ODex-PAMAM / NONOates nanogel for releasing NO based on acid response as well as preparation method and application of ODex-PAMAM / NONOates nanogel
ODex-PAMAM/NONOates nanogels that release NO in response to acid utilize enzyme-mediated endocytosis and dual acid-responsive properties to solve the problem of Streptococcus mutans biofilm penetration, achieving highly efficient caries prevention and control.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-22
- Publication Date
- 2026-03-17
AI Technical Summary
Existing technologies are unable to effectively penetrate Streptococcus mutans biofilms, resulting in poor caries prevention and treatment, and commonly used methods carry risks of drug resistance and side effects.
We developed an ODex-PAMAM/NONOates nanogel based on acid-responsive NO release. The gel matrix was formed by crosslinking partially oxidized dextran ODex with dendritic molecules PAMAM and loaded with NONOates-like NO donors. The targeting capability and dual acid-responsive properties were achieved by utilizing enzyme-mediated endocytosis.
It significantly improves the permeability of NO in Streptococcus mutans biofilm, rapidly removes biofilm, inhibits glycolysis and lactic acid production, prevents dental caries, and has a good oral hygiene maintenance effect.
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Figure CN121668092A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of dental caries prevention and treatment materials, and particularly relates to an ODex-PAMAM / NONOates nanogel based on acid-responsive NO release, its preparation method and application. Background Technology
[0002] Dental caries is one of the most common human diseases, and its pathogenesis involves complex interactions between bacteria, diet, and host factors, with dental plaque biofilm playing a crucial role. Streptococcus mutans (S. mutans) is one of the main cariogenic pathogens. It can utilize carbohydrates in food debris to synthesize insoluble glucans, thereby enhancing its adhesion to the tooth surface, gradually forming dental plaque biofilm, and promoting the aggregation of various bacteria on the tooth surface. These acid-producing bacteria ferment sugars to produce organic acids such as lactic acid, creating a highly acidic microenvironment within the biofilm, with a pH ranging from 4.5 to 5.0. When the pH falls below 5.5, the demineralization and remineralization balance of the tooth is disrupted, leading to gradual demineralization of enamel, which, over time, eventually develops into dental caries.
[0003] While mechanical cleaning, antibiotics, and fluoride are currently the most commonly used methods for preventing and treating dental caries, they have significant limitations. For mechanical cleaning, mature biofilms have extremely strong adhesion, significantly limiting the effectiveness of mechanical removal. For antibiotics, the complex three-dimensional structure of biofilms, composed of extracellular polymeric substances (EPS) and efflux pumps, forms a strong permeability barrier, making it difficult for antibiotics, such as chlorhexidine, to completely penetrate the Streptococcus mutans biofilm, potentially causing side effects such as drug resistance, mucosal irritation, and tooth staining. Regarding fluoride, although it can enhance enamel's acid resistance and reduce plaque adhesion, increasing evidence suggests that fluoride exposure is associated with fluorosis, thus requiring cautious use. These factors indicate the need to develop safer and less drug-resistant anti-biofilm strategies to achieve effective prevention and treatment of dental caries.
[0004] Nitric oxide (NO) is a crucial signaling molecule regulating cardiovascular function, metabolism, nerve conduction, and immunity, participating in various physiological processes in mammals. Furthermore, NO possesses significant broad-spectrum antibacterial activity. NO and its metabolites, such as nitrogen trioxide and peroxynitrite, can disrupt bacterial outer membranes, interfere with protein function, and cause DNA damage, ultimately leading to bacterial death and inhibiting the development of drug resistance. Notably, NO can regulate bacterial phosphodiesterase (PDE) activity, accelerating the degradation of cyclic diguanylate (c-di-GMP), thereby inhibiting EPS synthesis and promoting biofilm dissociation. Therefore, NO holds great potential in eliminating Streptococcus mutans biofilms.
[0005] However, as a gaseous molecule, NO is highly unstable and has a short half-life in physiological environments, which limits its applications. Currently, small-molecule NO donors mainly include organic nitrates, nitrosothiols (RSNO), nitrobenzenes, metal nitrosyl compounds, and azomonium glycolates (NONOates). Among these, NONOates have been extensively studied due to their ability to spontaneously release NO in aqueous solutions. In acidic environments, the biological half-life of NONOates rapidly shortens to seconds to minutes, thus quickly releasing NO and improving its therapeutic effect. Compared to small-molecule donors, macromolecular materials such as dendritic polyamide-amines (PAMAM) have higher NO loading efficiency due to their numerous primary and secondary amine sites, and also possess high modifiability, making them ideal nanomedical materials. Although PAMAM has good NO loading capacity, the passive permeation of water-soluble drugs into biofilms is severely hindered, significantly reducing its effectiveness. Therefore, it is necessary to develop a more stable and efficient material to achieve effective penetration of Streptococcus mutans biofilms. Summary of the Invention
[0006] The present invention aims to develop a more stable and efficient material to achieve effective penetration of Streptococcus mutans biofilm, providing a new treatment strategy for oral hygiene maintenance and caries prevention.
[0007] In view of this, the present invention provides an ODex-PAMAM / NONOates nanogel based on acid-responsive NO release, wherein the ODex-PAMAM / NONOates nanogel comprises a gel matrix and NONOates-like NO donors loaded in the gel matrix, wherein the gel matrix is formed by crosslinking partially oxidized dextran ODex with dendritic molecules PAMAM.
[0008] Furthermore, the PAMAM is a third-generation PAMAM.
[0009] This invention also provides a method for preparing ODex-PAMAM / NONOates nanogels based on acid-responsive NO release. The method is used to prepare the aforementioned ODex-PAMAM / NONOates nanogels based on acid-responsive NO release, and the method includes: Step S1, Synthesis of ODex: Using dextran as raw material and sodium periodate as oxidant, partially oxidized dextran ODex is prepared; Step S2, Preparation of gel matrix solution: A gel matrix solution is prepared by crosslinking PAMAM with ODex using PAMAM as a crosslinking agent; Step S3, NONOates loading: Weigh 10-15 mg sodium methoxide and dissolve it in 15-25 mL of methanol. Then add 0.8-1.3 mL of gel matrix solution with a concentration of 30-70 mg / mL. After ultrasonic dispersion, transfer the mixture to a high-pressure reactor and react at 70-90 psi NO pressure for 2-4 days. Subsequently, concentrate by ultrafiltration and centrifugation and wash with methanol 2-3 times. Store the product in methanol solution below -10℃.
[0010] Furthermore, in step S1, the synthesis process of ODex is as follows: Dissolve 0.5–2 g of dextran in 5–20 mL of ultrapure water, and add 5–15 mL of a solution with a concentration of 0.3–1 mol·L⁻¹ under light-protected conditions. -1 The sodium periodate solution was stirred for 3-5 hours, and then 0.8-1.2 mL of ethylene glycol was added to terminate the reaction. After the reaction was completed, the impurities were removed by dialysis, and the ODex was obtained by freeze-drying and stored at a low temperature below -10℃.
[0011] Furthermore, in step S1, the oxidation degree of the prepared ODex is 30-40%.
[0012] Furthermore, the oxidation degree of ODex was determined by titration with hydroxylamine hydrochloride.
[0013] Furthermore, in step S2, the preparation process of the gel matrix solution is as follows: Prepare aqueous solutions of ODex and PAMAM obtained in step S1 with a concentration of 8-12 mg / mL. Add 3-8 mL of ODex solution to 5-8 mL of PAMAM solution at a rate of 0.1-0.3 mL / min, stirring thoroughly at 1000-2000 rpm during the addition. After the addition is completed, continue stirring at 25-35℃ for 10-15 h. After the reaction is completed, centrifuge to remove free ODex and PAMAM to obtain the gel matrix solution, and store it at 1-5℃.
[0014] Furthermore, in step S2, the mass ratio of ODex to PAMAM is 1:(0.9~1.3).
[0015] Furthermore, the preparation method includes: Step S1, Synthesis of ODex: Dissolve 1g of dextran in 10mL of ultrapure water, and add 8mL of 0.5mol·L⁻¹ sodium hydroxide solution under light-protected conditions. -1 The sodium periodate solution was stirred for 4 hours, and then 1 mL of ethylene glycol was added to terminate the reaction. After the reaction was completed, the impurities were removed by dialysis, and then ODex was obtained by freeze-drying and stored at -20℃. Step S2, preparation of gel matrix solution: Prepare aqueous solutions of ODex and PAMAM obtained in step S1 with a concentration of 10 mg / mL. Add 5 mL of ODex solution to 6.5 mL of PAMAM solution at a rate of 0.2 mL / min, stirring thoroughly at 1500 rpm during the addition. After the addition is completed, continue stirring at 30℃ for 12 h. After the reaction is completed, centrifuge to remove free ODex and PAMAM to obtain the gel matrix solution, and store it at 4℃. Step S3, NONOates loading: Weigh 12.5 mg sodium methoxide and dissolve it in 19 mL of methanol. Then add 1 mL of gel matrix solution with a concentration of 50 mg / mL. After ultrasonic dispersion, transfer the mixture to a high-pressure reactor and react for 3 days at 80 psi NO pressure. Subsequently, concentrate by ultrafiltration and centrifugation and wash with methanol 2-3 times. Store the product in methanol solution at -20℃. Before use, separate by centrifugation at 15000 rpm for 10 min and dissolve in DMSO.
[0016] Another object of the present invention is to provide the application of the above-mentioned ODex-PAMAM / NONOates nanogel based on acid-responsive NO release in the preparation of dental caries prevention and treatment products.
[0017] Compared with existing technologies, the ODex-PAMAM / NONOates nanogel based on acid-responsive NO release, its preparation method, and its application described in this invention have the following advantages: 1. In the NG / NO nanogel material provided by the present invention, NO can cause nitrosylation of the thiol group of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key glycolytic enzyme of Streptococcus mutans, thereby inhibiting glycolysis and lactic acid production.
[0018] 2. In the NG / NO nanogel material provided by this invention, the partially oxidized dextran (ODex) can undergo a Schiff base reaction with the amino groups in the dendritic molecule PAMAM to form a nanogel, thereby improving the loading stability of NONOates-type NO donors and enhancing their permeability in Streptococcus mutans biofilms. More importantly, the dextran component gives the NG / NO nanogel material selective targeting capabilities, allowing it to be actively taken up by Streptococcus mutans through enzyme-mediated endocytosis, thus significantly improving its ability to penetrate biofilms.
[0019] 3. The NG / NO nanogel prepared in this invention can remain stable for a long time under normal physiological conditions. When the NG / NO nanogel is adsorbed onto dental plaque biofilm through its positive charge, the expression of glucosyltransferase genes (gtfs) in the Streptococcus mutans biofilm is upregulated, and the secretion of related transport proteins increases, thereby promoting the active uptake of NG / NO nanogel. Subsequently, the acidic environment of the biofilm triggers the rapid degradation of Schiff base bonds in the NG / NO nanogel, increasing its contact area with the biofilm and further accelerating the release of NO from acid-responsive NONOates. The released NO can penetrate the biofilm more deeply and directly kill bacteria, exhibiting dual acid-responsive characteristics. In addition, NO can promote the activity of PDEs, accelerate the degradation process of c-di-GMP, thereby reducing extracellular polysaccharide synthesis and promoting biofilm dissociation. In summary, the dextran component in the ODex-PAMAM / NONOates nanogel based on acid-responsive NO release, as described in this invention, endows the NG / NO nanogel with selective targeting capabilities. It can be actively taken up by Streptococcus mutans through enzyme-mediated endocytosis, thereby significantly enhancing its ability to penetrate biofilms. The Schiff base bond in the NG / NO nanogel and the NONOates-like NO donor give it a dual-acid-responsive NO release characteristic, releasing millimoles of NO within minutes in cariogenic environments, achieving rapid biofilm removal. Furthermore, low concentrations of NO can inhibit glycolysis and lactic acid production in Streptococcus mutans, thereby improving the acidic environment and possessing the potential to inhibit enamel demineralization and prevent caries. Therefore, the NG / NO nanogel provided by this invention has promising applications in daily oral hygiene maintenance and caries prevention. Attached Figure Description
[0020] Figure 1 This is a schematic diagram of the preparation method of ODex-PAMAM / NONOates (i.e., NG / NO) nanogel based on acid-responsive NO release as described in this invention. Figure 2 These are photographs and UV-Vis spectra of four test samples: ODex, PAMAM, NG, and NG / NO. Figure 3 These are the Fourier transform infrared spectra of four test samples: ODex, PAMAM, NG, and NG / NO. Figure 4 These are the potential values of four test samples: ODex, PAMAM, NG, and NG / NO. Figure 5 These are the particle size distribution map and polydispersity index test results for NG / NO; Figure 6 These are TEM images of NG / NO at two different magnifications; Figure 7 These are TEM images and EDS spectra of NG; Figure 8 These are TEM images and EDS spectra of NG / NO; Figure 9 These are bright-field and fluorescence images of treated Streptococcus mutans; Figure 10 These are the absorbance test results of bacterial suspensions treated with different concentrations of NG and NG / NO at a wavelength of 600 nm. Figure 11 These are the statistical results of bacterial viability after treatment with different concentrations of NG and NG / NO. Figure 12 These are the bacterial colony counts after treating bacteria with different concentrations of NG and NG / NO. Figure 13 These are images of pre-cultured mature biofilms treated with different materials; Figure 14 The results show the statistical results of biofilm content in 64-well plates after pre-cultured mature biofilms were treated with different materials. Figure 15 The results are statistical results of biofilm content after pre-cultured mature biofilms on the surface of extracted teeth treated with different materials. Figure 16 This is a statistical result of the relative activity of PDEs in Streptococcus mutans after treatment with different materials; Figure 17 The results show the statistical results of c-di-GMP concentration in the biofilm of mature Streptococcus mutans after treatment with different materials. Figure 18 These are statistical results of the total sugar concentration in the biofilm of mature Streptococcus mutans after treatment with different materials. Figure 19 These are statistical results of bacterial protein leakage concentrations after treating Streptococcus mutans with different materials. Figure 20 These are statistical results of the ATP concentration in Streptococcus mutans after treatment with different materials. Detailed Implementation
[0021] The technical solutions in this application will be clearly described below with reference to specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments in this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application are within the scope of protection of this application.
[0022] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.
[0023] Addressing the problem of severely impaired passive permeation of water-soluble drugs in biofilms, this invention constructs an ODex-PAMAM / NONOates nanogel (PAMAM-ODex / NONOates, hereinafter referred to as NG / NO) based on acid-responsive NO release. The NG / NO nanogel comprises a gel matrix and a small molecule NO donor, azomonium glycol salt (NONOates), loaded in the gel matrix. The gel matrix is formed by crosslinking partially oxidized dextran (ODex) with dendritic molecules PAMAM. The NG / NO is used for the removal of Streptococcus mutans biofilms.
[0024] Preferably, the PAMAM is a third-generation PAMAM (G3-PAMAM).
[0025] It should be noted that the third-generation PAMAM mentioned in this invention refers to a third-generation polyamide-amine dendritic macromolecule, which is a hyperbranched polymer with a precise hierarchical structure. Structurally, when ethylenediamine is used as the core, the third-generation PAMAM undergoes three rounds of branching reactions to form a third-generation structure, with 32 primary amine end groups on the surface. If other cores are used, such as triamine, the number of end groups will be adjusted accordingly, resulting in a symmetrical spherical configuration. Third-generation PAMAM exhibits low toxicity, good biocompatibility, and moderate drug loading capacity, demonstrating excellent overall performance when used to prepare NO-loaded partially oxidized dextran / PAMAM nanogels in this invention.
[0026] The NG / NO nanogel prepared in this invention remains stable under normal physiological conditions. When the NG / NO nanogel is adsorbed onto dental plaque biofilm via its positive charge, the expression of glucosyltransferase genes (gtfs) in the Streptococcus mutans biofilm is upregulated, and the secretion of related transport proteins increases, thereby promoting the active uptake of NG / NO nanogel. Subsequently, the acidic environment of the biofilm triggers the rapid degradation of Schiff base bonds in the NG / NO nanogel, increasing its contact area with the biofilm and further accelerating the release of NO from acid-responsive NONOates. The released NO can penetrate the biofilm more deeply and directly kill bacteria, exhibiting dual acid-responsive characteristics. In addition, NO can accelerate the degradation process of c-di-GMP by promoting the activity of PDEs, thereby reducing extracellular polysaccharide synthesis and promoting biofilm dissociation.
[0027] Furthermore, in the NG / NO nanogel material provided by this invention: NO can cause nitrosation of the sulfhydryl groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key glycolytic enzyme in Streptococcus mutans, thereby inhibiting glycolysis and lactic acid production. Simultaneously, dextran, a polysaccharide derived from microorganisms, is actively absorbed by Streptococcus mutans using enzymes such as glucosyltransferases (GtfB, GtfC, GtfD) and dextranase (DexA), synthesizing extracellular polysaccharides in EPS. This process enables the active uptake of dextran-containing substances by the biofilm. Moreover, dextran is an FDA-approved polymer with high biocompatibility. The partially oxidized dextran (ODex) used in this invention can undergo a Schiff base reaction with the amino groups in the dendritic molecule PAMAM to form a nanogel, thereby improving the loading stability of NONOates-like NO donors and enhancing their permeability in Streptococcus mutans biofilms. More importantly, the dextran component gives the NG / NO nanogel material selective targeting capabilities, allowing it to be actively taken up by Streptococcus mutans through enzyme-mediated endocytosis, thereby significantly enhancing its ability to penetrate biofilms.
[0028] Overall, the NG / NO nanogel material provided by this invention exhibits excellent removal effect on Streptococcus mutans biofilm, providing a new treatment strategy for oral hygiene maintenance and caries prevention.
[0029] This invention also provides a method for preparing the above-mentioned ODex-PAMAM / NONOates nanogel based on acid-responsive NO release, specifically, the preparation method includes: Step S1, Synthesis of partially oxidized dextran (ODex): Using dextran as raw material and sodium periodate as oxidant, partially oxidized dextran ODex is prepared; Step S2, Preparation of gel matrix (NG) solution: Gel matrix solution is prepared by crosslinking PAMAM with ODex using PAMAM as a crosslinking agent; Step S3, NONOates loading: Weigh 10-15 mg sodium methoxide and dissolve it in 15-25 mL of methanol, then add 0.8-1.3 mL of LNG solution (concentration of 30-70 mg / mL). After ultrasonic dispersion, transfer the mixture to a high-pressure reactor and react at 70-90 psi NO pressure for 2-4 days. Subsequently, concentrate by ultrafiltration and centrifugation and wash with methanol 2-3 times. Store the product in methanol solution below -10°C.
[0030] As some examples of the present invention, such as Figure 1 A schematic diagram of the preparation process of ODex-PAMAM / NONOates nanogel based on acid response to release NO as described in this invention is given. In the preparation process, the aldehyde group in ODex first forms a Schiff base cross-linking structure with the amino group in PAMAM, and then further loads NONOates-type NO donors to form NG / NO nanogel.
[0031] As a preferred example of the present invention, in step S1, the degree of oxidation of the partially oxidized dextran ODex is 30-40%.
[0032] As a preferred example of the present invention, in step S1, the oxidation degree of ODex is determined by titration with hydroxylamine hydrochloride.
[0033] The degree of oxidation of the partially oxidized dextran ODex should be appropriate. If the oxidation of ODex is too high, the ODex will be more toxic. However, if the oxidation of ODex is too low, it will affect the reaction yield.
[0034] As a preferred example of the present invention, the synthesis process of partially oxidized dextran ODex in step S1 is as follows: Dissolve 0.5–2 g of dextran in 5–20 mL of ultrapure water, and add 5–15 mL of a solution with a concentration of 0.3–1 mol·L⁻¹ under light-protected conditions. -1 The sodium periodate (NaIO4) solution was added and the reaction was stirred for 3-5 hours. Then, 0.8-1.2 mL of ethylene glycol was added to terminate the reaction. After the reaction was completed, the impurities were removed by dialysis, and the ODex was obtained by freeze-drying and stored at a low temperature below -10℃.
[0035] As a preferred example of the present invention, in step S1, a dialysis bag with a retention capacity of 3500 Da can be used for dialysis for 40 to 60 hours to remove impurities, followed by freeze-drying and storage at -20°C.
[0036] As a preferred example of the present invention, the preparation process of the gel matrix solution in step S2 is as follows: Prepare aqueous solutions of ODex and PAMAM obtained in step S1 with a concentration of 8-12 mg / mL. Add 3-8 mL of ODex solution to 5-8 mL of PAMAM solution at a rate of 0.1-0.3 mL / min, stirring thoroughly at 1000-2000 rpm during the addition. After the addition is completed, continue stirring at 25-35℃ for 10-15 h. After the reaction is completed, centrifuge to remove free ODex and PAMAM to obtain the gel matrix solution, and store it at 1-5℃.
[0037] As a preferred example of the present invention, in step S2, the mass ratio of ODex to PAMAM is 1:(0.9~1.3).
[0038] As some examples of the present invention, the PAMAM is a commercially available product.
[0039] As a preferred example of the present invention, in step S2, the method for removing free ODex and PAMAM by centrifugation is as follows: free ODex and PAMAM are removed using a 10kDa ultrafiltration centrifuge tube.
[0040] As a preferred example of the present invention, in step S3, before use, the prepared product is separated by centrifugation at 10,000 to 20,000 rpm for 8 to 15 minutes and dissolved in DMSO. Since DMSO has better biocompatibility than methanol, this can improve the biocompatibility of the final product.
[0041] The ODex-PAMAM / NONOates nanogel based on acid-responsive NO release provided by this invention can be used to prepare dental caries prevention and treatment products for humans and animals.
[0042] As some examples of the present invention, the ODex-PAMAM / NONOates nanogel can be diluted with water or other suitable solvents to prepare a low-concentration aqueous solution, such as 0.01~0.5mg / mL, for use as oral care products, such as mouthwashes for caries prevention, oral sprays, etc.
[0043] The following specific examples illustrate the ODex-PAMAM / NONOates nanogel based on acid-responsive NO release and its preparation method as described in this invention: First, the materials and reagents used in the following examples, comparative examples, and experimental cases will be described: G3-PAMAM was purchased from Weihai Chenyuan Molecular (China). α-1,6-glucan (10kDa) was purchased from Maclean's (China); Cy5.5-NHS ester, crystal violet, and anthrone were purchased from Aladdin Company (China). Streptococcus mutans was purchased from the American Type Culture Collection (ATCC 25175). The LIVE / DEAD™ BacLight™ bacterial activity assay kit was purchased from ThermoFisher (USA). Cell counting kit-8 (CCK8), DAF-FMDA (3-amino-4-aminomethyl-2',7'-difluorofluorescein diacetic acid), goat anti-rabbit IgG / HRP, goat anti-mouse IgG / HRP, BCA protein quantification kit, and ATP detection kit were all purchased from Beyotime (China). Griess reagent, lactate content test kit, and brain heart infusion culture medium (BHI) were purchased from Solarbio (China). The glyceraldehyde-3-phosphate assay kit was purchased from Abcam (UK). The 1,3-diphosphoglycerate rat ELISA kit was purchased from ZCIBIO (China). GAPDH monoclonal antibody and Beta-actin monoclonal antibody were purchased from proteintech (China). The cyclic diguanosine monophosphate (c-di-GMP) ELISA kit was purchased from Pantech Biotech (China). Penicillin, streptomycin, fetal bovine serum (FBS), and Duchenne modified Eagle Medium (DMEM) were purchased from Sigma-Aldrich (USA). The phosphodiesterase (PDEs) activity assay kit was purchased from GraceBiotechnology (China).
[0044] Example 1 Synthesis of NG / NO nanogels: The synthesis steps of ODex are as follows: Dissolve 1.0 g of dextran in 10 mL of ultrapure water, and add 8.0 mL of 0.5 mol·L⁻¹ solution under light-protected conditions. -1 The sodium periodate (NaIO4) solution was stirred for 4 hours; then 1.0 mL of ethylene glycol was added to terminate the reaction. The mixture was dialyzed for 48 hours using a 3500 Da cutoff dialysis bag to remove impurities. After impurity removal, ODex with an oxidation degree of 35.66% ± 2.24% was obtained. It was then lyophilized and stored at -20℃. The oxidation degree of ODex was determined by titration with hydroxylamine hydrochloride. Subsequently, a gel matrix was formed with ODex using G3-PAMAM as a crosslinking agent: First, ODex and G3-PAMAM were each prepared as 10 mg / mL aqueous solutions. Then, 5 mL of the ODex solution was added dropwise to 6.5 mL of the G3-PAMAM solution at a rate of 0.2 mL / min, with thorough stirring at 1500 rpm during the addition. After the addition was completed, stirring was continued at 30 °C for 12 h. After the reaction was completed, free ODex and PAMAM were removed using a 10 kDa ultrafiltration centrifuge tube to obtain the NG solution, which was then stored at 4 °C. Loading of NONOates: Weigh 12.5 mg sodium methoxide and dissolve it in 19 mL of methanol, then add 1 mL of concentrated NG solution (50 mg / mL), sonicate to disperse evenly, then transfer the mixture to a high-pressure reactor and react for 3 days at 80 psi NO pressure. Subsequently, concentrate by ultrafiltration and centrifugation and wash twice with methanol. The product is stored in methanol solution at -20℃. Before use, separate by centrifugation at 15000 rpm for 10 min and dissolve in DMSO.
[0045] Example 2 Synthesis of NG / NO nanogels: The synthesis steps of ODex are as follows: Dissolve 0.5 g of dextran in 5 mL of ultrapure water, and add 5.0 mL of 0.3 mol·L⁻¹ solution under light-protected conditions. -1 The sodium periodate (NaIO4) solution was stirred and reacted for 3 hours; then 0.8 mL of ethylene glycol was added to terminate the reaction. The mixture was dialyzed for 40 hours using a 3500 Da cutoff dialysis bag to remove impurities. After impurity removal, partially oxidized ODex was obtained, which was then freeze-dried and stored at -15°C. Subsequently, a gel matrix was formed with ODex using G3-PAMAM as a crosslinking agent: First, ODex and G3-PAMAM were each prepared as 8 mg / mL aqueous solutions. Then, 3 mL of the ODex solution was added dropwise to 2.7 mL of the G3-PAMAM solution at a rate of 0.3 mL / min, with thorough stirring at 1000 rpm during the addition. After the addition was completed, stirring was continued at 25 °C for 10 h. After the reaction was completed, free ODex and PAMAM were removed using a 10 kDa ultrafiltration centrifuge tube to obtain the NG solution, which was then stored at 5 °C. NONOates loading: Weigh 10.0 mg sodium methoxide and dissolve it in 15 mL of methanol, then add 0.8 mL of LNG solution (concentration 30 mg / mL), sonicate to disperse evenly, then transfer the mixture to a high-pressure reactor and react at 70 psi NO pressure for 2 days. Subsequently, concentrate by ultrafiltration and centrifugation and wash three times with methanol. The product is stored in methanol solution at -20℃. Before use, separate by centrifugation at 10000 rpm for 8 min and dissolve in DMSO.
[0046] Example 3 Synthesis of NG / NO nanogels: The synthesis steps of ODex are as follows: Dissolve 2.0 g of dextran in 20 mL of ultrapure water, and add 15.0 mL of 1.0 mol·L⁻¹ solution under light-protected conditions. -1 The sodium periodate (NaIO4) solution was stirred and reacted for 5 hours; then 1.2 mL of ethylene glycol was added to terminate the reaction. The mixture was dialyzed for 55 hours using a dialysis bag with a 3500 Da cutoff to remove impurities. After impurity removal, partially oxidized ODex was obtained, which was then lyophilized and stored at -20°C. Subsequently, a gel matrix was formed with ODex using G3-PAMAM as a crosslinking agent: First, ODex and G3-PAMAM were prepared into 12 mg / mL aqueous solutions. Then, 8 mL of ODex solution was added dropwise to 8.0 mL of G3-PAMAM solution at a rate of 0.3 mL / min. During the addition, the mixture was stirred thoroughly at 2000 rpm. After the addition was completed, the mixture was stirred at 35 °C for 15 h. After the reaction was completed, free ODex and PAMAM were removed using a 10 kDa ultrafiltration centrifuge tube to obtain an NG solution, which was then stored at 1 °C. NONOates loading: Weigh 15.0 mg sodium methoxide and dissolve it in 25 mL of methanol, then add 1.3 mL of concentrated NG solution (70 mg / mL), sonicate to disperse evenly, then transfer the mixture to a high-pressure reactor and react at 90 psi NO pressure for 4 days. Subsequently, concentrate by ultrafiltration and centrifugation and wash three times with methanol. The product is stored in methanol solution at -20℃. Before use, separate by centrifugation at 20000 rpm for 15 min and dissolve in DMSO.
[0047] Comparative Example 1 Synthesis of PAMAM / NO: Weigh 12.5 mg sodium methoxide and dissolve it in 19 mL of methanol. Then add 50 mg G3-PAMAM and ultrasonically disperse it evenly. Then transfer the mixture into a high-pressure reactor and react it under 80 psi NO pressure for 3 days. Subsequently, concentrate it by ultrafiltration and centrifugation and wash it twice with methanol. Store the product in methanol solution at -20℃. Before use, separate it by centrifugation at 15000 rpm for 10 min and dissolve it in DMSO.
[0048] Experimental Example 1: ODex, PAMAM, NG, and NG / NO from Example 1 were used as test samples and characterized using a UV-Vis spectrophotometer and FTIR. Particle size and zeta potential were determined using dynamic light scattering (DLS), and the three-dimensional structure was analyzed by transmission electron microscopy (TEM) combined with energy dispersive spectroscopy (EDS).
[0049] The results obtained through testing are as follows: Figures 2-8 As shown, where: Figure 2 These are photographs and UV-Vis spectra of four test samples: ODex, PAMAM, NG, and NG / NO. Figure 2 It can be observed that NG and NG / NO exhibit a maximum absorption peak at 315 nm, which is consistent with the characteristic absorption of Schiff bases (C=N). Furthermore, since C=N is a chromophore, aqueous solutions of NG and NG / NO exhibit a yellow characteristic.
[0050] Figure 3 The Fourier transform infrared (FTIR) spectra of four test samples—ODex, PAMAM, NG, and NG / NO—further validated the composition of NG / NO. The FTIR spectra show a value at 1628.11 cm⁻¹. -1 The absorption peak corresponds to the stretching vibration of the Schiff base C=N, indicating that the Schiff base cross-linked structure was successfully constructed. (1269.90 cm⁻¹) -1 The absorption peak is a characteristic peak of NONOates, indicating that NO has been successfully loaded into the nanogel.
[0051] Figure 4 The results show the potential values of four test samples: ODex, PAMAM, NG, and NG / NO. The zeta potential test results show that NG / NO has a high positive charge, which is beneficial to its stability in aqueous solution and enhances its adsorption to bacterial biofilm.
[0052] Figure 5 The results show the particle size distribution and polydispersity index (PDI) of NG / NO. The particle size analysis results show that NG / NO has a moderate particle size and a small PDI, which helps it to penetrate bacterial biofilms.
[0053] Figure 6 These are TEM images of NG / NO at two different magnifications. The TEM detection results show that NG / NO has a regular spherical structure.
[0054] Figure 7 These are TEM images and EDS spectra of NG, among which... Figure 7 The first vertical column shows TEM images of NG at two different magnifications. The top image in the second column shows the EDS energy spectrum of nitrogen in NG, and the bottom image in the second column shows the EDS energy spectrum of oxygen in NG. The top image in the third column shows the overlay of dark-field imaging of NG with carbon, nitrogen, and oxygen, and the bottom image in the third column shows the signal intensity of carbon, nitrogen, and oxygen in NG.
[0055] Figure 8 These are TEM images and EDS spectra of NG / NO, where... Figure 8The first vertical column shows TEM images of NG / NO at two different magnifications. The top image in the second column is the EDS energy spectrum of nitrogen in NG / NO, and the bottom image in the second column is the EDS energy spectrum of oxygen in NG / NO. The top image in the third column is the overlay image of dark-field imaging of NG / NO with carbon, nitrogen, and oxygen elements, and the bottom image in the third column is the signal intensity of carbon, nitrogen, and oxygen elements in NG / NO.
[0056] according to Figure 7 and 8 As can be seen from the TEM images, both NG and NG / NO exhibit regular spherical structures. The EDS analysis results show that the nitrogen-oxygen ratio in NG indicates a PAMAM to ODex monomer mass ratio of approximately 1:7.05, meaning PAMAM accounts for approximately 82.85% of the total mass. In summary, this invention successfully prepared Schiff base-crosslinked nanogels and successfully loaded NO.
[0057] Experimental Example 2: In vitro antibacterial activity test of NG / NO Test method: The DAF-FMDA fluorescent probe was used to detect whether NO released from NG / NO entered the bacteria. 1 μL DAF-FMDA (5 mM) was added to 1 mL of Streptococcus mutans suspension (10... 8 The bacteria were incubated at 37°C for 15 min with 2.5 μL of LNG / NO (1 mg / mL) for another 2 h. Fluorescence signals inside the bacteria were observed using a fluorescence microscope. The minimum inhibitory concentration (MIC) was determined using the broth dilution method: NG or NG / NO was diluted in BHI broth and mixed with an equal volume of Streptococcus mutans suspension (2 × 10⁵ CFU / mL). The mixture was incubated at 37°C for 24 h, and the OD₆₀ was measured. The minimum bactericidal concentration (MBC) was determined using the colony counting method: the bacterial suspension after the MIC experiment was serially diluted 10²-10⁵ times, and 5 μL of each solution was plated on agar plates for colony counting.
[0058] Test results: In vitro antibacterial activity test such as Figure 9-12 As shown, where, Figure 9 Bright-field and fluorescence images of processed Streptococcus mutans, Figure 9 The first two images in the horizontal row are bright-field micrographs of Streptococcus mutans after treatment with NG and NG / NO, respectively. The two images in the second horizontal row are fluorescence images of DAF-FMDA added after the experimental material was treated with the bacteria. Strong fluorescence signals were observed inside the bacteria after NG / NO treatment using the DAF-FMDA probe, indicating that the released NO can penetrate the bacteria and exert its effect.
[0059] Figure 10The results are obtained by measuring the absorbance of bacterial suspensions at a wavelength of 600 nm after dilution with broth and treatment with different concentrations of NG and NG / NO. Figure 11 These are the statistical results of bacterial viability after treatment with different concentrations of NG and NG / NO. Figure 12 These are the bacterial colony counts after treating bacteria with different concentrations of NG and NG / NO.
[0060] In vitro antibacterial activity tests showed that NG / NO has significant antibacterial ability, which stems from its high positive charge and the synergistic effect of NO.
[0061] Experimental Example 3: NG / NO Biofilm Scavenging Activity Test Test method: After preparing mature biofilms in 96-well plates, the membranes were treated with NG / NO (32 μg / mL) for 12 h, washed with PBS, and stained with 0.1% crystal violet. After dissolving the dye in 100 μL of ethanol, the absorbance was measured at 570 nm.
[0062] Tooth samples were obtained from Pingyang Hospital Affiliated to Wenzhou Medical University (with ethical approval). After sterilization, the samples were placed in PBS. The teeth were then immersed in a solution containing Streptococcus mutans (10 μg / mL). 6 Biofilms were formed by culturing in BHI + 1% sucrose medium (CFU / mL) for 24 h. After washing with PBS, NG / NO (32 μg / mL) was added and treated for 12 h. After crystal violet staining, the solution was dissolved in 1.5 mL of ethanol, and the absorbance at 595 nm was measured.
[0063] To investigate the biofilm dissociation mechanism, mature biofilms were treated with NG / NO (4 μg / mL) for 4 h. Bacteria were collected, and c-di-GMP levels were measured using ELISA, while extracellular polysaccharides were detected using the sulfuric acid-anthrone method. PDEs activity was detected using a kit: after treatment, bacteria were collected, lysed, and reacted with reagents for 15 min, and OD was measured. 405 To verify whether NO induces oxidative stress, bacteria were isolated after treating the biofilm with NG / NO (32 μg / mL), and cytoplasmic protein leakage was measured using BCA assay; ATP content was measured using an ATP kit.
[0064] Test results: The results of the NG / NO biofilm scavenging activity test are as follows: Figure 13-20 As shown, where, Figure 13 Image A shows images of mature biofilms pre-cultured in 64-well plates after treatment with different materials (stained with crystal violet), and image B shows images of mature biofilms pre-cultured on the surface of extracted teeth after treatment with different materials (stained with crystal violet). Figure 14 The results show the statistical results of biofilm content in 64-well plates after pre-cultured mature biofilms were treated with different materials. Figure 15The results are statistical results of biofilm content after pre-cultured mature biofilms on the surface of extracted teeth treated with different materials. Figure 16 This is a statistical result of the relative activity of PDEs in Streptococcus mutans after treatment with different materials; Figure 17 The results show the statistical results of c-di-GMP concentration in the biofilm of mature Streptococcus mutans after treatment with different materials. Figure 18 These are statistical results of the total sugar concentration in the biofilm of mature Streptococcus mutans after treatment with different materials. Figure 19 These are statistical results of bacterial protein leakage concentrations after treating Streptococcus mutans with different materials. Figure 20 These are statistical results of the ATP concentration in Streptococcus mutans after treatment with different materials.
[0065] Based on the biofilm removal activity test results of NG / NO, it can be concluded that compared with other groups, NG / NO can achieve a biofilm removal rate of >80% on the 64-well plate and crown surface. Although NG can penetrate the biofilm, its bactericidal effect is insufficient; although PAMAM / NO can kill bacteria, it is difficult to penetrate; only NG / NO simultaneously satisfies the requirements of "penetration + sterilization", and has the strongest effect.
[0066] Meanwhile, mechanistic studies showed that PDEs activity significantly increased after NG / NO treatment (see details). Figure 16 The level of c-di-GMP in biofilms decreased significantly (see details). Figure 17 ); Extracellular polysaccharides in biomembranes are significantly reduced (see details) Figure 18 This indicates that NG / NO accelerates biomembrane disintegration by reducing c-di-GMP and decreasing polysaccharide synthesis.
[0067] Further testing ( Figures 19-20 The results showed that bacterial cytoplasmic protein leakage was significantly increased and ATP content was significantly decreased in the NG / NO treatment group. However, considering that the low dose did not cause a large number of bacterial deaths, it can be concluded that the main role of NG / NO is to induce oxidative stress in biofilm bacteria, thereby promoting biofilm dissociation, rather than directly killing large numbers of bacteria.
[0068] In summary, the dextran component in the ODex-PAMAM / NONOates nanogel based on acid-responsive NO release, as described in this invention, endows the NG / NO nanogel with selective targeting capabilities. It can be actively taken up by Streptococcus mutans through enzyme-mediated endocytosis, thereby significantly enhancing its ability to penetrate biofilms (>70% penetration depth in just 4 hours). The Schiff base bonds and NONOates-like NO donors in the NG / NO nanogel give it dual-acid-responsive NO release characteristics, enabling the release of millimoles of NO within minutes in cariogenic environments (pH 4.5–5.5), achieving rapid biofilm removal (>80% biomass reduction).
[0069] Furthermore, low concentrations of NO can inhibit glycolysis and lactic acid production in Streptococcus mutans, thereby improving the acidic environment and possessing the potential to inhibit enamel demineralization and prevent dental caries. In summary, the NG / NO nanogel provided by this invention has promising application prospects in daily oral hygiene maintenance and dental caries prevention. The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application is not limited to the specific embodiments described above; the specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art, under the guidance of this application, can make many other forms without departing from the spirit and scope of the claims, all of which fall within the protection scope of this application.
Claims
1. An ODex-PAMAM / NONOates nanogel based on acid-responsive release of NO, characterized in that, The ODex-PAMAM / NONOates nanogel comprises a gel matrix and NONOates NO donors loaded in the gel matrix, and the gel matrix is formed by cross-linking partially oxidized dextran ODex and dendrimer PAMAM.
2. The ODex-PAMAM / NONOates nanogel based on acid-responsive release of NO according to claim 1, characterized in that, The PAMAM is a third-generation PAMAM.
3. A method for preparing an ODex-PAMAM / NONOates nanogel based on acid-responsive release of NO, characterized in that, The preparation method for preparing the ODex-PAMAM / NONOates nanogel based on acid-responsive release of NO according to any one of claims 1-2 comprises the following steps: Step S1, synthesis of ODex: using dextran as a raw material and sodium periodate as an oxidant, partially oxidized dextran ODex is prepared; Step S2, preparation of a gel matrix solution: using PAMAM as a cross-linking agent, a gel matrix solution is prepared by cross-linking with ODex; Step S3, loading of NONOates: 10-15 mg of sodium methoxide is dissolved in 15-25 mL of methanol, and then 0.8-1.3 mL of the gel matrix solution with a concentration of 30-70 mg / mL is added, and the mixture is uniformly dispersed by ultrasonic dispersion, and then the mixture is transferred into a high-pressure reaction kettle, and reacted at a NO gas pressure of 70-90 psi for 2-4 days, and then concentrated by ultrafiltration centrifugation and washed with methanol for 2-3 times, and the product is stored in the form of a methanol solution below -10℃.
4. The method for preparing ODex-PAMAM / NONOates nanogel based on acid- responsive release of NO according to claim 3, characterized in that, In step S1, the synthesis process of ODex is as follows: Dextrans of 0.5-2 g were dissolved in 5-20 mL of ultrapure water, 5-15 mL of 0.3-1 mol·L -1 of sodium periodate solution was added under light shielding conditions, and the reaction was stirred for 3-5 h. Then, 0.8-1.2 mL of ethylene glycol was added to terminate the reaction. After the reaction was completed, impurities were removed by dialysis, and ODex was obtained by freeze-drying and stored at a temperature below -10°C.
5. The method for preparing the ODex-PAMAM / NONOates nanogel based on acid- responsive release of NO according to claim 3 or 4, characterized in that, In step S1, the oxidation degree of the prepared ODex is 30-40%.
6. The method for preparing ODex-PAMAM / NONOates nanogel based on acid- responsive release of NO according to claim 5, characterized in that, The oxidation degree of ODex is determined by hydroxylamine hydrochloride titration.
7. The method for preparing ODex-PAMAM / NONOates nanogel based on acid- responsive release of NO according to claim 3, characterized in that, In step S2, the preparation process of the gel matrix solution is as follows: In step S1, the ODex and PAMAM are respectively prepared into aqueous solutions with a concentration of 8-12 mg / mL, 3-8 mL of the ODex solution is added dropwise into 5-8 mL of the PAMAM solution at a speed of 0.1-0.3 mL / min, and the stirring is fully carried out at 1000-2000 rpm during the dropwise addition; after the dropwise addition is completed, the stirring is continued at 25-35℃ for 10-15 h, and then the free ODex and PAMAM are removed by centrifugation to obtain the gel matrix solution, which is stored at 1-5℃.
8. The method for preparing ODex-PAMAM / NONOates nanogel based on acid- responsive release of NO according to claim 7, characterized in that, In step S2, the mass ratio of the ODex to the PAMAM is 1: (0.9-1.3).
9. The method for preparing ODex-PAMAM / NONOates nanogel based on acid- responsive release of NO according to claim 3, characterized in that, The preparation method comprises: Step S1, synthesis of ODex: 1 g of dextran was dissolved in 10 mL of ultrapure water, 8 mL of sodium periodate solution with a concentration of 0.5 mol·L -1 was added under light shielding conditions, and the reaction was stirred for 4 h, then 1 mL of ethylene glycol was added to terminate the reaction. After the reaction was completed, impurities were removed by dialysis, and ODex was obtained by freeze-drying and stored at -20 °C. Step S2, preparation of a gel matrix solution: the ODex and PAMAM prepared in step S1 are respectively prepared into aqueous solutions with a concentration of 10 mg / mL, 5 mL of the ODex solution is added dropwise into 6.5 mL of the PAMAM solution at a speed of 0.2 mL / min, and the stirring is fully carried out at 1500 rpm during the dropwise addition; after the dropwise addition is completed, the stirring is continued at 30℃ for 12 h, and then the free ODex and PAMAM are removed by centrifugation to obtain the gel matrix solution, which is stored at 4℃; Step S3, loading of NONOates: 12.5 mg of sodium methoxide was dissolved in 19 mL of methanol, and then 1 mL of the gel matrix solution with a concentration of 50 mg / mL was added. After ultrasonic dispersion, the mixture was transferred into a high-pressure reactor, and reacted under 80 psi of NO pressure for 3 days. Subsequently, the product was concentrated by ultrafiltration centrifugation and washed with methanol for 2-3 times. The product was stored in methanol solution at -20℃, and before use, was separated by centrifugation at 15000 rpm for 10 min and dissolved in DMSO.
10. Use of the acid-responsive NO-releasing ODex-PAMAM / NONOates nanogel according to any one of claims 1-2 in the preparation of a product for preventing and treating dental caries.