Preparation method of sucrose-modified polyethylene glycol nanometer ferroferric oxide anticaries gel

Abstract

The present application relates to the technical field of caries-preventing gel, and particularly discloses a preparation method of sucrose-modified polyethylene glycol nanometer ferroferric oxide caries-preventing gel, wherein sucrose is used to modify ferroferric oxide, so that the adsorption of the material to bacteria is improved, and the killing capacity of the material to Streptococcus mutans is further improved compared with dextran material.The caries-preventing gel material has good biological safety, is not prone to drug resistance, has good acid-producing bacteria adsorption and killing effect on Streptococcus mutans; meanwhile, the material has a temperature-sensitive gel dosage form, has fluidity when stored at low temperature, forms a colloidal gel in an oral environment, can stay on the tooth surface for a long time, and effectively inhibits plaque microorganisms.

Description

Technical Field

[0001] This invention relates to the field of anti-caries gel technology, specifically a method for preparing sucrose-modified polyethylene glycol nano-ferric oxide anti-caries gel. Background Technology

[0002] Dental caries is considered a disease caused by microecological imbalance. Bacteria, such as *Streptococcus mutans*, which are acid-producing, acid-resistant, and capable of forming biofilms, accumulate and colonize on the tooth surface, leading to damage to the hard tissues of the teeth. For some individuals susceptible to caries, brushing, flossing, and rinsing alone are insufficient for effective prevention. However, the use of antibiotics to suppress the overall bacterial flora can easily lead to drug resistance and microecological imbalance. Overuse of antibiotics can cause oral-gut flora dysbiosis, easily resulting in the proliferation of superbugs and fungal infections, among other serious adverse effects.

[0003] The prior art discloses a dextran-modified topical ferumoxytol nanoparticles that disrupt biofilms and prevent tooth decay in vivo via intrinsic catalytic activity. Utilizing FDA-approved iron oxide nanoparticles—ferumoxytol—it disrupts oral biofilms and prevents tooth decay through its intrinsic peroxidase-like activity. A combination therapy using ferumoxytol nanoparticles and hydrogen peroxide (H2O2) is employed to disrupt oral biofilms and prevent tooth decay. The ferumoxytol nanoparticles can bind to the biofilm structure and generate free radicals from H2O2 under acidic conditions, leading to bacterial death and degradation of the EPS matrix.

[0004] 1: Nanoparticle preparation: FDA-approved ferumoxytol nanoparticles were used, consisting of an iron oxide core and a carboxymethyl-dextran coating.

[0005] 2: pH-dependent catalytic activity: Ferumoxytol nanoparticles exhibit high catalytic activity at acidic pH values ​​(e.g., 4.5), while their activity is minimal at near-neutral pH values ​​(e.g., 6.5).

[0006] 3: Biomembrane binding and catalysis: Ferumoxytol nanoparticles can bind to biomembranes and exhibit catalytic activity within them, generating free radicals through H2O2 to disrupt bacterial cell membranes and degrade EPS matrix.

[0007] 4. In vitro and in vivo models: The effects of ferumoxytol nanoparticles and H2O2 combined therapy on biofilm disruption and dental caries prevention were verified using in vitro biofilm models and in vivo animal models.

[0008] 5. Therapeutic application: By applying ferumoxytol nanoparticles and H2O2 topically, the development of dental caries was inhibited without causing adverse effects on the diversity of the oral microbiome or the surrounding soft tissues.

[0009] However, the existing technology has the following drawbacks: the preparation is a solution that needs to be used with hydrogen peroxide, and its residence time on the tooth surface is short, requiring repeated application, which is not conducive to long-term retention on the tooth surface. The reason for this is that the dextran-modified material has insufficient adsorption capacity for acid-producing bacteria, especially Streptococcus mutans, resulting in insufficient iron ion content on the bacterial surface based on the Fenton reaction. Therefore, hydrogen peroxide is needed to enhance the Fenton reaction. The material is in the form of drops, which does not easily remain on the tooth surface, thus requiring repeated use, making the operation cumbersome and complex.

[0010] Therefore, a method for preparing sucrose-modified polyethylene glycol nano-ferric oxide anticaries gel is provided. Summary of the Invention

[0011] The purpose of this invention is to address the deficiencies of the prior art by providing a method for preparing sucrose-modified polyethylene glycol nano-ferric oxide anticaries gel, thereby solving the problems mentioned in the background art.

[0012] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing sucrose-modified polyethylene glycol nano-ferric oxide anti-caries gel, the specific preparation method of which is as follows:

[0013] Step 1: Preparation of polyethylene glycol-nano iron oxide particles by high-temperature thermal decomposition: Weigh 15 g of polyethylene glycol and transfer it to a 50 mL three-necked flask. Heat to 80 °C until the polyethylene glycol is completely melted, then add 0.7 g of acetylacetone iron. Reflux with cooling water and maintain this temperature under nitrogen protection while stirring for 10 min. Then heat to 260 °C and react for 1 h. Stop heating and cool to 60 °C to obtain a black mixture containing the reactants and products. Wash the mixture three times with excess toluene and acetone respectively to remove unreacted polyethylene glycol and acetylacetone iron. Collect the polyethylene glycol-nano iron oxide particles with a magnet.

[0014] Step 2: Preparation of sucrose-modified polyethylene glycol-nano iron oxide particles: The polyethylene glycol-nano iron oxide particles obtained in Step 1 were dispersed in an aqueous solution, sucrose was added, and the mixture was placed in a shaker at room temperature and 400-600 rpm for 8-12 hours. The resulting black solution was then dialyzed with water using a dialysis bag with a molecular weight of 3 kDa at room temperature for 3 days to remove excess sucrose. The collected solution was the sucrose-modified polyethylene glycol-nano iron oxide aqueous solution. The aqueous solution was dried at high temperature to remove moisture, thus obtaining sucrose-modified polyethylene glycol-nano iron oxide particles.

[0015] Step 3: Prepare an aqueous solution of the particles obtained in Step 2 using sterile water at a concentration of 15-30 mg / ml, resuspend, sterilize using a 0.22 μm filter, add methylcellulose at a concentration of 10% by weight / volume, and stir at 800-1200 rpm for more than 15 minutes at room temperature to obtain sucrose-modified polyethylene glycol nano-ferric oxide anticaries gel.

[0016] Step 4: After fully dissolving, place the resulting sucrose-modified polyethylene glycol nano-ferric oxide anti-caries gel in a 4°C refrigerator for storage.

[0017] 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.

[0018] As a further technical solution of the present invention, the shaking temperature in step 2 is 20-30℃, the rotation speed is 400-600rpm, and the time is 8-12 hours.

[0019] As a further technical solution of the present invention, the mass concentration of the sucrose-modified polyethylene glycol nano-iron oxide aqueous solution in step 3 is 15-30 mg / ml.

[0020] Application of a sucrose-modified polyethylene glycol nano-ferric oxide anti-caries gel prepared by the above method in the fight against Streptococcus mutans.

[0021] A sucrose-modified polyethylene glycol nano-ferric oxide anti-caries gel, wherein the gel is polymethylcellulose.

[0022] Compared with the prior art, the beneficial effects of the present invention are: the anti-caries gel material of the present invention has good biocompatibility, is not prone to drug resistance, has good adsorption of acid-producing bacteria and killing effect on Streptococcus mutans; at the same time, the material has a temperature-sensitive gel formulation, is fluid when stored at low temperature, forms a colloid-like substance in the oral environment, can stay on the tooth surface for a long time, and effectively inhibits plaque microorganisms.

[0023] This invention modifies iron(III) oxide with sucrose, which improves the material's adsorption of bacteria and further enhances its killing ability against Streptococcus mutans compared to dextran materials.

[0024] This invention designs a temperature-sensitive gel formulation, which improves ease of use; and the gel can remain at the site of action for a long time, achieving good bactericidal effect even without the use of H2O2, thus reducing its side effects. Attached Figure Description

[0025] Figure 1 This diagram illustrates the material morphology, temperature-sensitive properties, and injectability of the present invention.

[0026] Figure 2 This is a diagram showing the adsorption capacity of common oral acid-producing bacteria according to the present invention.

[0027] Figure 3 This diagram illustrates the biofilm-killing ability of Streptococcus mutans according to the present invention. Detailed Implementation

[0028] The preferred embodiments of the present invention will now be described in detail with reference to 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 providing a clearer and more explicit definition of the scope of protection of the present invention.

[0029] Example 1: The preparation method of the sucrose-modified polyethylene glycol nano-ferric oxide anti-caries gel provided by the present invention is as follows:

[0030] Step 1: Preparation of polyethylene glycol-nano iron oxide particles by high-temperature thermal decomposition: Weigh 15 g of polyethylene glycol and transfer it to a 50 mL three-necked flask. Heat to 80 °C until the polyethylene glycol is completely melted, then add 0.7 g of acetylacetone iron. Reflux with cooling water and maintain this temperature under nitrogen protection while stirring for 10 min. Then heat to 260 °C and react for 1 h. Stop heating and cool to 60 °C to obtain a black mixture containing the reactants and products. Wash the mixture three times with excess toluene and acetone respectively to remove unreacted polyethylene glycol and acetylacetone iron. Collect the polyethylene glycol-nano iron oxide particles with a magnet.

[0031] Step 2: Preparation of sucrose-modified polyethylene glycol-nano iron oxide particles: The polyethylene glycol-nano iron oxide particles obtained in Step 1 were dispersed in an aqueous solution, sucrose was added, and the mixture was placed in a shaker at room temperature and 400-600 rpm for 8-12 hours. The resulting black solution was then dialyzed with water using a dialysis bag with a molecular weight of 3 kDa at room temperature for 3 days to remove excess sucrose. The collected solution was the sucrose-modified polyethylene glycol-nano iron oxide aqueous solution. The aqueous solution was dried at high temperature to remove moisture, thus obtaining sucrose-modified polyethylene glycol-nano iron oxide particles.

[0032] Step 3: Prepare an aqueous solution of the particles obtained in Step 2 using sterile water at a concentration of 15-30 mg / ml, resuspend, sterilize using a 0.22 μm filter, add methylcellulose at a concentration of 10% by weight / volume, and stir at 800-1200 rpm for more than 15 minutes at room temperature to obtain sucrose-modified polyethylene glycol nano-ferric oxide anticaries gel.

[0033] Step 4: After fully dissolving, place the resulting sucrose-modified polyethylene glycol nano-ferric oxide anti-caries gel in a 4°C refrigerator for storage.

[0034] like Figure 1 As shown: This sucrose-modified polyethylene glycol nano-ferric oxide anti-caries gel has a gel formulation, is injectable, and can remain on the tooth surface at room temperature to body temperature.

[0035] Example 2: The standard strains used in this experiment were *Streptococcus mutans* (UA159 & ATCC 700610), *Candida albicans* (SC5314), and *Escherichia coli* (Top10). Other strains were clinical isolates of *Streptococcus salivarius* and *Streptococcus oralis*, preserved by the National Key Laboratory for Prevention and Treatment of Oral Diseases. *Streptococcus mutans*, *Streptococcus salivarius*, and *Streptococcus oralis* were revived in BHI broth and cultured under microaerophilic conditions at 37°C with 5% CO2 for 24 h. *Escherichia coli* was revived in LB broth and cultured aerophilically at 37°C and 150 rpm for 24 h. *Candida albicans* was revived in YPD medium and cultured aerophilically at 30°C for 24 h. BHI agar was used as the solid medium for the growth of all monoclonal bacterial / fungal colonies used.

[0036] Bacterial adsorption colorimetric assay: Add 1 mL of bacterial suspension (5 × 10⁻⁶) to each EP tube. 8 Centrifuge at 4000 rpm for 4 minutes (CFU / mL), wash repeatedly, and then 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 solutions, respectively. After homogenization, incubate for 1 h or 24 h. After each incubation period, centrifuge and transfer the supernatant to a sample vial. On a white background, compare the color with 0%, 20%, 40%, 60%, 80%, and 100% concentrations of nano-ferric oxide solutions. The closest color to the 100% solution is recorded as 0, the closest as 80%, and so on, with the highest concentration (0%) recorded as 5.

[0037] like Figure 2As shown, the sucrose-modified polyethylene glycol nano-iron oxide (PEG) of this gel exhibits superior adsorption capacity for oral acid-producing bacteria such as *Streptococcus stomatitis*, *Streptococcus salivati*, and *Streptococcus mutans* compared to PEG-modified PEG-iron oxide. Specifically: after 1 hour of treatment, the adsorption capacity of sucrose-modified PEG-iron oxide for *Streptococcus stomatitis*, *Streptococcus salivati*, and *Streptococcus mutans* was 1, while the adsorption capacity of PEG-iron oxide for PEG-iron oxide was 0; after 24 hours of treatment, the adsorption capacity of sucrose-modified PEG-iron oxide for *Streptococcus stomatitis*, *Streptococcus salivati*, and *Streptococcus mutans* was 2, while the adsorption capacity of PEG-iron oxide for *Streptococcus stomatitis* and *Streptococcus salivati* was 0, and the adsorption capacity for *Streptococcus mutans* was 1.

[0038] Example 3: Combating biofilm-forming Streptococcus mutans: Streptococcus mutans bacterial culture grown to OD600≈0.5 was diluted 1:100 in BHI medium containing 1% sucrose. The resulting medium was added to 48-well plates, 475 µL per well, and incubated at 37°C and 5% CO2 for 24 h. The medium was removed, and the plate was washed three times with PBS. 200 µL of PBS gel was added to each well. Figure 3 The PBS-IONP-gel and dextran-modified polyethylene glycol nano-iron oxide gel shown are examples of this. Figure 3 The Dex-IONP-gel shown), sucrose-modified polyethylene glycol nano-iron oxide gel ( Figure 3 The Suc-IONP-gel and polyethylene glycol nano iron oxide gel shown are shown. Figure 3 The material was cultured in PEG-IONP-gel (as shown) for 24 hours. After removing the material and washing three times with PBS, bacterial colony counting was performed.

[0039] like Figure 3 As shown, this gel material, namely sucrose-modified polyethylene glycol nano-iron oxide gel, has a stronger killing effect on Streptococcus mutans in biofilms.

[0040] The above embodiments merely illustrate implementation methods of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. The application of sucrose-modified polyethylene glycol nano-ferric oxide anti-caries gel in the preparation of anti-mutagenic streptococcal products, characterized in that: The preparation method of the sucrose-modified polyethylene glycol nano-ferric oxide anti-caries gel is as follows: Step 1: Preparation of polyethylene glycol-nano iron oxide particles by high-temperature thermal decomposition: Weigh 15g of polyethylene glycol and transfer it to a 50mL three-necked flask. Heat to 80℃ until the polyethylene glycol is completely melted, then add 0.7g of acetylacetone iron. Reflux with cooling water and maintain this temperature under nitrogen protection while stirring for 10min. Then heat to 260℃ and react for 1h. Stop heating and cool to 60℃ to obtain a black mixture containing the reactants and products. Wash the mixture three times with excess toluene and acetone respectively to remove unreacted polyethylene glycol and acetylacetone iron. Collect the polyethylene glycol-nano iron oxide particles with a magnet. Step 2: Preparation of sucrose-modified polyethylene glycol-nano iron oxide particles: The polyethylene glycol-nano iron oxide particles obtained in Step 1 are dispersed in an aqueous solution, sucrose is added, and the mass concentration of the sucrose aqueous solution is 5-10 mg / ml. The solution is placed in a shaker at a temperature of 20-30℃ and a speed of 400-600 rpm for 8-12 hours. The resulting black solution is then dialyzed with water using a dialysis bag with a molecular weight of 3 kDa at room temperature for 3 days to remove excess sucrose. The collected solution is the sucrose-modified polyethylene glycol-nano iron oxide aqueous solution. The aqueous solution is dried at high temperature to remove moisture, thus obtaining sucrose-modified polyethylene glycol-nano iron oxide particles. Step 3: Prepare an aqueous solution of the particles obtained in Step 2 using sterile water at a concentration of 15-30 mg / ml, resuspend, and sterilize using a 0.22 μm filter. The mass concentration of the sucrose-modified polyethylene glycol nano-ferric oxide aqueous solution is 15-30 mg / ml. Add methylcellulose at a mass-volume ratio of 10%, and stir at 800-1200 rpm for more than 15 minutes at room temperature to obtain sucrose-modified polyethylene glycol nano-ferric oxide anti-caries gel. Step 4: After fully dissolving, place the resulting sucrose-modified polyethylene glycol nano-ferric oxide anti-caries gel in a 4°C refrigerator for storage.

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