFigure 2 and Figure 3 .

[0065] Figure 2 In the diagram, a and c represent the surfaces of normal tooth enamel samples. B and d represent the surfaces of acid-etched tooth enamel samples. Figure 2 It can be seen that the surface of acid-etched enamel becomes rougher compared to normal enamel.

[0066] Figure 3 A represents the surface of acid-etched tooth enamel samples treated with HAP-adsorbed peptides after artificial saliva remineralization (A1 is treated for 1 day, A2 for 7 days, and A3 for 14 days).

[0067] B represents the surface of the tooth enamel sample after artificial saliva remineralization treatment and tea polyphenol treatment (B1 is after 1 day of treatment, B2 is after 7 days, and B3 is after 14 days).

[0068] C represents the surface of an untreated acid-etched tooth enamel sample after artificial saliva remineralization (C1 is treated for 1 day, C2 for 7 days, and C3 for 14 days).

[0069] Depend on Figure 3 The results showed that the surface of acid-etched tooth enamel samples treated with HAP-adsorbed peptides (Group A) exhibited a greater amount of newly formed minerals after artificial saliva remineralization than Groups B and C. Furthermore, the amount of regenerated crystals increased in all samples across Groups A, B, and C with prolonged incubation time. After 14 days, the surface of the untreated group (Group C3) was covered with irregularly regenerated crystals, while the surface of the HAP-adsorbed peptide-treated sample group (Group A3) became smooth and exhibited a more ordered nanorod structure than Groups B and C.

[0070] The oral disintegrating membrane was cut into rectangular sheets of 20mm × 30mm. It was then immersed in 40mL of artificial saliva and kept at 37℃ for 15 minutes. The sheets were then magnetically stirred at 400rpm, and the time taken for the membrane to completely disintegrate was recorded.

[0071] Table 1. Disintegration time of the disintegrating film ; As shown in Table 1, the oral disintegrating membrane can disintegrate rapidly in a short period of time.

Claims

1. A method of preparing an oral disintegrating film for promoting remineralization of dental enamel, characterized in that, The preparation method comprises the following steps: adding hydroxypropyl methyl cellulose into deionized water, stirring, then adding sorbitol, stirring, then adding starch and microcrystalline cellulose, stirring and uniformly dispersing, finally adding HAP adsorption peptide, stirring and uniformly dissolving, vacuum degassing, spraying the solution on the continuously rolling silicone film by using a pressurized spray gun, and drying to form the oral disintegration film for promoting enamel remineralization on the surface of the silicone film.

2. The method for preparing an oral disintegrating film that promotes enamel remineralization according to claim 1, characterized in that, The mass ratio of the hydroxypropyl methyl cellulose, sorbitol, corn starch, microcrystalline cellulose and HAP adsorption peptide is (10-30):(10-30):(6-18):(4-12):(0.1-10).

3. The method for preparing an oral disintegrating film that promotes enamel remineralization according to claim 2, characterized in that, The preparation method of the HAP adsorption peptide comprises the following steps: (1) adding HAP adsorption peptide gene, PCR buffer, Taq Plus DNA polymerase, deoxyribonucleotide triphosphate, E-VHback, E-VLfor into a PCR tube, adding sterilized distilled water, placing in a PCR instrument, denaturing and then performing cyclic reaction, and after heat preservation, performing electrophoresis on the product, recovering the target band by cutting the gel to obtain a purified PCR product; (2) adding the PCR product, phagemid vector pCANTAB5E and restriction endonuclease Sfi I into enzyme cutting buffer, adding sterilized distilled water, placing in a PCR instrument, performing enzyme cutting reaction, cooling to room temperature, centrifugally separating, recovering by using a DNA recovery kit to obtain scFv fragment carrier DNA; (3) adding the scFv fragment carrier DNA, bovine serum albumin solution, triton X-100 solution and restriction endonuclease Not I solution into enzyme cutting buffer, placing in a PCR instrument, performing enzyme cutting reaction, performing electrophoresis on the product to recover to obtain an enzyme cutting product; (4) adding the phagemid vector pCANTAB5E, the enzyme cutting product and T4 DNA ligase into ligase buffer, adding sterilized distilled water, placing in a PCR instrument, performing ligation reaction, and after separation, obtaining a ligation product; (5) adding competent E. coli into a test tube in an ice water bath, adding the ligation product, uniformly mixing, then ice-bath standing, heat treatment, ice water bath cooling, then adding YT culture medium, and after shaking culture, removing the bacterial liquid to spread on a YT-Amp agar plate for culture to obtain ligation product transformed E. coli; (6) inoculating the ligation product transformed E. coli in YT-G-Amp liquid culture medium containing ampicillin and glucose, shaking culture, after culture, diluting the bacterial liquid in YT-G-Amp liquid culture medium containing ampicillin and glucose, shaking culture, adding helper phage M13KO7, after culture, centrifugally separating, adding the precipitate into YT-Amp-Kan liquid culture medium containing ampicillin and kanamycin, shaking culture, adding polyethylene glycol-containing NaCl solution, low-temperature preservation, centrifugally separating, resuspending the centrifugal precipitate by using PBS buffer to obtain purified phage suspension; (7) adding the purified phage suspension and enterokinase into a PCR tube, uniformly mixing, then incubating in a PCR instrument, filtering by using a filter, centrifugally separating, freeze-drying the centrifugal supernatant to obtain HAP adsorption peptide.

4. The method for preparing an oral disintegrating film that promotes enamel remineralization according to claim 3, characterized in that, The cycle reaction in the (1) is performed at 94℃ for 1 min, then at 60℃ for 1 min, and finally at 72℃ for 1.5 min; the cycle number is 5 times.

5. The method for preparing an oral disintegrating film that promotes enamel remineralization according to claim 3, characterized in that, The enzyme digestion reaction in the (2) is performed at 50℃ for 4 h.

6. The method of making an oral disintegrating film for promoting enamel remineralization according to claim 3, wherein, The enzyme digestion reaction in the (3) is performed at 37℃ for 4 h.

7. The method of making an oral disintegrating film for promoting enamel remineralization according to claim 3, wherein, The ligation reaction in the (4) is performed at 16℃ for 12 h.

8. The method for preparing an oral disintegrating film that promotes enamel remineralization according to claim 3, characterized in that, The concentration of ampicillin in the YT-G-Amp liquid medium in the (6) is 100 μg / mL, and the mass fraction of glucose is 2%; the concentration of ampicillin in the YT-Amp-Kan liquid medium is 100 μg / mL, and the concentration of kanamycin is 50 μg / mL.

9. The method of making an oral disintegrating film for promoting enamel remineralization according to claim 3, wherein, The concentration of NaCl in the NaCl solution in the (6) is 2.5 mol / L, and the mass fraction of polyethylene glycol is 20%.

10. The method of making an oral disintegrating film for promoting enamel remineralization according to claim 3, wherein, The incubation in the (7) is performed at 25℃ for 12 h.

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