pH-Responsive Nanocomposites, Their Preparation Methods and Applications

Abstract

The present invention discloses a pH-responsive nanocomplex, a preparation method thereof and applications, belonging to the technical field of biomedicine. The preparation method of the pH-responsive nanocomplex disclosed by the present invention includes: S1. Using N-(2-hydroxyethyl) acrylamide, PPTS and allyl vinyl ether as raw materials to react to generate AEEAA; S2. Reacting PAMAM with AEEAA to generate PAMAM-acetal-ene; S3. Reacting benzoin diethyl ether, cysteamine hydrochloride with PAMAM-acetal-ene to synthesize PAMAM-acetal-NH2; S4. Mixing and reacting PAMAM-acetal-NH2 with VDQACs in water to synthesize a pH-responsive nanocomplex. The present invention also discloses the pH-responsive nanocomplex prepared by this method and its application in the preparation of drugs for preventing and / or treating dental caries. The nanocomplex of the present invention releases the antibacterial drug VDQACs in response to pH changes to kill pathogenic bacteria; at the same time, the nanocomplex also has an unexpected remineralization induction function.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a pH-responsive nanocomplex, a preparation method thereof, and an application thereof. Background Art

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

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

[0004] Existing anti-caries drugs are mostly antibacterial agents. However, the long-term use of antibacterial agents will induce the generation of drug-resistant bacteria, and at the same time, the indiscriminate killing of oral microorganisms will cause microecological imbalance. Developing an antibacterial complex with high biosafety and low drug resistance induction is one of the key points. In addition, a "smart" drug that responds to the most critical pH fluctuations during the occurrence and development of dental caries to achieve antibacterial effects is another key to solving the above problems. Therefore, providing a drug with pH-responsive antibacterial and remineralization functions has become an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a preparation method of a pH-responsive nanocomplex.

[0006] Another purpose of the present invention is to provide a pH-responsive nanocomplex prepared by the above preparation method.

[0007] The third purpose of the present invention is to provide an application of the pH-responsive nanocomplex.

[0008] To achieve the above purposes, the technical solutions adopted by the present invention are as follows:

[0009] A preparation method of a pH-responsive nanocomplex disclosed by the present invention includes the following steps:

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

[0011] S2. Synthesize PAMAM molecules with acetal bonds and olefin end groups: React the third-generation PAMAM with AEEAA prepared in step S1 to generate PAMAM molecules with acetal bonds and olefin end groups, abbreviated as PAMAM-acetal-ene;

[0012] S3. Synthesize PAMAM molecules with acetal bonds and amino end groups: React benzoin diethyl ether, cysteamine hydrochloride with PAMAM-acetal-ene prepared in step S2 to construct an amino group at the end of PAMAM-acetal-ene, and synthesize PAMAM molecules with acetal bonds and amino end groups, abbreviated as PAMAM-acetal-NH2;

[0013] S4. Synthesize pH-responsive nanocomposites: Mix and react PAMAM-acetal-NH2 with VDQACs in water to synthesize pH-responsive nanocomposites 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, under a protective gas atmosphere, add N-(2-hydroxyethyl) acrylamide and PPTS to the first solvent, stir at low temperature, and then dropwise add the allyl vinyl ether solution to this solution for reaction;

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

[0020] Preferably, the first solvent includes dichloromethane;

[0021] Preferably, stir at 0 °C for 5-120 min, more preferably 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, and the mixture is stirred and reacted;

[0025] Preferably, the second solvent includes an aqueous methanol solution. Further preferably, the volume ratio of methanol to water in the aqueous methanol solution is 6 - 8:2 - 4, and more preferably 7:3;

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

[0027] Preferably, the reaction mixture is dialyzed using a dialysis bag in deionized water, 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 the PAMAM-acetal-ene prepared in step S2 are reacted under ultraviolet light irradiation 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 includes methanol;

[0031] Preferably, under stirring conditions, it is irradiated under an ultraviolet lamp for 2 - 8 h, preferably 4 h;

[0032] Preferably, the reaction mixture is dialyzed using a dialysis bag in deionized water, 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] A pH-responsive nanocomplex disclosed by the present invention is prepared by the above method.

[0035] The application of the above pH-responsive nanocomplex disclosed by the present invention is the 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] In vivo, VDQACs can be metabolized by carboxylesterase widely present in tissues and organs such as serum, gastric juice, liver, small intestine, and colon. The end metabolites have no other biological activities and are non-toxic, with high biological safety, avoiding the long-term interaction between quaternary ammonium salts and bacteria in the body. Therefore, the possibility of inducing bacterial drug resistance is relatively low.

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

[0040] The present invention is scientifically designed and ingeniously conceived. The present invention creatively designs a nano-carrier loaded with VDQACs with pH-responsive antibacterial and remineralization functions, so as to achieve the purpose of "intelligent" caries prevention. When pathogenic bacteria overgrow and the local pH decreases, the nano-complex of the present invention responds to the pH change and releases the antibacterial drug VDQACs to kill pathogenic bacteria; at the same time, the nano-complex also has an unexpected remineralization induction function. The present invention acts on two key links in the prevention and treatment of caries, which is beneficial to the efficient prevention and treatment of caries.

[0041] The Chinese names corresponding to the English abbreviations in the present invention are as follows:

[0042] PPTS: Pyridinium p-toluenesulfonate

[0043] DCM: Dichloromethane

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

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

[0046] Att Figure 2 is the structure and binding energy diagram of the nano-complex V@P-A of the present invention; among them, Figure A is the molecular simulation model of V@P-A, and Figure B is the binding energy calculation model between the carrier PAMAM-acetal and the monomer molecule of the loaded drug VDQACs. The result shows that the binding energy between the two is -8.85ev, indicating the theoretical feasibility of the synthesis of the nano-complex;

[0047] Appendix Figure 3 It is the inspection result graph of the antibacterial effect of VDQACs. Among them, Figure A is the growth curve of cariogenic bacteria Streptococcus mutans UA159 (S. mutans UA159) co-cultured with VDQACs at different concentrations. Figure B is the detection of the composition and proportion of salivary biofilm strains by 16S RNA sequencing. Figure C is the analysis of the abundance and diversity of the microbial community. The analysis of the total number of species index (Chao Index) indicates that VDQACs can increase the community abundance of oral microorganisms, and the Alpha diversity OTU index (OTUs Index) confirms that the species diversity has also been improved.

[0048] Appendix Figure 4 It is the transmission electron microscope image of the nano-complex V@P-A of the present invention under different pH conditions;

[0049] Appendix Figure 5 It is the inspection result graph of the antibacterial effect and remineralization effect of the nano-complex V@P-A of the present invention under different pH conditions in an in vitro caries model. Among them, Figure A is the inspection result graph of the antibacterial effect, and Figure B is the inspection result graph of the remineralization effect. Detailed implementation mode

[0050] To further illustrate the present invention, the nano-complex and its application provided by the present invention will be described in detail below in conjunction with embodiments.

[0051] Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0052] Example 1

[0053] This example discloses a preparation method of a pH-responsive antibacterial and remineralizing caries prevention and treatment nano-complex of the present invention, specifically as follows:

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

[0055] Under nitrogen protection, N-(2-hydroxyethyl)acrylamide (24 mmol) and PPTS (2 mmol) were dissolved in 20 mL of anhydrous DCM and stirred at 0 °C for 30 min. Anhydrous DCM (10 mL) containing allyl vinyl ether (20 mmol) was slowly added dropwise to the mixture, and the reaction was stirred overnight at room temperature. 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, whose English name is N-(2-(1-(allyloxy)ethoxy)ethyl)-acrylamide, abbreviated 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 of methanol / water (v / v = 7:3) and stirred at 70 °C for 48 h. Then, the mixture was dialyzed in deionized water using a dialysis bag (molecular cut-off 1 kDa), with the water changed every 6 h for multiple times, and freeze-dried to obtain a viscous liquid, the synthesized PAMAM molecules with acetal bonds and olefin terminal groups, abbreviated as PAMAM-acetal-ene, about 450 mg.

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

[0059] React benzoin diethyl ether, cysteamine hydrochloride with PAMAM-acetal-ene to construct an amino group at the end of PAMAM-acetal-ene, and synthesize PAMAM molecules with acetal bonds and amino terminal groups: PAMAM-acetal-NH2, specifically as follows:

[0060] Mix PAMAM-acetal-ene (60 μmol), DMPA (40 μmol) and cysteamine hydrochloride (8.4 mmol) in 40 mL of methanol. The reaction mixture was irradiated with a UV lamp at 365 nm and stirred at room temperature for 4 hours. Then, it was dialyzed in deionized water using a dialysis bag (molecular cut-off 1 kDa) and freeze-dried to obtain a pale yellow powder, the PAMAM molecules with acetal bonds and amino terminal groups: PAMAM-acetal-NH2, about 300 mg.

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

[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@P-A).

[0064] The structure and binding energy of the nanocomposite V@P-A are shown in the appendix Figure 2 As can be seen from this figure, two VDQACs monomers can stably bind to the carrier PAMAM, and the binding energies are -8.89 eV and -8.85 eV.

[0065] Example 2

[0066] This example discloses a preparation method of the pH-responsive antibacterial and remineralizing nanocomposite for preventing and treating dental caries of the present invention, specifically:

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

[0068] Under nitrogen protection, N-(2-hydroxyethyl)acrylamide (20 mmol) and PPTS (2.4 mmol) were dissolved in 20 mL of anhydrous DCM and stirred at 0 °C for 120 min; anhydrous DCM (10 mL) containing allyl vinyl ether (20 mmol) 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, with the English name N-(2-(1-(allyloxy)ethoxy)ethyl)-acrylamide, abbreviated 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 of methanol / water (v / v = 7:3), and stirred at 70 °C for 48 h. Then the mixture was dialyzed in deionized water using a dialysis bag (molecular cut-off 1 kDa), changing the water every 6 h, changing the water multiple times, and freeze-dried to obtain a viscous liquid to synthesize a PAMAM molecule with acetal bonds and olefin end groups, abbreviated as PAMAM-acetal-ene, about 450 mg.

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

[0072] React benzoin diethyl ether, cysteamine hydrochloride with PAMAM-acetal-ene to construct an amino group at the end of PAMAM-acetal-ene, and synthesize a PAMAM molecule with acetal bonds and amino end groups: PAMAM-acetal-NH2, specifically as follows:

[0073] Mix PAMAM-acetal-ene (30 μmol), DMPA (20 μmol) and cysteamine hydrochloride (12 mmol) in 40 mL of methanol. The reaction mixture was irradiated with a UV lamp at 365 nm and stirred at room temperature for 4 hours, then dialyzed in deionized water using a dialysis bag (molecular cut-off 1 kDa), and freeze-dried to obtain a pale yellow powder of a PAMAM molecule with acetal bonds and amino end groups: PAMAM-acetal-NH2, about 300 mg.

[0074] S4. Synthesis of pH-responsive nanocomposites

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

[0076] Example 3

[0077] This example discloses a preparation method of the pH-responsive antibacterial and remineralizing nanocomposite for preventing and treating dental caries of the present invention, specifically as follows:

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

[0079] Under nitrogen protection, N-(2-hydroxyethyl)acrylamide (28 mmol) and PPTS (1.6 mmol) were dissolved in 20 mL of anhydrous DCM, and the mixture was stirred at 0 °C for 120 min; 10 mL of anhydrous DCM containing allyl vinyl ether (24 mmol) 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, whose English name is N-(2-(1-(allyloxy)ethoxy)ethyl)-acrylamide, abbreviated 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 of methanol / water (v / v = 7:3), and the mixture was stirred at 70 °C for 48 h. Then the mixture was dialyzed in deionized water using a dialysis bag (molecular cut-off 1 kDa), the water was changed every 6 h, and the water was changed multiple times, and then freeze-dried to obtain a viscous liquid, the synthesized PAMAM molecules with acetal bonds and olefin terminal groups, abbreviated as PAMAM-acetal-ene, about 450 mg.

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

[0083] Benzoin diethyl ether, cysteamine hydrochloride were reacted with PAMAM-acetal-ene to construct an amino group at the end of PAMAM-acetal-ene, and PAMAM molecules with acetal bonds and amino terminal groups: PAMAM-acetal-NH2 were synthesized as follows:

[0084] PAMAM-acetal-ene (120 μmol), DMPA (60 μmol) and cysteamine hydrochloride (4.0 mmol) were mixed in 40 mL of methanol. The reaction mixture was irradiated with a UV lamp at 365 nm and stirred at room temperature for 4 hours. Then it was dialyzed in deionized water using a dialysis bag (molecular cut-off 1 kDa) and freeze-dried to obtain a pale yellow powder, PAMAM molecules with acetal bonds and amino terminal groups: 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@P-A).

[0087] Example 4

[0088] This example discloses a preparation method of the pH-responsive antibacterial and remineralizing nanocomposite for preventing and treating dental caries of the present invention, specifically as follows:

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

[0090] Under nitrogen protection, N-(2-hydroxyethyl)acrylamide (24 mmol) and PPTS (2.4 mmol) were dissolved in 20 mL of anhydrous DCM, and stirred at 0 °C for 120 min; anhydrous DCM (10 mL) containing allyl vinyl ether (16 mmol) was slowly added dropwise to the mixture, and stirred at room temperature overnight, 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 acetal bond, with the English name N-(2-(1-(allyloxy)ethoxy)ethyl)-acrylamide, abbreviated as AEEAA;

[0091] S2. Synthesis of PAMAM molecules with acetal bond 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 of methanol / water (v / v = 7:3), and stirred at 70 °C for 48 h. Then the mixture was dialyzed in deionized water using a dialysis bag (molecular cut-off 1 kDa), and the water was changed every 6 h, and the water was changed multiple times, and freeze-dried to obtain a viscous liquid to synthesize PAMAM molecules with acetal bond and olefin terminal groups, abbreviated as PAMAM-acetal-ene, about 450 mg.

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

[0094] Benzoin diethyl ether, cysteamine hydrochloride were reacted with PAMAM-acetal-ene to construct an amino group at the end of PAMAM-acetal-ene to synthesize PAMAM molecules with acetal bond and amino terminal groups: PAMAM-acetal-NH2, specifically 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 of a 365-nm ultraviolet lamp, then dialyzed in deionized water using a dialysis bag (molecular cut-off 1 kDa), and freeze-dried to obtain a pale yellow powder of PAMAM molecules with acetal bonds and amino-terminal groups: 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@P-A).

[0098] Experimental Example 1

[0099] This experimental example discloses an investigation of the antibacterial effect of VDQACs. The VDQACs in this experiment are prior art, and their synthesis is carried out according to the method described in the literature "Design, synthesis, antibacterial activity and toxicity of novel quaternary ammonium compounds based on pyridoxine and fatty acids" (Sapozhnikov S V, Sabirova A E, Shtyrlin N V, et al.. European Journal of Medicinal Chemistry, 2020, 211:113100. DOI: 10.1016 / j.ejmech.2020.113100.).

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

[0101] 1. In a 96-well plate, S. mutans UA159 with a final concentration of 1×10 6 was cultured in 100 μL of medium containing different concentrations of VDQACs for 24 h. 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 using a microplate reader every 1 h until 24 h to obtain the bacterial growth curve.

[0102] 2. The saliva-derived oral biofilm was cultured under anaerobic conditions for 48 h. After treating the biofilm with VDQACs, it was cultured for another 24 h, and 16S RNA sequencing was used to detect the proportion of bacterial species in the biofilm and related parameters of the microbial community composition.

[0103] The results are shown in the Figure 3 attachment. As can be seen from this figure, VDQACs can effectively inhibit the growth of cariogenic bacterium S. mutans UA 159, while improving the oral microecological balance, increasing the biodiversity of the oral biofilm, and reducing the proportion of cariogenic bacteria.

[0104] Test Example 2

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

[0106] 1. Characterization of the nanocomposite V@P-A. Transmission electron microscopy was used to observe the molecular morphology of the nanocomposite in a neutral environment (pH = 7.0) and an acidic environment at the critical pH of caries occurrence (pH = 5.5).

[0107] The results are shown in the Figure 4 attachment. As can be seen from this figure, the molecular morphology of V@P-A varies in different environments. In the neutral environment, the composite shows a relatively compact molecular structure, while in the acidic environment, it becomes more porous, facilitating the release of the loaded drug.

[0108] 2. Detection of pH-responsive antibacterial effect

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

[0110] The results are shown in Figure A of the Figure 5 attachment. As can be seen from this figure, V@P-A shows a better antibacterial effect in the 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@P-A in an in vitro caries model and its comparison with the unmodified empty carrier PAMAM. Specifically 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 preparation method of a pH-responsive nanocomposite, characterized in that It includes the following steps: S1. Preparation of the precursor product of the acetal bond, N-(2-(1-(allyloxy)ethoxy)ethyl)acrylamide: Using N-(2-hydroxyethyl)acrylamide, PPTS, and allyl vinyl ether as raw materials, reacting at room temperature to generate the precursor product of the acetal bond, N-(2-(1-(allyloxy)ethoxy)ethyl)acrylamide, abbreviated as AEEAA; the molar ratio of N-(2-hydroxyethyl)acrylamide, PPTS, and allyl vinyl ether is 10-14:0.8-1.2:8-12; S2. Synthesis of PAMAM molecules with acetal bonds and olefin terminal groups: Reacting the third-generation PAMAM with AEEAA prepared in step S1 at 60-80 °C to generate PAMAM molecules with acetal bonds and olefin terminal groups, abbreviated as PAMAM-acetal-ene; the molar ratio of AEEAA to PAMAM is 150-200:1; S3. Synthesis of PAMAM molecules with acetal bonds and amino terminal groups: Reacting benzoin diethyl ether, cysteamine hydrochloride with PAMAM-acetal-ene prepared in step S2 under ultraviolet light irradiation to construct an amino group at the end of PAMAM-acetal-ene, and synthesizing PAMAM molecules with acetal bonds and amino terminal groups, abbreviated as PAMAM-acetal-NH2; the molar ratio of PAMAM-acetal-ene, benzoin diethyl ether, and cysteamine hydrochloride is 1.5-6:1-4:200-600; S4. Synthesis of the nanocomposite VDQACs@PAMAM-acetal-NH2: Mixing and reacting PAMAM-acetal-NH2 with VDQACs in water at room temperature to synthesize the pH-responsive nanocomposite VDQACs@PAMAM-acetal-NH2; the mass ratio of VDQACs to PAMAM-acetal-NH2 is 0.8-1.2:1.5-2.

5.

2. The preparation method of 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 12:1:10; In step S2, the molar ratio of AEEAA to PAMAM is 180:1; In step S3, the molar ratio of PAMAM-acetal-ene, benzoin diethyl ether, and cysteamine hydrochloride is 3:2:420; In step S4, the mass ratio of VDQACs to PAMAM-acetal-NH2 is 1:

2.

3. The preparation method of a pH-responsive nanocomposite according to claim 1 or 2, characterized in that, In step S1, under a protective gas atmosphere, adding N-(2-hydroxyethyl)acrylamide and PPTS to the first solvent, stirring at low temperature, and then dropping the allyl vinyl ether solution into this solution for reaction; The solvent in the allyl vinyl ether solution is the first solvent; The first solvent includes dichloromethane.

4. The preparation method of a pH-responsive nanocomposite according to claim 3, characterized in that, In step S1, under a protective gas atmosphere, adding N-(2-hydroxyethyl)acrylamide and PPTS to the first solvent, stirring at 0 °C for 5-120 min.

5. The preparation method of a pH-responsive nanocomposite according to claim 3, characterized in that, Stirring at 0 °C for 30 min.

6. The preparation method of a pH-responsive nanocomposite according to claim 3, characterized in that, In step S1, 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.

7. The preparation method of a pH-responsive nanocomposite according to claim 1 or 2, characterized in that, In step S2, under a protective gas atmosphere, PAMAM and AEEAA are placed in a container, a second solvent is added, and the mixture is stirred and reacted. The second solvent includes an aqueous methanol solution.

8. The preparation method of a pH-responsive nanocomposite according to claim 7, characterized in that, In the aqueous methanol solution, the volume ratio of methanol to water is 6 - 8:2 - 4.

9. The preparation method of a pH-responsive nanocomposite according to claim 7, characterized in that, In the aqueous methanol solution, the volume ratio of methanol to water is 7:

3.

10. The preparation method of a pH-responsive nanocomposite according to claim 7, characterized in that, In step S2, the reaction temperature is 70 °C.

11. The preparation method of a pH-responsive nanocomposite according to claim 7, characterized in that, In step S2, the reaction mixture is dialyzed using a dialysis bag in deionized water, and then the dialyzed product is freeze-dried to obtain PAMAM-acetal-ene.

12. The preparation method of a pH-responsive nanocomposite according to claim 1 or 2, characterized in that, In step S3, benzoin diethyl ether, cysteamine hydrochloride, and the PAMAM-acetal-ene prepared in step S2 are dissolved in a third solvent and irradiated under an ultraviolet lamp. The third solvent includes methanol.

13. The preparation method of a pH-responsive nanocomposite according to claim 12, wherein, In step S3, irradiation is carried out under an ultraviolet lamp for 2 - 8 h under stirring conditions.

14. The preparation method of a pH-responsive nanocomposite according to claim 12, characterized in that, Irradiation is carried out under an ultraviolet lamp for 4 h.

15. The preparation method of a pH-responsive nanocomposite according to claim 12, characterized in that, In step S3, the reaction mixture is dialyzed using a dialysis bag in deionized water, and then the dialyzed product is freeze-dried to obtain PAMAM-acetal-NH2.

16. A pH-responsive nanocomposite, characterized in that, Prepared by the method according to any one of claims 1 - 15.

17. Use of the pH-responsive nanocomposite according to claim 16, characterized in that, Use in the preparation of a drug for preventing and / or treating dental caries.

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