Silver ion-nucleoside supramolecular hydrogel as well as preparation method and application thereof

Abstract

The invention provides silver ion-nucleoside supramolecular hydrogel as well as a preparation method and application thereof, and belongs to the field of biological materials. The nucleoside supramolecular hydrogel with a three-dimensional space structure is prepared from 2-amino adenosine and silver ions. The hydrogel has good biocompatibility, stability, biodegradability, injectability and antibacterial property, and can be used for injection administration in a periodontal pocket. The hydrogel is further coated with a photo-thermal reagent to obtain the composite hydrogel, and the composite hydrogel can play a role in chemotherapy-photo-thermal synergistic treatment, has excellent antibacterial and anti-biofilm capabilities, and has a good application prospect in treatment of periodontitis.

Description

Technical Field

[0001] The invention belongs to the field of material processing, and in particular relates to a silver ion-nucleoside supramolecular hydrogel and a preparation method and application thereof. Background Art

[0002] Periodontitis is one of the most common chronic non-communicable diseases in the world. Its main manifestations include red, swollen and bleeding gums, periodontal pocket formation, alveolar bone resorption, loose teeth and even tooth loss. It is the main cause of tooth loss in adults. Periodontitis also has a certain impact on normal physiological functions such as chewing, pronunciation, and eating. In addition, periodontitis is a risk factor for many systemic diseases. The probability of patients with periodontitis suffering from cardiovascular diseases, Alzheimer's disease and diabetes increases significantly. Therefore, due to its high incidence and great impact on the local and systemic systems of the human body, periodontitis has become a global health problem.

[0003] At present, the main method for clinical treatment of periodontitis is to remove plaque biofilm through mechanical therapy such as mechanical scaling and root planing to control infection. However, due to the irregular shape of periodontal pockets, it is difficult to completely remove bacteria deep in the periodontal pockets using only mechanical therapy. Therefore, the application of local or systemic drug therapy is a common adjuvant treatment for periodontitis. However, the amount of drug that reaches the periodontal pocket lesion area with systemic drug therapy is very small, and it is easy to produce systemic adverse reactions. Local drug therapy is a major choice for drug treatment of periodontitis because of its advantages such as high local drug concentration in the periodontal pocket and low systemic adverse reactions. At present, the commonly used local drug treatment methods in clinical practice are mainly gargle, local irrigation, and periodontal pocket administration. These local drug administration methods mainly face two problems: ① It is difficult for drugs to penetrate into the periodontal pocket, and the local retention time is not long; ② Traditional antibiotics have limited bactericidal effects on bacteria in plaque biofilms. Therefore, in order to overcome the above-mentioned defects of local treatment drugs for periodontitis, it is imperative to develop new local treatment strategies for the treatment of periodontitis. Summary of the invention

[0004] The purpose of the present invention is to provide a silver ion-nucleoside supramolecular hydrogel and a preparation method and application thereof.

[0005] The invention provides a nucleoside supramolecular hydrogel. The nucleoside supramolecular hydrogel is prepared by using 2-aminoadenosine, a silver ion compound, a boron-containing acid, a base and water as raw materials; the mass molar ratio of the 2-aminoadenosine, the silver ion compound, the boron-containing acid and the base is 20-40 mg: 0.02-0.2 mmol: 0.02-0.2 mmol: 0.02-0.2 mmol.

[0006] Furthermore, the silver ion compound is silver nitrate; the boron-containing acid is boric acid; the base is cesium hydroxide; and the mass molar ratio of the 2-aminoadenosine, the silver ion compound, the boron-containing acid and the base is 28.3 mg:0.05 mmol:0.05 mmol:0.05 mmol.

[0007] The present invention also provides a method for preparing the nucleoside supramolecular hydrogel, which comprises the following steps: dissolving 2-aminoadenosine in water, adding a silver ion compound, a boron-containing acid and a base, mixing and heating for reaction, and cooling to obtain the nucleoside supramolecular hydrogel.

[0008] Furthermore, the temperature of the heating reaction is 80-140° C., and the time is 1-10 min. Preferably, the temperature of the heating reaction is 100-120° C., and the time is 5 min.

[0009] The present invention also provides a composite hydrogel, which is obtained by encapsulating the photothermal agent in the nucleoside supramolecular hydrogel; wherein the mass ratio of 2-aminoadenosine to the photothermal agent is 20-35 mg:1-100 μg.

[0010] Furthermore, the mass ratio of the 2-aminoadenosine to the photothermal agent is 28.3 mg:30 μg;

[0011] The photothermal agent is gold nanoparticles.

[0012] The present invention also provides a method for preparing the composite hydrogel, which comprises the following steps: dissolving 2-aminoadenosine in water, adding a silver ion compound, a boron-containing acid and a base, mixing and heating for reaction, adding a photothermal reagent, mixing evenly, and cooling to obtain the composite hydrogel.

[0013] Furthermore, the temperature of the heating reaction is 80-140° C., and the time is 1-10 min. Preferably, the temperature of the heating reaction is 100-120° C., and the time is 5 min.

[0014] The present invention also provides the use of the above-mentioned nucleoside supramolecular hydrogel and composite hydrogel in the preparation of antibacterial agents or drugs for preventing and / or treating periodontitis;

[0015] Preferably, the antibacterial agent is an anti-Streptococcus mutans or anti-Porphyromonas gingivalis preparation, and the antibacterial agent is a preparation that destroys bacterial biofilm.

[0016] The present invention also provides the use of the above-mentioned nucleoside supramolecular hydrogel and photothermal therapy device in combination to prepare antibacterial equipment;

[0017] And / or, use of the above composite hydrogel and photothermal therapy device in combination to prepare an antibacterial device.

[0018] The present invention also provides the use of the above-mentioned nucleoside supramolecular hydrogel and a photothermal therapy device in combination to prepare a device for preventing and / or treating periodontitis;

[0019] And / or, use of the above composite hydrogel and photothermal therapy device in combination to prepare a device for preventing and / or treating periodontitis.

[0020] The present invention uses 2-aminoadenosine and silver ions to prepare a nucleoside supramolecular hydrogel with a three-dimensional spatial structure. The hydrogel has good biocompatibility, stability, biodegradability, injectability and antibacterial properties, and can be used for injection into periodontal pockets. The present invention further encapsulates the hydrogel with a photothermal agent to obtain a composite hydrogel, which can play a role in chemotherapy-photothermal synergistic therapy, has excellent antibacterial and anti-biofilm capabilities, and has good application prospects in the treatment of periodontitis.

[0021] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0022] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Structural characterization of NA hydrogel: (A) NA hydrogel; (B) AFM image of NA hydrogel; (C) TEM image of NA hydrogel; (D): SEM image of NA hydrogel with a scale bar of 5 μm; (E): SEM image of NA hydrogel with a scale bar of 0.5 μm.

[0024] Figure 2 Characterization of the gelation mechanism of NA hydrogel: (A) NA hydrogel 11 B NMR spectrum; (B) NA hydrogel 1 H NMR spectrum; (C) NA hydrogel IR spectrum; (D) NA hydrogel PXRD spectrum.

[0025] Figure 3Rheological results of NA hydrogel and NA@Au-NPs hydrogel: (A) frequency scanning graph of NA hydrogel; (B) strain scanning graph of NA hydrogel; (C) shear thinning graph of NA hydrogel; (D) self-healing graph of NA hydrogel; (E) frequency scanning graph of NA@Au-NPs hydrogel; (F) strain scanning graph of NA@Au-NPs hydrogel; (G) shear thinning graph of NA@Au-NPs hydrogel; (H) self-healing graph of NA@Au-NPs hydrogel.

[0026] Figure 4 The hydrogel is cytotoxic.

[0027] Figure 5 The results of bacterial plate spread colony counting after S. mutans was co-cultured with hydrogel.

[0028] Figure 6 The results of colony counting on bacterial plates after co-culture of P.gingivalis and hydrogel.

[0029] Figure 7 The results of colony counting on plates after co-culture of S. mutans biofilm and hydrogel.

[0030] Figure 8 In vivo study of hydrogel treatment of periodontitis in rats: (A) Micro-CT three-dimensional images and sagittal tomographic images of representative maxillary bones in each group; (B) CEJ-ABC distance of maxillary bones in each group. DETAILED DESCRIPTION

[0031] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0032] Example 1. Preparation of NA hydrogel

[0033] 28.3 mg of 2-aminoadenosine (NA) was added to 1700 μL of deionized water, heated to dissolve, and 100 μL of 0.5 M boric acid (H 3 BO 3 ) solution and 100 μL 0.5 M CsOH solution, mix well, continue heating at 100-120 °C for 5 minutes, add 100 μL 0.5 M AgNO 3 The solution was mixed and cooled to room temperature to obtain NA hydrogel.

[0034] Example 2: Preparation of NA@Au-NPs photothermal water gel

[0035] 28.3 mg of 2-aminoadenosine (NA) was added to 1700 μL of deionized water, heated to dissolve, and 100 μL of 0.5 M boric acid (H 3 BO 3) solution and 100 μL 0.5 M CsOH solution, mix well, continue heating for 5 minutes, add 100 μL 0.5 M AgNO 3 Solution, mix well. Add 100 μL of gold nanoparticles (Au-NPs) (300 μg / mL) and mix well. Cool to room temperature to obtain NA@Au-NPs hydrogel loaded with photothermal agent (final concentration of photothermal nanoparticles is 15 μg / mL).

[0036] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0037] Experimental Example 1: Structural Characterization of Hydrogel

[0038] 1. Experimental methods

[0039] The formation of NA hydrogel was confirmed by inverting the vial, observing and taking photos. The NA hydrogel was taken for atomic force microscopy (AFM), transmission electron microscopy (TEM), scanning electron microscopy (SEM), 1 H NMR, 11 B NMR, infrared spectroscopy (IR), and powder X-ray diffraction analysis (PXRD) characterization.

[0040] 2. Experimental results

[0041] Depend on Figure 1 A shows that after the vial is inverted, the NA hydrogel remains stable ( Figure 1 A), indicating that the NA hydrogel was successfully prepared. AFM, TEM and SEM results showed that the NA hydrogel was a loose porous structure composed of fibers ( Figure 1 B-1E). 11 B NMR experimental results show that there are boric acid diester bonds in NA hydrogels ( Figure 2 A), 1 The above results were further confirmed by H NMR ( Figure 2 B); IR analysis found that the hydroxyl peak ν (-OH, 3500cm -1 ) signal is significantly weaker than that of NA, suggesting that the hydroxyl groups at the 2' and 3' positions of the NA molecule may be involved in the formation of the boric acid diester bond ( Figure 2 C) PXRD results show that the freeze-dried sample of NA hydrogel has a 2θ≈26.68° There is a peak ( Figure 2 D), indicating that NA hydrogels are formed by π-π stacking.

[0042] Experimental Example 2: Characterization of Mechanical Properties of Hydrogels

[0043] 1. Experimental methods

[0044] Rheological tests were performed using an Anton Paar modular intelligent rotational rheometer (MCR302). At 25°C, the rheological properties of NA hydrogels were analyzed by frequency sweep, strain sweep, shear rate, and self-healing tests.

[0045] 2. Experimental results

[0046] The frequency scanning results show that the storage modulus G′ of NA hydrogel is much larger than the loss modulus G″, indicating that the sample is in a gel state and has good solid-like mechanical properties ( Figure 3 A). Figure 3 As shown in Figure B, when the strain is less than 15%, G′ is greater than G″, and the NA hydrogel is in a gel state; when the strain is greater than 40%, G′ is less than G″, and the NA hydrogel is in a sol state. The sample was subjected to high strain-low strain alternating behavior, and the results showed that the G′ and G″ of the sample can basically recover to the initial state, indicating that the NA hydrogel has good self-healing properties ( Figure 3 C). Shear rate experiments showed that the viscosity of NA hydrogels gradually decreased with increasing shear rate ( Figure 3 D), indicating that NA hydrogel has shear thinning properties. Figure 3 EH results showed that NA@Au-NPs hydrogels loaded with photothermal agents could maintain good mechanical properties.

[0047] Experimental Example 3: Cytotoxicity test of hydrogel

[0048] 1. Experimental methods

[0049] 10 mg of hydrogel was added to 10 mL of DMEM and cultured for 24 hours to prepare the mother solution of the leaching solution. Well-growing L929 cells were collected and the cell density was adjusted to 1*10 4 After the cells were attached to the wall, 100 μl of hydrogel extract was added. The photothermal treatment (680 nm, 1 W / cm 2 After incubation at 37°C for 24 h, 10% CCK8 reagent was added and incubated for 1 h. The absorbance was detected at a wavelength of 450 nm.

[0050] 2. Experimental results

[0051] The results showed that the cell viability of the NA hydrogel and NA@Au-NPs hydrogel groups was almost the same as that of the control group ( Figure 4 ), indicating that the NA hydrogel and NA@Au-NPs hydrogel illumination groups have good cell biocompatibility.

[0052] Experimental Example 4: Hydrogels to Fit Photothermal Therapy for Antibacterial Activity in Vitro

[0053] 1. Experimental methods

[0054] (1) Hydrogel combined with photothermal therapy against Streptococcus mutans (S. mutans) in vitro

[0055] Take the frozen stock of S. mutans and thaw it quickly in a 37℃ water bath. Add 10mL of BHI medium to a 15ml sterile centrifuge tube, then add 100μL of thawed S. mutans strains to it. After culturing at 37℃ overnight, take 10μL of bacterial solution and drop it on the surface of the BHI solid medium plate. Use a sterile inoculation loop to spread it on the plate. After anaerobic culture at 37℃ for 1-3 days, pick a single colony and add it to the BHI medium. Culture at 37℃ until the logarithmic growth phase, and adjust the bacterial concentration to 10 6 CFU / ml. Take 1mL of bacterial solution and add 50mg of the following substances to each group: PBS, Ag + Solution, Au-NPs, NA hydrogel, and NA@Au-NPs hydrogel. Au-NPs and NA@Au-NPs hydrogel groups were subjected to photothermal therapy (680nm, 1W / cm 2 After culturing for 24 h, the bacteria were diluted with PBS and plated for colony counting.

[0056] (2) Hydrogel combined with photothermal therapy against Porphyromonas gingivalis in vitro

[0057] Take the frozen stock of P.gingivalis and thaw it quickly in a 37℃ water bath. Add 10mL of BHI medium (containing 5μg / mL heme and 1μg / mL vitamin K) to a 15ml sterile centrifuge tube, then add 100μL of thawed P.gingivalis strains and culture at 37℃ anaerobically (10% H 2 , 10% CO 2 and 80% N 2 ) 1-2 days later, take 10 μL of bacterial liquid and drop it on the surface of the blood plate, and spread it with a sterile inoculation loop plate streaking method. After anaerobic culture at 37°C for 3-5 days, pick a single colony and add it to BHI medium (containing 5 μg / mL heme and 1 μg / mL vitamin K). Incubate at 37°C anaerobicly until the logarithmic growth phase, and adjust the bacterial concentration to 10 6 CFU / ml. Take 1mL of bacterial solution and add 50mg of the following substances to each group: PBS, Ag + Solution, Au-NPs, NA hydrogel, and NA@Au-NPs hydrogel. Au-NPs and NA@Au-NPs hydrogel groups were subjected to photothermal therapy (680nm, 1W / cm 2 After culturing for 24 h, the bacteria were diluted with PBS and plated for colony counting.

[0058] 2. Experimental results

[0059] (1) Hydrogel combined with photothermal therapy against Streptococcus mutans (S. mutans) in vitro

[0060] The results of S. mutans bacterial plate counts showed Ag + The colony counts of the NA hydrogel and Au-NPs (NIR+) groups decreased by 2 to 3 orders of magnitude ( Figure 5 ), indicating that both the photothermal therapy and the NA hydrogel group containing silver ions have certain antibacterial properties. The number of colonies in the NA@Au-NPs (NIR+) group decreased by about 4 orders of magnitude, and its antibacterial properties were better than those of the Au-NPs (NIR+), NA hydrogel group, and minocycline group. In other words, the antibacterial effect of the same dose of NA@Au-NPs (NIR+) was better than that of Au-NPs (NIR+) and NA hydrogel, indicating that the NA@Au-NPs hydrogel of the present invention combined with the photothermal therapy device exerted a synergistic antibacterial effect.

[0061] (2) Hydrogel combined with photothermal therapy against Porphyromonas gingivalis in vitro

[0062] The bacterial plate count results showed that Ag + , NA hydrogel and NA@Au-NPs groups could kill 100% of P.gingivalis ( Figure 6 ).

[0063] Experimental Example 5: Anti-biofilm effect of hydrogel combined with photothermal therapy

[0064] 1. Experimental methods

[0065] Adjust the bacterial concentration of S. mutans to 1*10 5 Place a sterile glass slide with a diameter of 18 mm in a 12-well plate, add 2 mL of bacterial solution, and culture for 48 hours to form a biofilm. Add 50 mg of the following substances to the 12-well plate for culturing biofilms: PBS, Ag + Solution, minocycline, Au-NPs, NA hydrogel, and NA@Au-NPs hydrogel. Au-NPs and NA@Au-NPs hydrogel groups were subjected to photothermal therapy (680 nm, 1 W / cm 2 After culturing for 24 h, the bacteria on the slide were scraped into PBS, diluted with PBS, and plated for colony counting.

[0066] 2. Experimental results

[0067] The biofilm elimination efficiency directly affects the therapeutic effect of periodontitis. Therefore, we established a biofilm of S. mutans to evaluate the anti-biofilm ability of NA@Au-NPs hydrogel. The plate count results showed that Ag + The colony counts of the NA hydrogel and Au-NPs (NIR+) groups decreased by about 2 orders of magnitude ( Figure 7 ), indicating that both the photothermal therapy and the silver ion-containing NA hydrogel group have certain anti-biofilm properties. The number of colonies in the NA@Au-NPs (NIR+) group decreased by about 3 orders of magnitude, and its antibacterial property was better than that of the Au-NPs (NIR+), NA hydrogel group, and minocycline group. In other words, the antibacterial effect of the same dose of NA@Au-NPs (NIR+) was better than that of Au-NPs (NIR+), and at the same time, it was better than that of NA hydrogel, indicating that the NA@Au-NPs hydrogel of the present invention combined with the photothermal therapy device played a synergistic antibacterial effect. It shows that the synergistic effect of photothermal therapy and silver ion chemotherapy will enhance the anti-biofilm effect.

[0068] Experimental Example 6: Evaluation of the therapeutic effect on periodontitis in vivo

[0069] 1. Experimental methods

[0070] The rat periodontitis model was established using the ligature method: a 3-0 silk thread (pre-soaked in Porphyromonas gingivalis solution) was used to wrap around the neck of the right maxillary second molar of the rat and ligatured. The silk thread was placed in the gingival sulcus and tied on the palate. After 2 weeks, the gums around the maxillary second molar of the rat were red and swollen, bleeding on probing, deep periodontal pockets, and a large amount of plaque and soft scale accumulation were found. The periodontitis model was successfully established. The ligature was removed, and 20 μL of the following substances were injected into the periodontal pocket in each group: PBS, Ag + Solution (25 mM AgNO 3 solution), minocycline hydrochloride ointment (2%), NA hydrogel (containing 25mM Ag + ), NA@Au-NPs (containing 25 mM Ag + The NA@Au-NPs hydrogel group was treated with photothermal therapy (680 nm, 1 W / cm 2 , 5min). The rats were killed on the 14th day of treatment, and the maxilla of the rats was taken, one part was fixed with 4% paraformaldehyde, and the other part was stored at -80°C for future use. The maxilla of the rats was scanned using a Micro CT system, and the image analysis of the alveolar bone of the rats was performed.

[0071] 2. Experimental results

[0072] Micro CT analysis results showed that after silk thread ligation, significant alveolar bone resorption occurred on the buccal and palatal sides of the maxillary second molars. After photothermal treatment, partial alveolar bone recovery was observed in the NA@Au-NPs hydrogel group ( Figure 8 A). The corresponding alveolar bone resorption is represented by the distance from the cementoenamel junction to the alveolar bone crest (CEJ-ABC). The results showed that the CEJ-ABC distance in the NA@Au-NPs photothermal therapy group was the shortest and was significantly shorter than that in the other groups (PBS, Ag + solution, minocycline, and NA hydrogel groups) showed significant differences ( Figure 8 B), indicating that NA@Au-NPs photothermal therapy has a good therapeutic effect on periodontitis.

[0073] In summary, the present invention provides a silver ion-nucleoside supramolecular hydrogel and a preparation method and use thereof. The present invention uses 2-aminoadenosine and silver ions to prepare a nucleoside supramolecular hydrogel with a three-dimensional spatial structure. The hydrogel has good biocompatibility, stability, biodegradability, injectability and antibacterial properties, and can be used for injection into periodontal pockets. The present invention further encapsulates the hydrogel with a photothermal agent to obtain a composite hydrogel, which can play the role of chemotherapy-photothermal synergistic therapy, has excellent antibacterial and anti-biofilm capabilities, and has good application prospects in the treatment of periodontitis.

Claims

1. A nucleoside supramolecular hydrogel, characterized in that: The nucleoside supramolecular hydrogel is prepared from 2-aminoadenosine, a silver ion compound, a boron-containing acid, a base and water as raw materials; the mass molar ratio of the 2-aminoadenosine, the silver ion compound, the boron-containing acid and the base is 20-40 mg: 0.02-0.2 mmol: 0.02-0.2 mmol: 0.02-0.2 mmol.

2. The nucleoside supramolecular hydrogel according to claim 1, characterized in that: The silver ion compound is silver nitrate; the boron-containing acid is boric acid; the base is cesium hydroxide; the mass molar ratio of the 2-aminoadenosine, the silver ion compound, the boron-containing acid and the base is 28.3 mg: 0.05 mmol: 0.05 mmol: 0.05 mmol.

3. A method for preparing the nucleoside supramolecular hydrogel according to claim 1 or 2, characterized in that: The method comprises the following steps: dissolving 2-aminoadenosine in water, adding a silver ion compound, a boron-containing acid and a base, mixing and heating for reaction, and cooling to obtain the product.

4. The method according to claim 3, characterized in that The temperature of the heating reaction is 80-140° C., and the time is 1-10 min. Preferably, the temperature of the heating reaction is 100-120° C., and the time is 5 min.

5. A composite hydrogel, characterized in that: The composite hydrogel is obtained by encapsulating the photothermal agent in the nucleoside supramolecular hydrogel according to claim 1 or 2; wherein the mass ratio of 2-aminoadenosine to the photothermal agent is 20-35 mg:1-100 μg.

6. The composite hydrogel according to claim 5, characterized in that The mass ratio of 2-aminoadenosine to the photothermal reagent is 28.3 mg:30 μg; The photothermal agent is gold nanoparticles.

7. A method for preparing the composite hydrogel according to claim 5 or 6, characterized in that: The method comprises the following steps: dissolving 2-aminoadenosine in water, adding a silver ion compound, a boron-containing acid and a base, mixing and heating for reaction, adding a photothermal reagent, mixing evenly, and cooling to obtain the product.

8. Use of the nucleoside supramolecular hydrogel according to claim 1 or 2, or the composite hydrogel according to claim 5 or 6 in the preparation of an antibacterial agent or a drug for preventing and / or treating periodontitis; Preferably, the antibacterial agent is an anti-Streptococcus mutans or anti-Porphyromonas gingivalis preparation, and the antibacterial agent is a preparation that destroys bacterial biofilm.

9. Use of the nucleoside supramolecular hydrogel according to claim 1 or 2 in combination with a photothermal therapy device in the preparation of an antibacterial device; And / or, use of the composite hydrogel according to claim 5 or 6 in combination with a photothermal therapy device in the preparation of an antibacterial device.

10. Use of the nucleoside supramolecular hydrogel according to claim 1 or 2 in combination with a photothermal therapy device in the preparation of a device for preventing and / or treating periodontitis; And / or, use of the composite hydrogel according to claim 5 or 6 in combination with a photothermal therapy device in the preparation of a device for preventing and / or treating periodontitis.

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