PH responsive nano-composite, and preparation method and application thereof

Abstract

The invention discloses a pH responsive nano-composite as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The invention discloses a preparation method of a pH responsive nanocomposite. The preparation method comprises the following steps: S1, taking N-(2-ethoxyl) acrylamide, PPTS and allyl vinyl ether as raw materials, and reacting to generate AEEAA; s2, carrying out a reaction on the PAMAM and AEAA (Acetyl Ether Acrylic Acid) to generate PAMAM-acetal-ene; s3, benzoin diethyl ether, cysteamine hydrochloride and the PAMAM-acetal-ene are subjected to a reaction, and PAMAM-acetal-NH2 is synthesized; and S4, carrying out mixing reaction on the PAMAM-aceto-NH2 and the VDQACs in water, so as to synthesize the pH (Potential of Hydrogen) responsive nano composite. The invention also discloses the pH-responsive nano-complex prepared by the method and application of the pH-responsive nano-complex in preparation of drugs for preventing or / and treating caries. The nano-composite disclosed by the invention responds to pH change to release an antibacterial drug VDQACs, and pathogenic bacteria are killed; and meanwhile, the nano-composite also has an unexpected remineralization induction function.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a pH-responsive nanocomposite, a preparation method and an application thereof. Background Art

[0002] Dental caries is a disease of chronic damage to dental hard tissues mediated by dental plaque biofilm. It is a global public health problem and has caused a serious medical and economic burden. Microorganisms in dental plaque biofilm can produce excessive organic acids through glycolysis, forming an acidic microenvironment, causing a continuous decrease in local pH, triggering demineralization of dental hard tissues and developing into dental caries. Therefore, antibacterial treatment is an important part of dental caries prevention and treatment.

[0003] The progression of dental caries is a dynamic process characterized by alternating demineralization and remineralization. In the early stages of dental caries, this process can be prevented or reversed by promoting remineralization. Therefore, dental caries prevention and treatment drugs need to take into account both antibacterial and remineralization functions of dental tissues.

[0004] Most of the existing anti-caries drugs are antibacterial agents, but the long-term effects of antibacterial agents can induce the production of drug-resistant bacteria, and at the same time indiscriminately kill oral microorganisms, causing microecological imbalance. The development of antibacterial complexes with high biosafety and low drug resistance induction is one of the key points. In addition, "smart" drugs that respond to pH fluctuations, which are the most critical for the occurrence and development of caries, to achieve antibacterial effects are another key to solving the above problems. Therefore, providing a pH-responsive antibacterial and remineralizing drug has become a problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0005] One of the objectives of the present invention is to provide a method for preparing a pH-responsive nanocomposite.

[0006] The second object of the present invention is to provide a pH-responsive nanocomposite prepared by the preparation method.

[0007] The third object of the present invention is to provide applications of the pH-responsive nanocomposite.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] The present invention discloses a method for preparing a pH-responsive nanocomposite, comprising the following steps:

[0010] S1. Preparation of the precursor product N-(2-(1-(allyloxy)ethoxy)ethyl)acrylamide of acetal bond: using N-(2-hydroxyethyl)acrylamide, PPTS and allyl vinyl ether as raw materials, reacting to generate the precursor product N-(2-(1-(allyloxy)ethoxy)ethyl)acrylamide with acetal bond, referred to as AEEAA;

[0011] S2. Synthesis of PAMAM molecules with acetal bonds and olefin terminal groups: reacting the third-generation PAMAM with AEEAA obtained in step S1 to generate PAMAM molecules with acetal bonds and olefin terminal groups, referred to as PAMAM-acetal-ene;

[0012] S3. Synthesis of a PAMAM molecule with an acetal bond and an amino terminal group: reacting benzoin diethyl ether and cysteamine hydrochloride with the PAMAM-acetal-ene prepared in step S2 to construct an amino group at the end of the PAMAM-acetal-ene to synthesize a PAMAM molecule with an acetal bond and an amino terminal group, referred to as PAMAM-acetal-NH2;

[0013] S4. Synthesis of pH-responsive nanocomplex: PAMAM-acetal-NH2 and VDQACs were mixed and reacted in water to synthesize pH-responsive nanocomplex VDQACs@PAMAM-acetal-NH2.

[0014] In some embodiments of the present invention, in step S1, the molar ratio of N-(2-hydroxyethyl)acrylamide, PPTS and allyl vinyl ether is 10-14:0.8-1.2:8-12, preferably 12:1:10;

[0015] In step S2, the molar ratio of AEEAA to PAMAM is 150-200:1, preferably 180:1;

[0016] In step S3, the molar ratio of PAMAM-acetal-ene, benzoin diethyl ether and cysteamine hydrochloride is 1.5-6:1-4:200-600, preferably 3:2:420;

[0017] In step S4, the mass ratio of VDQACs to PAMAM-acetal-NH2 is 0.8-1.2:1.5-2.5; preferably 1:2.

[0018] In some embodiments of the present invention, in step S1, N-(2-hydroxyethyl)acrylamide and PPTS are added to a first solvent under a protective gas atmosphere, stirred at low temperature, and then an allyl vinyl ether solution is added dropwise to the solution to react;

[0019] Preferably, the solvent in the allyl vinyl ether solution is a first solvent;

[0020] Preferably, the first solvent comprises dichloromethane;

[0021] Preferably, stirring is performed at 0°C for 5 to 120 min, more preferably for 30 min;

[0022] Preferably, the reaction is carried out at room temperature;

[0023] Preferably, after the reaction, the mixture is filtered, and the filtrate is concentrated under reduced pressure to obtain the precursor product AEEAA with an acetal bond.

[0024] In some embodiments of the present invention, in step S2, under a protective gas atmosphere, the third generation PAMAM and AEEAA are placed in a container, a second solvent is added, stirred, and reacted;

[0025] Preferably, the second solvent comprises a methanol aqueous solution, and further preferably, the volume ratio of methanol to water in the methanol aqueous solution is 6 to 8:2 to 4, more preferably 7:3;

[0026] Preferably, the reaction temperature is 60 to 80°C, more preferably 70°C;

[0027] Preferably, the reaction mixture is dialyzed in deionized water using a dialysis bag, and then the dialyzed product is freeze-dried to obtain PAMAM-acetal-ene.

[0028] In some embodiments of the present invention, in step S3, benzoin diethyl ether, cysteamine hydrochloride and PAMAM-acetal-ene prepared in step S2 are reacted under ultraviolet light to synthesize PAMAM-acetal-NH2;

[0029] Preferably, benzoin diethyl ether, cysteamine hydrochloride and the PAMAM-acetal-ene prepared in step S2 are dissolved in a third solvent and then irradiated under an ultraviolet lamp;

[0030] Further preferably, the third solvent comprises methanol;

[0031] Preferably, irradiating under ultraviolet light with stirring for 2 to 8 hours, preferably 4 hours;

[0032] Preferably, the reaction mixture is dialyzed in deionized water using a dialysis bag, and then the dialyzed product is freeze-dried to obtain PAMAM-acetal-ene.

[0033] In some embodiments of the present invention, in step S4, PAMAM-acetal-NH2 and VDQACs are mixed and reacted in water at room temperature.

[0034] The pH-responsive nanocomposite disclosed by the invention is prepared by the above method.

[0035] The application of the pH-responsive nanocomposite disclosed in the present invention is application in the preparation of drugs for preventing and / or treating dental caries.

[0036] The VDQACs of the present invention are a series of novel quaternary ammonium salts based on vitamin B6 derivatives (VitaminB6derivatives based quaternary ammonium compounds, VDQACs), and their structures are as follows:

[0037]

[0038] VDQACs can be metabolized in the body by carboxylesterases that are widely present in tissues and organs such as serum, gastric juice, liver, small intestine, and colon. The metabolic end products have no other biological activity and are non-toxic, with high biosafety, avoiding long-term interaction between quaternary ammonium salts and bacteria in the body. Therefore, the possibility of inducing bacterial resistance is low.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The invention is scientifically designed and ingeniously conceived. The invention creatively designs a nano-carrier loaded with VDQACs with pH-responsive antibacterial and remineralization properties, thereby achieving the purpose of "intelligent" dental caries prevention. When pathogenic bacteria overgrow and the local pH decreases, the nano-composite of the invention responds to pH changes to release the antibacterial drug VDQACs to kill the pathogenic bacteria; at the same time, the nano-composite also has an unexpected remineralization induction function. The invention acts on the two key links of dental caries prevention and treatment, which is conducive to the efficient prevention and treatment of dental caries.

[0041] The Chinese names corresponding to the English abbreviations in this invention are:

[0042] PPTS: Pyridinium p-toluenesulfonate

[0043] DCM: dichloromethane

[0044] PAMAM: Polyamidoamine dendrimer BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Attached Figure 1 The hydrogen nuclear magnetic resonance spectra of PAMAM (G3-NH2), PAMAM-acetal-en (G3-acetal-en), and PAMAM-acetal-NH2 (G3-acetal-NH2) of the present invention;

[0046] Attached Figure 2 The structure and binding energy diagram of the nanocomposite V@PA of the present invention; Figure A is a molecular simulation model of V@PA, and Figure B is a calculation model of the binding energy between the carrier PAMAM-acetal and the loaded drug VDQACs monomer molecule. The results show that the binding energy between the two is -8.85 eV, indicating the theoretical feasibility of the synthesis of the nanocomposite;

[0047] Attached Figure 3 Figure 1 is the result of the investigation of the antibacterial effect of VDQACs, where Figure A is the growth curve of the cariogenic bacteria Streptococcus mutans UA159 (S.mutans UA159) under co-culture with different concentrations of VDQACs, and Figure B is the composition and proportion of salivary biofilm bacteria detected by 16S RNA sequencing; Figure C is the abundance and diversity analysis of the microbial community, and the Chao Index analysis suggests that VDQACs can increase the abundance of oral microbial communities, and the Alpha diversity OTU index (OTUs Index) confirms that species diversity has also been improved;

[0048] Attached Figure 4 The transmission electron microscopy images of the nanocomposite V@PA of the present invention under different pH conditions;

[0049] Attached Figure 5 The graphs show the results of investigating the antibacterial effect of the nanocomposite V@PA of the present invention under different pH conditions and the remineralization effect in an in vitro caries model; wherein, Graph A shows the results of investigating the antibacterial effect; and Graph B shows the results of investigating the remineralization effect. DETAILED DESCRIPTION

[0050] In order to further illustrate the present invention, the nanocomposite provided by the present invention and its application are described in detail below in conjunction with the embodiments.

[0051] Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0052] Example 1

[0053] This embodiment discloses a method for preparing the pH-responsive antibacterial and remineralizing dental caries prevention and treatment nanocomposite of the present invention, specifically:

[0054] S1. Preparation of acetal bond precursor N-(2-(1-(allyloxy)ethoxy)ethyl)acrylamide

[0055] Under nitrogen protection, N-(2-hydroxyethyl)acrylamide (24mmol) and PPTS (2mmol) were dissolved in 20mL of anhydrous DCM and stirred at 0°C for 30min; anhydrous DCM (10mL) containing allyl vinyl ether (20mmol) was slowly added dropwise to the mixture, and the reaction was stirred overnight at room temperature, and then an excess of K2CO3 was added to quench the reaction. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the precursor product N-(2-(1-(allyloxy)ethoxy)ethyl)acrylamide with an acetal bond, the English name of which is N-(2-(1-(allyloxy)ethoxy)ethyl)-acrylamide, referred to as AEEAA;

[0056] S2. Synthesis of PAMAM molecules with acetal bonds and olefin terminal groups

[0057] Under nitrogen protection, PAMAM dendrimer G3-NH2 (75 μmol) and AEEAA (2.7 g, 13.5 mmol) were dissolved in 10 mL methanol / water (v / v=7:3) and stirred at 70°C for 48 h. The mixture was then dialyzed in deionized water using a dialysis bag (molecular cutoff 1 kDa), with the water changed every 6 h, multiple times, and freeze-dried to obtain a viscous liquid to synthesize a PAMAM molecule with an acetal bond and an olefin terminal group, referred to as PAMAM-acetal-ene, about 450 mg.

[0058] S3. Synthesis of PAMAM molecules with acetal bonds and amino terminal groups

[0059] Benzoin diethyl ether and cysteamine hydrochloride are reacted with PAMAM-acetal-ene to construct an amino group at the end of PAMAM-acetal-ene to synthesize a PAMAM molecule with an acetal bond and an amino terminal group: PAMAM-acetal-NH2, as follows:

[0060] PAMAM-acetal-ene (60 μmol), DMPA (40 μmol) and cysteine ​​hydrochloride (8.4 mmol) were mixed in 40 mL of methanol. The reaction mixture was stirred at room temperature for 4 hours under irradiation with a 365 nm ultraviolet lamp, and then dialyzed in deionized water using a dialysis bag (molecular cutoff 1 kDa) and freeze-dried to obtain a light yellow powder of a PAMAM molecule with an acetal bond and an amino terminal group: PAMAM-acetal-NH2, about 300 mg.

[0061] The H NMR spectra of PAMAM(G3-NH2), PAMAM-acetal-en(G3-acetal-en), and PAMAM-acetal-NH2(G3-acetal-NH2) are shown in the attached figure. Figure 1 As shown in the figure, it can be seen that the present invention successfully synthesized PAMAM-acetal-NH2.

[0062] S4. Synthesis of pH-responsive nanocomposites

[0063] VDQACs and PAMAM-acetal-NH2 were mixed in deionized water at a mass ratio of 1:2 to synthesize pH-responsive VDQACs@PAMAM-acetal-NH2 (V@PA).

[0064] The structure and binding energy of the nanocomposite V@PA are shown in the attached Figure 2 As shown in the figure, the two VDQACs monomers can stably bind to the carrier PAMAM with binding energies of -8.89ev and -8.85ev.

[0065] Example 2

[0066] This embodiment discloses a method for preparing the pH-responsive antibacterial and remineralizing dental caries prevention and treatment nanocomposite of the present invention, specifically:

[0067] S1. Preparation of acetal bond precursor N-(2-(1-(allyloxy)ethoxy)ethyl)acrylamide

[0068] Under nitrogen protection, N-(2-hydroxyethyl)acrylamide (20mmol) and PPTS (2.4mmol) were dissolved in 20mL of anhydrous DCM and stirred at 0°C for 120min; anhydrous DCM (10mL) containing allyl vinyl ether (20mmol) was slowly added dropwise to the mixture, and the reaction was stirred overnight at room temperature, and then an excess of K2CO3 was added to quench the reaction. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the precursor product N-(2-(1-(allyloxy)ethoxy)ethyl)acrylamide with an acetal bond, the English name of which is N-(2-(1-(allyloxy)ethoxy)ethyl)-acrylamide, referred to as AEEAA;

[0069] S2. Synthesis of PAMAM molecules with acetal bonds and olefin terminal groups

[0070] Under nitrogen protection, PAMAM dendrimer G3-NH2 (75 μmol) and AEEAA (2.25 g, 11.25 mmol) were dissolved in 10 mL methanol / water (v / v=7:3) and stirred at 70°C for 48 h. The mixture was then dialyzed in deionized water using a dialysis bag (molecular cutoff 1 kDa), with the water changed every 6 h, multiple times, and freeze-dried to obtain a viscous liquid to synthesize a PAMAM molecule with an acetal bond and an olefin terminal group, referred to as PAMAM-acetal-ene, about 450 mg.

[0071] S3. Synthesis of PAMAM molecules with acetal bonds and amino terminal groups

[0072] Benzoin diethyl ether and cysteamine hydrochloride are reacted with PAMAM-acetal-ene to construct an amino group at the end of PAMAM-acetal-ene to synthesize a PAMAM molecule with an acetal bond and an amino terminal group: PAMAM-acetal-NH2, as follows:

[0073] PAMAM-acetal-ene (30 μmol), DMPA (20 μmol) and cysteine ​​hydrochloride (12 mmol) were mixed in 40 mL of methanol. The reaction mixture was stirred at room temperature for 4 hours under irradiation with a 365 nm ultraviolet lamp, and then dialyzed in deionized water using a dialysis bag (molecular cutoff 1 kDa) and freeze-dried to obtain a light yellow powder of a PAMAM molecule with an acetal bond and an amino terminal group: PAMAM-acetal-NH2, about 300 mg.

[0074] S4. Synthesis of pH-responsive nanocomposites

[0075] VDQACs and PAMAM-acetal-NH2 were mixed in deionized water at a mass ratio of 0.8:2 to synthesize pH-responsive VDQACs@PAMAM-acetal-NH2 (V@PA).

[0076] Example 3

[0077] This embodiment discloses a method for preparing the pH-responsive antibacterial and remineralizing dental caries prevention and treatment nanocomposite of the present invention, specifically:

[0078] S1. Preparation of acetal bond precursor N-(2-(1-(allyloxy)ethoxy)ethyl)acrylamide

[0079] Under nitrogen protection, N-(2-hydroxyethyl)acrylamide (28mmol) and PPTS (1.6mmol) were dissolved in 20mL of anhydrous DCM and stirred at 0°C for 120min; anhydrous DCM (10mL) containing allyl vinyl ether (24mmol) was slowly added dropwise to the mixture, and the reaction was stirred overnight at room temperature, and then an excess of K2CO3 was added to quench the reaction. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the precursor product N-(2-(1-(allyloxy)ethoxy)ethyl)acrylamide with an acetal bond, the English name of which is N-(2-(1-(allyloxy)ethoxy)ethyl)-acrylamide, referred to as AEEAA;

[0080] S2. Synthesis of PAMAM molecules with acetal bonds and olefin terminal groups

[0081] Under nitrogen protection, PAMAM dendrimer G3-NH2 (75 μmol) and AEEAA (3.0 g, 15.0 mmol) were dissolved in 10 mL methanol / water (v / v=7:3) and stirred at 70°C for 48 h. The mixture was then dialyzed in deionized water using a dialysis bag (molecular cutoff 1 kDa), with the water changed every 6 h, multiple times, and freeze-dried to obtain a viscous liquid to synthesize a PAMAM molecule with an acetal bond and an olefin terminal group, referred to as PAMAM-acetal-ene, about 450 mg.

[0082] S3. Synthesis of PAMAM molecules with acetal bonds and amino terminal groups

[0083] Benzoin diethyl ether and cysteamine hydrochloride are reacted with PAMAM-acetal-ene to construct an amino group at the end of PAMAM-acetal-ene to synthesize a PAMAM molecule with an acetal bond and an amino terminal group: PAMAM-acetal-NH2, as follows:

[0084] PAMAM-acetal-ene (120 μmol), DMPA (60 μmol) and cysteine ​​hydrochloride (4.0 mmol) were mixed in 40 mL of methanol. The reaction mixture was stirred at room temperature for 4 hours under irradiation with a 365 nm ultraviolet lamp, and then dialyzed in deionized water using a dialysis bag (molecular cutoff 1 kDa) and freeze-dried to obtain a light yellow powder of a PAMAM molecule with an acetal bond and an amino terminal group: PAMAM-acetal-NH2, about 300 mg.

[0085] S4. Synthesis of pH-responsive nanocomposites

[0086] VDQACs and PAMAM-acetal-NH2 were mixed in deionized water at a mass ratio of 1.2:1.5 to synthesize pH-responsive VDQACs@PAMAM-acetal-NH2 (V@PA).

[0087] Example 4

[0088] This embodiment discloses a method for preparing the pH-responsive antibacterial and remineralizing dental caries prevention and treatment nanocomposite of the present invention, specifically:

[0089] S1. Preparation of acetal bond precursor N-(2-(1-(allyloxy)ethoxy)ethyl)acrylamide

[0090] Under nitrogen protection, N-(2-hydroxyethyl)acrylamide (24mmol) and PPTS (2.4mmol) were dissolved in 20mL of anhydrous DCM and stirred at 0°C for 120min; anhydrous DCM (10mL) containing allyl vinyl ether (16mmol) was slowly added dropwise to the mixture, and the reaction was stirred overnight at room temperature, and then an excess of K2CO3 was added to quench the reaction. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the precursor product N-(2-(1-(allyloxy)ethoxy)ethyl)acrylamide with an acetal bond, the English name of which is N-(2-(1-(allyloxy)ethoxy)ethyl)-acrylamide, referred to as AEEAA;

[0091] S2. Synthesis of PAMAM molecules with acetal bonds and olefin terminal groups

[0092] Under nitrogen protection, PAMAM dendrimer G3-NH2 (75 μmol) and AEEAA (2.4 g, 12.0 mmol) were dissolved in 10 mL methanol / water (v / v=7:3) and stirred at 70°C for 48 h. The mixture was then dialyzed in deionized water using a dialysis bag (molecular cutoff 1 kDa), with the water changed every 6 h, multiple changes of water, and freeze-dried to obtain a viscous liquid to synthesize a PAMAM molecule with an acetal bond and an olefin terminal group, referred to as PAMAM-acetal-ene, about 450 mg.

[0093] S3. Synthesis of PAMAM molecules with acetal bonds and amino terminal groups

[0094] Benzoin diethyl ether and cysteamine hydrochloride are reacted with PAMAM-acetal-ene to construct an amino group at the end of PAMAM-acetal-ene to synthesize a PAMAM molecule with an acetal bond and an amino terminal group: PAMAM-acetal-NH2, as follows:

[0095] PAMAM-acetal-ene (90 μmol), DMPA (80 μmol) and cysteine ​​hydrochloride (10.0 mmol) were mixed in 40 mL of methanol. The reaction mixture was stirred at room temperature for 4 hours under irradiation with a 365 nm ultraviolet lamp, and then dialyzed in deionized water using a dialysis bag (molecular cutoff 1 kDa) and freeze-dried to obtain a light yellow powder of a PAMAM molecule with an acetal bond and an amino terminal group: PAMAM-acetal-NH2, about 300 mg.

[0096] S4. Synthesis of pH-responsive nanocomposites

[0097] VDQACs and PAMAM-acetal-NH2 were mixed in deionized water at a mass ratio of 0.8:2.5 to synthesize pH-responsive VDQACs@PAMAM-acetal-NH2 (V@PA).

[0098] Test Example 1

[0099] This test example discloses the investigation of the antibacterial effect of VDQACs. The VDQACs in this test are prior art, and their synthesis is carried out according to the method described in the document "Design, synthesis, antibacterial activity and toxicity of novel quaternaryammonium compounds based on pyridoxine and fatty acids" (Sapozhnikov SV, Sabirova AE, Shtyrlin NV, et al.. European Journal of Medicinal Chemistry, 2020, 211: 113100. DOI: 10.1016 / j.ejmech.2020.113100.).

[0100] This test example investigated the antibacterial effect of VDQACs on S. mutans UA159. The specific steps are as follows:

[0101] 1. In a 96-well plate, add a final concentration of 1 × 10 6 S. mutans UA159 was cultured with 100 μL of medium containing different concentrations of VDQACs for 24 h, and the blank control group (control) used BHI medium. Starting from the culture time of 0 h, the absorbance of the bacterial solution at 600 nm was measured with an ELISA instrument, and the measurement was performed every 1 h until 24 h to obtain the bacterial growth curve.

[0102] 2. Oral biofilms derived from saliva were cultured under anaerobic conditions for 48 h. The biofilms were treated with VDQACs and then cultured for another 24 h. 16S RNA sequencing was used to detect the proportion of biofilm bacterial species and related parameters of bacterial community composition.

[0103] The results are attached Figure 3 As shown in the figure, VDQACs can effectively inhibit the growth of cariogenic bacteria S. mutans UA 159, improve the balance of oral microecology, increase the biodiversity of oral biofilm, and reduce the proportion of cariogenic bacteria.

[0104] Test Example 2

[0105] In this test example, the nanocomposite V@PA was characterized under different pH conditions and its pH-responsive antibacterial effect was detected.

[0106] 1. Characterization of the nanocomposite V@PA. The molecular morphology of the nanocomposite was observed using a transmission electron microscope in a neutral environment (pH=7.0) and an acidic environment (pH=5.5), which is the critical pH for the occurrence of dental caries.

[0107] The results are attached Figure 4 As shown in the figure, the molecular morphology of V@PA is different in different environments. In a neutral environment, the complex presents a relatively compact molecular structure, while in an acidic environment it becomes looser, which is conducive to the release of loaded drugs.

[0108] 2. pH-responsive antibacterial effect detection

[0109] In a 96-well plate, BHI medium with pH = 5.5 and 7.0 was prepared, and the final concentration was 1×10 6 S. mutans UA159 was cultured with 100 μL of medium containing VDQACs for 24 h, and the blank control group was cultured with BHI medium of corresponding pH value. Starting from the culture time of 0 h, the absorbance of the bacterial solution at 600 nm was measured with an ELISA instrument, and the measurement was performed every 1 h until 24 h to obtain the bacterial growth curve.

[0110] The results are attached Figure 5 As shown in Figure A. As can be seen from the figure, V@PA exhibits better antibacterial effect in an acidic environment, thus confirming its pH-responsive antibacterial effect.

[0111] Test Example 3

[0112] This test example discloses the investigation of the remineralization effect of the nanocomposite V@PA in an in vitro dental caries model and compares it with the empty carrier PAMAM before modification. The details are as follows:

[0113] 1. Enamel demineralization treatment: 37% phosphoric acid etching for 60 seconds, DDW ultrasonic washing three times, 2 minutes each time, natural drying, and standby. Cover the rest of the sample with nail polish, leaving a 3×2mm window.

[0114] 2. pH cycle: 2 hours of demineralization and 22 hours of remineralization incubation shaker (37°C, 50rpm / min), 1 hour before and 1 hour after demineralization, that is, at 9:00 am and 17:00 pm, 100μL of deionized water, PAMAM and nanocomposite V@PA treatment solution were dropped on the exposed surface of demineralized enamel for 15 minutes to adsorb the experimental reagents onto the sample. The entire pH cycle time was 12 days, and all solutions were freshly replaced daily.

[0115] The results are attached Figure 5 As shown in Figure B. As can be seen from the figure, both V@PA and PAMAM can effectively induce the formation of remineralization crystals, have the same remineralization induction effect as , and have good remineralization effect.

[0116] In the field of tooth tissue remineralization research, PAMAM is recognized to have a good remineralization effect. The V@PA of the present invention has the same remineralization induction effect as PAMAM. In addition, the V@PA of the present invention also has a pH-responsive antibacterial effect. The present invention realizes pH-responsive antibacterial and remineralization induction effects for the first time.

[0117] The above embodiments are only used to help understand the method and core idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a pH-responsive nanocomposite, characterized in that: The steps include: S1. Preparation of the precursor product N-(2-(1-(allyloxy)ethoxy)ethyl)acrylamide of acetal bond: using N-(2-hydroxyethyl)acrylamide, PPTS and allyl vinyl ether as raw materials, reacting to generate the precursor product N-(2-(1-(allyloxy)ethoxy)ethyl)acrylamide with acetal bond, referred to as AEEAA; S2. Synthesis of PAMAM molecules with acetal bonds and olefin terminal groups: reacting the third-generation PAMAM with AEEAA obtained in step S1 to generate PAMAM molecules with acetal bonds and olefin terminal groups, referred to as PAMAM-acetal-ene; S3. Synthesis of a PAMAM molecule with an acetal bond and an amino terminal group: reacting benzoin diethyl ether and cysteamine hydrochloride with the PAMAM-acetal-ene obtained in step S2 to construct an amino group at the end of the PAMAM-acetal-ene to synthesize a PAMAM molecule with an acetal bond and an amino terminal group, referred to as PAMAM-acetal-NH2; S4. Synthesis of nanocomplex VDQACs@PAMAM-acetal-NH2: PAMAM-acetal-NH2 and VDQACs were mixed and reacted in water to synthesize pH-responsive nanocomplex VDQACs@PAMAM-acetal-NH2.

2. The method for preparing a pH-responsive nanocomposite according to claim 1, characterized in that: In step S1, the molar ratio of N-(2-hydroxyethyl)acrylamide, PPTS and allyl vinyl ether is 10-14:0.8-1.2:8-12, preferably 12:1:10; In step S2, the molar ratio of AEEAA to PAMAM is 150-200:1, preferably 180:1; In step S3, the molar ratio of PAMAM-acetal-ene, benzoin diethyl ether and cysteamine hydrochloride is 1.5-6:1-4:200-600, preferably 3:2:420; In step S4, the mass ratio of VDQACs to PAMAM-acetal-NH2 is 0.8-1.2:1.5-2.5; preferably 1:

2.

3. The method for preparing a pH-responsive nanocomposite according to claim 1 or 2, characterized in that: In step S1, under a protective gas atmosphere, N-(2-hydroxyethyl)acrylamide and PPTS are added to a first solvent, stirred at low temperature, and then an allyl vinyl ether solution is added dropwise to the solution to react; Preferably, the solvent in the allyl vinyl ether solution is a first solvent; Preferably, the first solvent comprises dichloromethane; Preferably, stirring is performed at 0°C for 5 to 120 min, more preferably for 30 min; Preferably, the reaction is carried out at room temperature; Preferably, after the reaction, the mixture is filtered, and the filtrate is concentrated under reduced pressure to obtain the precursor product AEEAA with an acetal bond.

4. The method for preparing a pH-responsive nanocomposite according to claim 1 or 2, characterized in that: In step S2, PAMAM and AEEAA are placed in a container under a protective gas atmosphere, a second solvent is added, and the mixture is stirred and reacted; Preferably, the second solvent comprises a methanol aqueous solution, and further preferably, the volume ratio of methanol to water in the methanol aqueous solution is 6 to 8:2 to 4, more preferably 7:3; Preferably, the reaction temperature is 60 to 80°C, more preferably 70°C; Preferably, the reaction mixture is dialyzed in deionized water using a dialysis bag, and then the dialyzed product is freeze-dried to obtain PAMAM-acetal-ene.

5. The method for preparing a pH-responsive nanocomposite according to claim 1 or 2, characterized in that: In step S3, benzoin diethyl ether, cysteamine hydrochloride and PAMAM-acetal-ene prepared in step S2 are reacted under ultraviolet light to synthesize PAMAM-acetal-NH2; Preferably, benzoin diethyl ether, cysteamine hydrochloride and the PAMAM-acetal-ene prepared in step S2 are dissolved in a third solvent and then irradiated under an ultraviolet lamp; Further preferably, the third solvent comprises methanol; Preferably, irradiating under ultraviolet light with stirring for 2 to 8 hours, preferably 4 hours; Preferably, the reaction mixture is dialyzed in deionized water using a dialysis bag, and then the dialyzed product is freeze-dried to obtain PAMAM-acetal-ene.

6. The method for preparing a pH-responsive nanocomposite according to claim 1 or 2, characterized in that: In step S4, PAMAM-acetal-NH2 and VDQACs are mixed and reacted in water at room temperature.

7. A pH-responsive nanocomposite, characterized in that: The method is prepared by the method described in any one of claims 1 to 6.

8. The use of the pH-responsive nanocomposite according to claim 7, characterized in that: Application in the preparation of medicines for preventing and / or treating dental caries.

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