A dynamic covalent colloidal material, its preparation method and application

Abstract

This invention discloses a dynamic covalent colloidal adhesive material, its preparation method, and its applications, belonging to the field of biomedical materials. This invention provides a novel method for constructing dynamic covalent colloidal adhesives (DCCGs) based on a reaction-induced self-assembly (RISA) strategy, and uses the prepared DCCGs for the combined treatment of periodontal inflammation and tooth demineralization. Experimental results show that the DCCGs prepared by this invention can adhere efficiently and exhibit outstanding functions in scavenging free radicals and pro-inflammatory molecules, inhibiting bacterial colonization, and promoting tooth remineralization. The DCCGs prepared by this invention can meet the needs of synergistic anti-inflammatory and promineralization treatment in oral cavity, providing a novel multifunctional integrated material for the treatment of periodontal diseases and laying the foundation for multi-disease co-treatment strategies.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical materials, in particular to a dynamic covalent colloidal hydrogel material and a preparation method and application thereof. BACKGROUND

[0002] At present, a dynamic covalent drug delivery nanonet preparation technology based on reaction-induced self-assembly has been developed, which is often used in the treatment of bacterial infectious diseases and inflammatory diseases, realizes the responsive release of active components or drugs in an acidic / high active oxygen environment, and has a targeted delivery property.

[0003] Periodontal disease is a combination of inflammation and infection of some or all tooth support structures (gingiva, cementum, periodontal ligament, alveolar bone and other tissues around the teeth). Periodontitis is the main form of periodontal disease, often combined with tooth demineralization. The dynamic covalent nanonet drug delivery system prepared by the prior art is in the form of a micellar suspension, which cannot play an interface regulation role and is difficult to be directly applied to the treatment of periodontal diseases. At present, there is no prior art that can perform combined treatment for periodontal inflammation combined with tooth demineralization.

[0004] Therefore, it is urgent to design a new dynamic covalent colloidal hydrogel structure with an interface regulation function, which can regulate local inflammatory response while synergistically promoting tooth remineralization. SUMMARY

[0005] The purpose of the present application is to provide a dynamic covalent colloidal hydrogel material and a preparation method and application thereof to solve the problems existing in the prior art. The DCCGs prepared by the present application can meet the demand for oral anti-inflammatory and remineralization synergistic treatment, provide a new multifunctional integrated material for the treatment of periodontal diseases, and lay a foundation for the multi-disease treatment strategy.

[0006] To achieve the above purpose, the present application provides the following solutions:

[0007] The present application provides a preparation method of a dynamic covalent colloidal hydrogel material, comprising the following steps:

[0008] Preparation of a polyamide-amine dendrimer mother liquor, epigallocatechin gallate mother liquor and 2-formylphenylboronic acid mother liquor, respectively;

[0009] Mixing the epigallocatechin gallate mother liquor, the 2-formylphenylboronic acid mother liquor and ultrapure water, stirring, adding dropwise the polyamide-amine dendrimer mother liquor, and performing a self-assembly reaction until a white colloidal hydrogel appears, to obtain the dynamic covalent colloidal hydrogel material.

[0010] Further, the solvents of the polyamide-amine dendrimer mother liquor and the epigallocatechin gallate mother liquor are ultrapure water.

[0011] The solvent of the 2-formylphenylboronic acid mother liquor is dimethyl sulfoxide.

[0012] Further, in the reaction system of the self-assembly reaction, the molar ratio of the polyamide-amine dendrimer, the 2-formylphenylboronic acid and the epigallocatechin gallate is 2:10:5.

[0013] The molar amount of the polyamide-amine dendrimer is calculated based on the amino groups in the polyamide-amine dendrimer.

[0014] The molar amount of the 2-formylphenylboronic acid is calculated based on the ortho-dihydroxy structure units in the 2-formylphenylboronic acid.

[0015] The molar amount of the epigallocatechin gallate is calculated based on the ortho-benzene diol structure units in the epigallocatechin gallate.

[0016] Further, the stirring speed is 2500 rpm, and the temperature of the self-assembly reaction is 25℃.

[0017] The application further provides a dynamic covalent colloidal material obtained by the preparation method.

[0018] The application further provides an application of the dynamic covalent colloidal material in preparing a medicine for treating periodontal diseases.

[0019] Further, the periodontal diseases include periodontitis and tooth demineralization.

[0020] Further, the periodontal disease is periodontitis combined with tooth demineralization.

[0021] The application further provides a medicine for treating periodontitis and / or tooth demineralization, wherein the dynamic covalent colloidal material is a main effective component.

[0022] Further, the medicine further comprises a pharmaceutically acceptable excipient.

[0023] The application discloses the following technical effects:

[0024] The application provides a method for constructing dynamic covalent colloidal gels (DCCGs) based on a reaction-induced self-assembly (RISA) strategy, and uses the prepared DCCGs for combined treatment of periodontal inflammation and tooth demineralization in the oral cavity. Experimental results show that the DCCGs prepared by the application can be efficiently adhered, and have outstanding functions in terms of removing free radicals and pro-inflammatory related molecules, inhibiting bacterial colonization and promoting tooth remineralization. The DCCGs prepared by the application can meet the needs of oral anti-inflammatory and pro-mineralization synergistic treatment, provide a new type of multifunctional integrated material for the treatment of periodontal diseases, and lay a foundation for the multi-disease treatment strategy. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0026] Figure 1 Synthetic route for preparing PFE CGs;

[0027] Figure 2 Particle size and TEM representative images at different time points in the preparation process of PFE CGs;

[0028] Figure 3 FT-IR spectra of PAMAM, EGCG, FPBA and PFE;

[0029] Figure 4 Experimental results of the influence of pH and H2O2 on the particle size of PFE;

[0030] Figure 5 Statistical graphs of the clearance rates of ABTS+free radicals (a) and DPPH free radicals (b) by different concentrations of PFE CGs;

[0031] Figure 6 Statistical graphs of the adsorption capacity of different materials on CpG and LPS; wherein a is the residual rate of CpG; b is the residual rate of LPS;

[0032] Figure 7 Quantitative statistical result graphs of the adhesion force of different materials on the surface of tooth grinding pieces;

[0033] Figure 8 Representative images of Micro-CT three-dimensional reconstruction of the upper jaw of rats after treatment with different materials and quantitative statistical graphs of gray values;

[0034] Figure 9Representative images of oral plaque staining in rats after treatment with different materials and quantitative plaque staining index; Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] Epigallocatechin gallate (EGCG), with the molecular formula C0.05 22 H 18 O 11 The CAS number is 989-51-5.

[0041] 2-Formylphenylboronic acid (2-FPBA), with the molecular formula C7H7BO3 and CAS number 40138-16-7.

[0042] Polyamide-amine dendritic polymer (PAMAM) is a nanomaterial with a three-dimensional dendritic structure, which is a colorless to pale yellow transparent liquid at room temperature; the PAMAM of this invention is an ethylenediamine core, generation 3.0; purchased from Maclean; CAS: 153891-46-4.

[0043] Example 1: Method for constructing DCCGs

[0044] In this embodiment, EGCG was selected as a polyphenol compound to construct DCCGs, and the constructed DCCGs were named PFECGs.

[0045] 10 mg PAMAM and 10 mg EGCG were dissolved in 1 mL of ultrapure water to prepare 10 mg / mL PAMAM stock solution and EGCG stock solution, respectively; 10 mg 2-FPBA was dissolved in 1 mL of dimethyl sulfoxide (DMSO) to prepare 10 mg / mL 2-FPBA stock solution. The preparation temperature was 25℃, and the solution was subjected to vortexing and ultrasonic vibration for 5 min.

[0046] Then, 725 μL of ultrapure water was taken, and 106 μL of EGCG stock solution and 70 μL of 2-FPBA stock solution were added. With magnetic stirring at 2500 rpm, 100 μL of PAMAM stock solution was added dropwise at a rate of 10 μL every 10 seconds. The reaction was carried out at 25°C for 5 min. The initial suspension stage was completed when the solution turned into transparent milky blue micelles, yielding PFE. The reaction time was extended to 30 min at 25°C until a white colloidal gel appeared, indicating the reaction was complete, yielding PFE CGs.

[0047] The synthetic route of the PFE CGs of the present invention is as follows: Figure 1 As shown.

[0048] Example 2 Material Characterization of DCCGs

[0049] The PFE CGs prepared in Example 1 were subjected to structural characterization studies.

[0050] 1. Particle size and material morphology

[0051] The detection steps are as follows: PFE / PFE CGs at different time points during the preparation process were diluted 10 times with ultrapure water, and the particle size was obtained using a nanoparticle size analyzer. The morphology of the material was then photographed using a transmission electron microscope (TEM).

[0052] Figure 2 The images show the particle size of PFE / PFE CGs at different time points during the preparation process and the corresponding TEM images. The results show that PFE / PFE CGs gradually assemble from the colloidal suspension into a three-dimensional network colloidal structure with increasing time.

[0053] 2. Chemical bond detection

[0054] The detection steps are as follows: After centrifuging PFE CGs at 5000 rpm, the precipitate was collected and freeze-dried. The freeze-dried PFE powder and PAMAM, EGCG, and 2-FPBA raw materials were subjected to Fourier transform infrared spectroscopy (FT-IR) testing.

[0055] Figure 3 The FT-IR plot of PFE shows that PAMAM is at 3271 cm⁻¹. -1 The characteristic peak values ​​of NH vibration, 2-FPBA, and EGCG at 3345 cm⁻¹ -1 Nearby OH vibration, 1673 cm -1 2-FPBA C=O vibration at 1362 cm -1 The characteristic peaks of BO vibration at 1615 cm⁻¹ all disappeared. In contrast, PFE at 1615 cm⁻¹... -1 (C=N), 1000 cm -1 (BOC) and 760 cm -1 Three new characteristic peaks were observed at (B←N), indicating the formation of imine bonds, borate ester bonds, and B←N coordination bonds in the PFE.

[0056] Example 3: Verification of the effect of DCCGs

[0057] The therapeutic efficacy of the PFE CGs prepared in Example 1 was verified.

[0058] 1. Response Release

[0059] The particle size variation of PFE was detected under different pH conditions and with or without hydrogen peroxide (H2O2).

[0060] The detection steps are as follows: PFE is added to phosphate buffer (10 mM) at pH 7.4 or 6.5, and then co-incubated with 100 μM H2O2 at 37°C. The particle size change under different conditions is detected using a nanoparticle size analyzer.

[0061] The results are as follows Figure 4 As shown, the presence of acidity and high ROS increases the particle size of PFE, proving that dynamic covalent bonds break.

[0062] 2. Free radical scavenging

[0063] The results of the determination of the ability of PFE CGs to scavenge ABTS+ and DPPH free radicals.

[0064] The detection steps are as follows: Mix equal volumes of different concentrations of PFE CGs with ABTS+ working solution, incubate in the dark, and then measure the absorbance at 734 nm using a microplate reader. Mix equal volumes of different concentrations of PFE CGs with DPPH working solution, incubate in the dark, and then measure the absorbance at 517 nm using a microplate reader.

[0065] The results are as follows Figure 5 As shown, PFE CGs possess concentration-dependent broad-spectrum free radical scavenging capabilities.

[0066] 3. Adsorption of inflammation-related molecules

[0067] The adsorption of oligonucleotides (CpG) and lipopolysaccharides (LPS) by different materials was detected.

[0068] The detection steps are as follows: Different materials were incubated with FITC-labeled CpG / LPS in the dark for 1 h, and then centrifuged at 10,000 rpm for 15 min to separate the complex bound to the materials from the free FITC-CpG / LPS. The fluorescence intensity of the supernatant was measured using an ELISA reader, which represents the unbound CpG / LPS.

[0069] The results are as follows Figure 6 As shown, PFE CGs exhibited a superior ability to adsorb inflammation-related molecules compared to other control groups.

[0070] 4. Highly efficient adhesion

[0071] The adhesion force distribution of different materials on the surface of dental grinding discs was detected.

[0072] The testing steps are as follows: different materials are dropped onto the surface of clinically obtained human extracted tooth sections, and the adhesion of the materials is examined using atomic force microscopy (AFM).

[0073] The results are as follows Figure 7 As shown, PFE CGs exhibit significantly better adhesion strength on the surface of dental radiographs than EGCG and the Mix group.

[0074] 5. Promotes remineralization

[0075] Micro-CT three-dimensional reconstruction results of rat teeth after treatment with different materials were detected.

[0076] The detection steps are as follows: The demineralized rat maxillary first molar was treated with the material for 14 consecutive days. After sacrifice, the rat maxillary teeth were photographed with Micro-CT.

[0077] The results are as follows Figure 8 As shown, the demineralized areas of the maxillary teeth of rats treated with PFE CGs appear whiter, indicating a higher degree of mineralization.

[0078] 6. Inhibits dental plaque biofilm

[0079] The detection steps are as follows: Rat teeth were ligated and bacteria were administered to establish an animal model. Different materials were used to treat the rats continuously for 14 days. Plaque indicator was used to stain the rat maxillary teeth.

[0080] Figure 9 Images of dental plaque indicator staining after treatment with different materials were obtained. The results showed that the plaque content in the oral cavity of rats was significantly reduced after PFE CGs treatment.

[0081] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method of making a dynamic covalent colloidal gel material, characterized in that, The method comprises the following steps: Preparation of polyamide-amine dendrimer mother liquor, epigallocatechin gallate mother liquor and 2-formylphenylboronic acid mother liquor respectively; Mixing, stirring, dropwise adding polyamide-amine dendrimer mother liquor, and self-assembly reaction of the epigallocatechin gallate mother liquor, the 2-formylphenylboronic acid mother liquor and ultrapure water to obtain the dynamic covalent hydrogel material.

2. The production method according to claim 1, wherein The solvent of the polyamide-amine dendrimer mother liquor and the epigallocatechin gallate mother liquor is ultrapure water; The solvent of the 2-formylphenylboronic acid mother liquor is dimethyl sulfoxide.

3. The production method according to claim 1, wherein The molar ratio of the polyamide-amine dendrimer, the 2-formylphenylboronic acid and the epigallocatechin gallate in the reaction system of the self-assembly reaction is 2:10:5; The molar amount of the polyamide-amine dendrimer is calculated based on the amino group in the polyamide-amine dendrimer; The molar amount of the 2-formylphenylboronic acid is calculated based on the ortho-dihydroxy structure unit in the 2-formylphenylboronic acid; The molar amount of the epigallocatechin gallate is calculated based on the ortho-benzene diol structure unit in the epigallocatechin gallate.

4. The production method according to claim 1, wherein The stirring speed is 2500 rpm, and the temperature of the self-assembly reaction is 25℃.

5. A dynamic covalent hydrogel material obtained by the preparation method according to any one of claims 1-4.

6. Use of a dynamic covalent hydrogel material according to claim 5 for the manufacture of a medicament for the treatment of periodontal disease. The periodontal disease is periodontitis or tooth demineralization.

7. Use of a dynamic covalent hydrogel material according to claim 5 for the manufacture of a medicament for the treatment of periodontal disease. The periodontal disease is periodontitis combined with tooth demineralization.

8. A medicament for treating periodontitis and / or tooth demineralization, characterized by, The dynamic covalent hydrogel material according to claim 5 is used as an effective component.

9. The medicament according to claim 8, wherein The pharmaceutically acceptable adjuvant is further included. The pharmaceutically acceptable adjuvant is further included.

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