Preparation method of sucrose modified polyethylene glycol nano ferroferric oxide anti-caries gel

Abstract

The invention relates to the technical field of anti-caries gels, and particularly discloses a preparation method of sucrose-modified polyethylene glycol nano ferroferric oxide anti-caries gels, and ferroferric oxide is modified by using sucrose, so that the adsorption of the material to bacteria is improved, and the anti-caries effect is improved. Compared with a glucan material, the killing capability on streptococcus mutans is further improved. The anti-caries gel material disclosed by the invention has good biological safety, is not easy to generate drug resistance, and has good acid-producing bacterium adsorption and killing effects on streptococcus mutans; meanwhile, the material has a temperature-sensitive gel dosage form, has fluidity during low-temperature storage, forms a colloid-like body in an oral environment, can stay on the surfaces of teeth for a long time, and effectively inhibits plaque microorganisms.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-caries gels, and specifically to a preparation method of a sucrose-modified polyethylene glycol nano-ferroferric oxide anti-caries gel. Background Art

[0002] Dental caries is considered a microecological imbalance disease. Bacteria with the ability to produce acid, tolerate acid, and form biofilms, represented by Streptococcus mutans, accumulate and colonize on the tooth surface, leading to damage to the hard tissues of the tooth. Some caries-susceptible populations still cannot effectively prevent dental caries only by brushing teeth, using dental floss, and gargling. However, using methods such as antibiotics to inhibit the overall flora is likely to cause adverse reactions such as drug resistance and microecological imbalance. The overuse of antibiotics leads to disorders of the oral-intestinal flora, easily causing serious adverse reactions such as the reproduction of super-resistant bacteria and fungal infections; A dextran-modified nano-ferroferric oxide drops (Topicalferumoxytol nanoparticles disrupt biofilms and prevent tooth decay in vivo via intrinsic catalytic activity) is disclosed in the prior art; using the iron oxide nanoparticles approved by the FDA - ferumoxytol, through its intrinsic peroxidase-like activity, to disrupt oral biofilms and prevent the occurrence of dental caries; using a combination therapy of ferumoxytol nanoparticles and hydrogen peroxide (H 2 O 2 ) to disrupt oral biofilms and prevent dental caries; Ferumoxytol nanoparticles can bind to the biofilm structure and generate free radicals from H 2 O 2 under acidic conditions, resulting in bacterial death and EPS matrix degradation.

[0003] 1: Nanoparticle preparation: Using ferumoxytol nanoparticles approved by the FDA, which consists of an iron oxide core and a carboxymethyl-dextran coating.

[0004] 2: pH-dependent catalytic activity: Ferumoxytol nanoparticles exhibit high catalytic activity at acidic pH values (such as 4.5), while having minimal activity at near-neutral pH values (such as 6.5).

[0005] 3: Biofilm binding and catalysis: Ferumoxytol nanoparticles can bind to the biofilm and exhibit catalytic activity within the biofilm, generating free radicals through H 2 O 2 to damage the bacterial cell membrane and degrade the EPS matrix.

[0006] 4: In vitro and in vivo models: The biofilm disruption and caries prevention effects of ferumoxytol nanoparticles and H 2 O 2 combination therapy were verified through in vitro biofilm models and in vivo animal models.

[0007] 5: Therapeutic applications: By topically applying ferumoxytol nanoparticles and H 2 O 2 , the development of dental caries was inhibited without adverse effects on the oral microbial community diversity or the surrounding soft tissues.

[0008] However, the disadvantages of the above existing technologies are as follows: The preparation is a solution that needs to be used in combination with hydrogen peroxide, and it has a short residence time on the tooth surface and needs to be applied repeatedly, which is not conducive to long-term residence on the tooth surface. Causes: The dextran-modified material has insufficient adsorption capacity for acid-producing bacteria, especially Streptococcus mutans, resulting in insufficient content of iron ions based on the Fenton reaction on the bacterial surface. Therefore, hydrogen peroxide is needed to enhance the Fenton reaction. The material is a drop solution and is not easy to stay on the tooth surface, so it needs to be used repeatedly, and the operation is cumbersome and complex.

[0009] Therefore, a preparation method of sucrose-modified polyethylene glycol nano-ferroferric oxide anti-caries gel is provided. Summary of the Invention

[0010] The object of the present invention is to address the deficiencies of the prior art and provide a preparation method of sucrose-modified polyethylene glycol nano-ferroferric oxide anti-caries gel to solve the problems raised in the above background technology.

[0011] To achieve the above object, the present invention provides the following technical solution: A preparation method of sucrose-modified polyethylene glycol nano-ferroferric oxide anti-caries gel, and the specific preparation method is as follows: Step 1: Preparation of polyethylene glycol-nano-ferric oxide particles by high-temperature thermal decomposition method: Weigh 15 g of polyethylene glycol and transfer it to a 50 mL three-necked flask. After heating to 80 °C until all the polyethylene glycol melts, add 0.7 g of iron acetylacetonate. Keep the condensate water refluxing and stir at this temperature for 10 min under nitrogen protection. Then heat to 260 °C and react for 1 h. Stop heating and cool to 60 °C to obtain a black mixture containing reaction raw materials and products; Wash the mixture three times with excessive toluene and acetone respectively to remove unreacted polyethylene glycol and iron acetylacetonate, and then collect the polyethylene glycol-nano-ferric oxide particles with a magnet; Step 2: Preparation of sucrose-modified polyethylene glycol-nanoferric oxide particles: Disperse the polyethylene glycol-nanoferric oxide particles obtained in Step 1 in an aqueous solution, add sucrose, place it in a shaker at room temperature, 400 - 600 rpm, and shake well for 8 - 12 hours; Dialyze the resulting black solution against water using a dialysis bag with a molecular weight cut-off of 3 KDa at room temperature for 3 days to remove excess sucrose. Collect the sucrose-modified polyethylene glycol-nanoferric oxide aqueous solution, and remove the water by high-temperature drying to obtain sucrose-modified polyethylene glycol-nanoferric oxide particles; Step 3: Prepare an aqueous solution of the particles obtained in Step 2 with a content of 15 - 30 mg / ml using sterile water, resuspend, sterilize through a 0.22 μm filter, and add methyl cellulose at a mass-volume ratio of 10%. Stir and dissolve at 800 - 1200 rpm at room temperature for more than 15 min to obtain a sucrose-modified polyethylene glycol nano-ferric oxide anti-caries gel; Step 4: After complete dissolution, store the obtained sucrose-modified polyethylene glycol nano-ferric oxide anti-caries gel in a 4°C refrigerator.

[0012] As a further technical solution of the present invention, the mass concentration of the sucrose aqueous solution in Step 2 is 5 - 10 mg / ml.

[0013] As a further technical solution of the present invention, the temperature of the shaker in Step 2 is 20 - 30°C, the rotation speed is 400 - 600 rpm, and the time is 8 - 12 hours.

[0014] As a further technical solution of the present invention, the mass concentration of the sucrose-modified polyethylene glycol nano-ferric oxide aqueous solution in Step 3 is 15 - 30 mg / ml.

[0015] Application of a sucrose-modified polyethylene glycol nano-ferric oxide anti-caries gel prepared by the above method in anti-Streptococcus mutans.

[0016] A sucrose-modified polyethylene glycol nano-ferric oxide anti-caries gel, wherein the gel is polymethyl cellulose.

[0017] Compared with the prior art, the beneficial effects of the present invention are: The anti-caries gel material of the present invention has good biosafety, is not prone to drug resistance, has good acid-producing bacteria adsorption and killing effect on Streptococcus mutans; At the same time, this material has a thermosensitive gel dosage form, has fluidity when stored at low temperature, forms a colloidal substance in the oral environment, can stay on the tooth surface for a long time, and effectively inhibits plaque microorganisms.

[0018] The present invention uses sucrose to modify ferric oxide, improving the adsorption of the material to bacteria, and further improving the killing ability against Streptococcus mutans compared with dextran materials.

[0019] The present invention designs a thermosensitive gel dosage form to improve the convenience of use; and the gel can remain at the action site for a long time, and even without using H 2 O 2 It can also achieve good bactericidal effects and reduce its side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a diagram of the material form, thermosensitive property and injectability of the present invention; Figure 2 It is a diagram of the adsorption ability of common acid-producing bacteria in the oral cavity of the present invention; Figure 3 It is a diagram of the killing ability of Streptococcus mutans in biofilms of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0021] The following elaborates on the preferred embodiments of the present invention in conjunction with the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0022] Example 1: The preparation method of the sucrose-modified polyethylene glycol nano-ferroferric oxide anti-caries gel provided by the present invention is as follows: Step 1: Preparation of polyethylene glycol-nano iron oxide particles by high-temperature thermal decomposition method: Weigh 15 g of polyethylene glycol and transfer it to a 50 mL three-necked flask. After heating to 80 °C until all the polyethylene glycol melts, add 0.7 g of iron acetylacetonate. Keep the condensate water refluxing and stir at this temperature for 10 min under nitrogen protection. Then heat to 260 °C and react for 1 h. Stop heating and cool to 60 °C to obtain a black mixture containing reaction raw materials and products; Wash the mixture three times with excessive toluene and acetone respectively to remove unreacted polyethylene glycol and iron acetylacetonate, and then collect the polyethylene glycol-nano iron oxide particles with a magnet; Step 2: Preparation of sucrose-modified polyethylene glycol-nano iron oxide particles: Disperse the polyethylene glycol-nano iron oxide particles obtained in Step 1 in an aqueous solution, add sucrose, place it in a shaker at room temperature, 400 - 600 rpm, and shake well for 8 - 12 hours; Dialyze the obtained black solution for 3 days at room temperature with a dialysis bag with a molecular weight of 3 KDa using water as the solution to remove excess sucrose, and collect it to obtain a sucrose-modified polyethylene glycol-nano iron oxide aqueous solution. Remove the water from the aqueous solution by high-temperature drying to obtain sucrose-modified polyethylene glycol-nano iron oxide particles; Step 3: The particles obtained in Step 2 are prepared into an aqueous solution with a content of 15 - 30 mg / ml using sterile water, resuspended, sterilized with a 0.22 μm filter, and methylcellulose is added at a concentration of 10% by mass - volume ratio, and stirred and dissolved at 800 - 1200 rpm at room temperature for more than 15 min to obtain a sucrose - modified polyethylene glycol nano - ferric oxide anti - caries gel; Step 4: After complete dissolution, the obtained sucrose - modified polyethylene glycol nano - ferric oxide anti - caries gel is placed in a 4°C refrigerator for storage.

[0023] As Figure 1 shown: This sucrose - modified polyethylene glycol nano - ferric oxide anti - caries gel has a gel dosage form, is injectable, and can stay on the tooth surface from room temperature to body temperature.

[0024] Example 2: The standard strains used in this experiment are Streptococcus mutans (UA159 & ATCC 700610), Candida albicans (SC5314), Escherichia coli (Top10), and other strains are Streptococcus salivarius and clinical isolates of Streptococcus oralis, which are preserved by the National Key Laboratory for Oral Disease Prevention and Control. Streptococcus mutans, Streptococcus salivarius, and Streptococcus oralis are resuscitated in BHI broth and cultured in a microaerophilic condition with 5% CO 2 in a 37°C incubator for 24 h. Escherichia coli is resuscitated in LB broth and cultured in an aerobic condition in a 37°C incubator at 150 rpm for 24 h. Candida albicans is resuscitated in YPD medium and cultured in an aerobic condition in a 30°C incubator for 24 h. BHI agar is used as a solid medium for the growth of all single - clone bacteria / fungal colonies used.

[0025] Bacterial adsorption colorimetry: Add 1 mL of bacterial suspension (5×10 8 CFU / mL) into an EP tube respectively, centrifuge at 4000 rpm for 4 minutes, and after repeated washing, add 200 μL of PBS, sucrose - modified polyethylene glycol nano - ferric oxide, dextran - modified polyethylene glycol nano - ferric oxide, and polyethylene glycol nano - ferric oxide solution respectively, blow evenly and culture for 1 h or 24 h. After reaching the corresponding time, centrifuge respectively and take out the supernatant and transfer it to an injection vial. Against a white background, colorimetry is carried out with 0%, 20%, 40%, 60%, 80%, 100% concentration nano - iron oxide solutions. The solution with the color closest to the 100% solution is recorded as 0, the closest to the 80% solution is recorded as 1, and so on, with the highest 0% recorded as 5.

[0026] As Figure 2As shown: The sucrose-modified polyethylene glycol nano-ferroferric oxide in this gel has a better adsorption capacity for oral acid-producing bacteria Streptococcus oralis, Streptococcus salivarius, and Streptococcus mutans than the dextran-modified polyethylene glycol nano-ferroferric oxide. Specifically: After acting for 1 hour, the adsorption capacities of the sucrose-modified polyethylene glycol nano-ferroferric oxide for Streptococcus oralis, Streptococcus salivarius, and Streptococcus mutans are 1, while the adsorption capacity of the dextran-modified polyethylene glycol nano-ferroferric oxide is 0; after acting for 24 hours, the adsorption capacities of the sucrose-modified polyethylene glycol nano-ferroferric oxide for Streptococcus oralis, Streptococcus salivarius, and Streptococcus mutans are 2, while the adsorption capacities of the dextran-modified polyethylene glycol nano-ferroferric oxide for Streptococcus oralis and Streptococcus salivarius are 0, and the adsorption capacity for Streptococcus mutans is 1.

[0027] Example 3: Against biofilm Streptococcus mutans: Dilute the Streptococcus mutans bacterial solution with an OD600≈0.5 to 1:100 in BHI medium containing 1% sucrose. Add the obtained medium to a 48-well plate, 475 µL per well, and culture at 37°C and 5% CO 2 for 24 h. Remove the medium and wash 3 times with PBS, and then add 200 µL of PBS gel ( Figure 3 the PBS-IONP-gel shown), dextran-modified polyethylene glycol nano-ferroferric oxide gel ( Figure 3 the Dex-IONP-gel shown), sucrose-modified polyethylene glycol nano-ferroferric oxide gel ( Figure 3 the Suc-IONP-gel shown), and polyethylene glycol nano-iron oxide gel ( Figure 3 the PEG-IONP-gel shown), and culture for 24 h. Remove the materials, wash 3 times with PBS, and then perform bacterial colony counting.

[0028] As Figure 3 shown: This gel material, namely the sucrose-modified polyethylene glycol nano-ferroferric oxide gel, has a stronger killing effect on Streptococcus mutans in the biofilm.

[0029] The above examples only illustrate the implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A method for preparing sucrose-modified polyethylene glycol nano-ferroferric oxide anti-caries gel, characterized in that: The specific preparation method is as follows: Step 1: Preparation of polyethylene glycol-nano iron oxide particles by high-temperature thermal decomposition method: 15 g polyethylene glycol was weighed and transferred to a 50 mL three-necked flask, heated to 80°C until the polyethylene glycol was completely melted, 0.7 g of acetylacetonate iron was added, condensed water was refluxed and stirred at this temperature for 10 min under nitrogen protection, then heated to 260°C for 1 h, heating was stopped, and cooled to 60°C to obtain a black mixture containing reaction raw materials and products; the mixture was washed three times with excess toluene and acetone respectively to remove unreacted polyethylene glycol and acetylacetonate iron, and then collected with a magnet to obtain polyethylene glycol-nano iron oxide particles; Step 2: Preparation of sucrose-modified polyethylene glycol-nano iron oxide particles: Disperse the polyethylene glycol-nano iron oxide particles obtained in step 1 in an aqueous solution, add sucrose, put into a shaker, shake at room temperature, 400-600 rpm, and shake for 8-12 hours; dialyze the obtained black solution with water as a solution using a dialysis bag with a molecular weight of 3 KDa at room temperature for 3 days to remove excess sucrose, and collect the sucrose-modified polyethylene glycol-nano iron oxide aqueous solution, and use high temperature baking to remove water from the aqueous solution to obtain sucrose-modified polyethylene glycol-nano iron oxide particles; Step 3: The particles obtained in step 2 are prepared into an aqueous solution at a content of 15-30 mg / ml using sterile water, resuspended, sterilized with a 0.22 μm filter, and methylcellulose is added at a concentration of 10% by mass volume ratio, and stirred and dissolved at 800-1200 rpm at room temperature for more than 15 minutes to obtain a sucrose-modified polyethylene glycol nano-iron tetroxide anti-caries gel; Step 4: After fully dissolved, the obtained sucrose-modified polyethylene glycol nano-iron tetroxide anti-caries gel is placed in a 4°C refrigerator for storage.

2. The preparation method according to claim 1, characterized in that: The mass concentration of the sucrose aqueous solution in step 2 is 5-10 mg / ml.

3. The preparation method according to claim 1, characterized in that: The shaking temperature in step 2 is 20-30°C, the rotation speed is 500 rpm, and the time is 12 hours.

4. The preparation method according to claim 1, characterized in that: The mass concentration of the sucrose-modified polyethylene glycol nano-ferroferric oxide aqueous solution in step 3 is 15-30 mg / ml.

5. Use of the sucrose-modified polyethylene glycol nano-ferroferric oxide anti-caries gel prepared by the method according to any one of claims 1 to 4 in fighting Streptococcus mutans.

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