Preparation method and application of pH-responsive double-synergistic-therapy hydrogel

Abstract

The invention discloses a preparation method and application of pH-responsive double-synergistic-therapy hydrogel. The hydrogel is mainly prepared from oxidized fucoidin (OFU), carboxymethyl chitosan (CMCs), zinc peroxide / copper nanoparticles (ZnO < 2-Cu >) and arginine (L-ARG). Under a gastric acid condition, the hydrogel generates H2O2 through zinc peroxide, copper ions catalyze H2O2 through a Fenton-like reaction to generate OH <->, arginine can catalyze H2O2 to generate NO, and the copper ions and the arginine act together to destroy a helicobacter pylori biological membrane and kill bacteria. The hydrogel has good injectability, self-healing property and adhesivity, is beneficial to intragastric injection application and can be adhered to the stomach, and the administration concentration of an infected part is improved; the antibacterial effect is generated only under the gastric acid condition, the helicobacter pylori biological membrane can be destroyed, and the helicobacter pylori antibacterial agent can be applied to the helicobacter pylori which is difficult to eradicate in the stomach of the human body.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a preparation method and application of a pH-responsive dual synergistic therapy hydrogel. Background Art

[0002] Helicobacter pylori (Hp) is one of the most common global pathogens that colonize the gastric mucosa, accounting for more than 50% of natural bacterial infections worldwide. The infection rate in developing countries exceeds that in developed countries. A large number of studies have shown that Hp can cause chronic inflammation of the gastric mucosa and is an important factor in causing digestive diseases such as gastric cancer, chronic gastritis and peptic ulcers. Hp infection is a major public health issue facing my country and the world today.

[0003] Currently, antibiotics are the main drugs for eradicating H. pylori, but due to the abuse of antibiotics, H. pylori resistance is increasing. Antibiotics also have other disadvantages: instability under acidic conditions, causing side effects such as nausea and vomiting and intestinal flora imbalance. Therefore, eradicating drug-resistant Helicobacter pylori is still a serious problem.

[0004] One of the important reasons why Hp easily develops drug resistance is its ability to form biofilms. When Hp senses an adverse external environment, it will embed itself into the extracellular polymers secreted by itself to form a multicellular three-dimensional structure, namely a biofilm. Biofilm is a complex bacterial micro-ecosystem composed of living microbial cells and dead cells and a wide range of self-produced extracellular polymers, including polysaccharides, nucleic acids and proteins, formed by bacteria adhering to the surface of biological or non-biological materials in order to adapt to the living environment during growth. Unlike planktonic bacteria, colonies that form biofilm structures are highly resistant to harsh external environments, including antibiotic exposure. According to studies, when bacteria produce biofilms, their resistance to antibiotics increases by 10-1000 times. Therefore, the formation of biofilms increases the difficulty of treating Hp and ultimately leads to chronic persistent infection of Hp.

[0005] Several studies have shown that disruption of the extracellular polymeric substance (EPS) matrix in established biofilms can significantly facilitate the penetration of fungicides, thereby improving bactericidal efficiency. For example, nitric oxide (NO) can downregulate the level of cyclic dimeric guanosine monophosphate (c-di-GMP) in EPS, leading to biofilm dispersal.

[0006] Hydroxyl radical (OH - ) is a kind of active oxygen, excess OH - It can directly mediate cell membrane damage and nucleic acid degradation, enhance membrane permeability and cause leakage of intracellular substances, ultimately leading to bacterial growth inhibition and death. -It has bactericidal effects on Gram-positive and Gram-negative bacteria, viruses and fungi, even multi-drug resistant strains. It can also destroy biofilms and prevent biofilm formation. Summary of the invention

[0007] In view of the existing problems, the present invention provides a preparation method and application of a pH-responsive dual synergistic therapy hydrogel based on the characteristics of acidic triggering and anti-Helicobacter pylori biofilm. The hydrogel has adhesive properties targeting the gastric mucus layer, increasing the drug concentration at the infection site, and releasing NO and OH in response to acid by loading antibacterial materials. - The biofilm is destroyed to achieve the eradication of Helicobacter pylori in vivo, and no toxic OH is produced under normal physiological conditions. - To protect the symbiotic intestinal flora.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions: A method for preparing a pH-responsive dual synergistic therapy hydrogel comprises the following steps: S1: Dissolve fucoidan in deionized water, add sodium periodate, react at room temperature in the dark for 6 hours, then add ethylene glycol, continue to react at room temperature in the dark for 1 hour to quench excess sodium periodate, dialyze the resulting reaction solution in deionized water for 2 days, and freeze-dry to obtain oxidized fucoidan; S2: Add zinc acetate to anhydrous ethanol, ultrasonically treat until completely dissolved, add polyvinyl pyrrolidone, treat again until completely dissolved, then add NH3·H2O under stirring, stir at room temperature for 10 minutes, then add H2O2 under stirring, stir at room temperature, continue stirring for 1 hour after the solution becomes milky white turbid, centrifuge and wash to collect the precipitate to obtain zinc peroxide; disperse zinc peroxide in anhydrous ethanol, add copper chloride, stir at room temperature for 1 hour, centrifuge and wash to collect the precipitate, and vacuum dry to obtain zinc peroxide / copper nanoparticles; S3: Oxidized fucoidan and carboxymethyl chitosan were respectively prepared into solutions with PBS buffer; the oxidized fucoidan solution and the carboxymethyl chitosan solution were mixed, zinc peroxide / copper nanoparticles and arginine were added, and stirred at room temperature until they were in a gel state, thereby obtaining a pH-responsive dual synergistic therapy hydrogel.

[0009] Furthermore, in the above step S1, the mass ratio of fucoidan to sodium periodate is 1:1.7.

[0010] Furthermore, in the above step S2, the mass ratio of zinc acetate to polyvinyl pyrrolidone is 0.566:1.4.

[0011] Furthermore, in the above step S2, the mass ratio of zinc peroxide to cupric chloride is 1:1.

[0012] Furthermore, in the above step S3, the concentration of the PBS buffer is 0.01 M and the pH value is 7.4.

[0013] Furthermore, in the above step S3, the concentrations of the oxidized fucoidan solution and the carboxymethyl chitosan solution are both 6 wt %.

[0014] Furthermore, in the above step S3, the mass ratio of oxidized fucoidan, carboxymethyl chitosan, zinc peroxide / copper nanoparticles and arginine in the hydrogel is 0.03:0.03:1:1.

[0015] A pH-responsive dual synergistic therapy hydrogel prepared by the preparation method.

[0016] The application of the above-mentioned dual synergistic therapy hydrogel in the preparation of anti-Helicobacter pylori drugs.

[0017] Furthermore, the above-mentioned anti-Helicobacter pylori drugs include drugs that kill Helicobacter pylori and drugs that destroy Helicobacter pylori biofilms.

[0018] The significant advantages of the present invention are: (1) ZCLA@OFC hydrogel has good adhesion, which is beneficial to increase the drug concentration in the gastric infection area, and ZCLA@OFC hydrogel can be degraded in the gastric fluid environment.

[0019] (2) ZCLA@OFC hydrogel has good injectability and self-healing properties, which is conducive to gastric injection administration.

[0020] (3) ZCLA@OFC hydrogel can be used without antibiotics and with NO and OH - Dual synergistic therapy achieves acid-responsive protection against Helicobacter pylori and its biofilm. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Infrared characterization images of oxidized fucoidan and carboxymethyl chitosan before and after cross-linking. Figure 2 : XRD pattern of Zn / Cu nanoparticles.

[0022] Figure 3 : Scanning electron microscopy images of zinc peroxide / copper nanoparticles.

[0023] Figure 4 : Injectability and self-healing properties of ZCLA@OFC hydrogel.

[0024] Figure 5 : Adhesion of ZCLA@OFC hydrogel to different materials.

[0025] Figure 6 : Photos of ZCLA@OFC hydrogel before and after gelation.

[0026] Figure 7 : Swelling property and degradation rate of ZCLA@OFC hydrogel at different pH values.

[0027] Figure 8 : Rheological properties of ZCLA@OFC hydrogel and OFC hydrogel.

[0028] Fig. 9 : Performance of ZCLA@OFC hydrogel in producing H2O2 at different pH values.

[0029] Fig.10 : OH production by ZCLA@OFC hydrogel at different pH values - performance.

[0030] Fig.11 : NO production performance of ZCLA@OFC hydrogel at different pH values.

[0031] Fig.12 : Killing performance of OFC hydrogel, ZC@OFC hydrogel and ZCLA@OFC hydrogel against Helicobacter pylori at different pH values.

[0032] Fig.13 : Performance of OFC hydrogel, ZC@OFC hydrogel, and ZCLA@OFC hydrogel in destroying Helicobacter pylori biofilm at different pH values. DETAILED DESCRIPTION

[0033] In order to better understand the present invention, the present invention is further described below in conjunction with the implementation modes. It should be noted that the implementation modes do not constitute a limitation on the protection scope of the present invention.

[0034] The fucoidan (FU) involved in the following examples has CAS: 9072-19-9.

[0035] The polyvinylpyrrolidone (PVP) involved in the following examples has CAS number: 9003-39-8.

[0036] The carboxymethyl chitosan (CMCs) involved in the following examples has CAS: 83512-85-0.

[0037] Embodiment 1: This embodiment provides a method for preparing a pH-responsive dual synergistic therapy hydrogel, which is carried out according to the following steps: S1: Dissolve 1g of fucoidan in 100mL of deionized water, add 1.7g of sodium periodate, react at room temperature in the dark for 6h, then add 10mL of ethylene glycol, continue to react at room temperature in the dark for 1h, transfer the resulting reaction solution to a dialysis bag with a molecular weight cutoff of 8000-14000g / mol, dialyze with deionized water for 2 days, and freeze-dry after dialysis to obtain oxidized fucoidan (OFU).

[0038] S2: Add 0.566g zinc acetate to 60mL anhydrous ethanol, ultrasonically treat until completely dissolved, then add 1.4g polyvinyl pyrrolidone, ultrasonically treat again until completely dissolved, add 4mL NH3·H2O under stirring, stir and react at room temperature for 10min, then add 800μL H2O2 under stirring, stir and react at room temperature, continue to stir and react for 1h after the solution becomes milky turbid, centrifuge, collect the precipitate, wash the collected precipitate with anhydrous ethanol by centrifugation 3 times to obtain zinc peroxide; add 100mg zinc peroxide to 60mL anhydrous ethanol, then add 100mg copper chloride, stir and react at room temperature for 1h, centrifuge, collect the precipitate, wash the collected precipitate with anhydrous ethanol by centrifugation 3 times, then vacuum dry at 60℃ for 12h, grind into powder to obtain zinc peroxide / copper nanoparticles (ZnO2-Cu).

[0039] S3: The oxidized fucoidan was prepared with PBS buffer (0.01 mol / L, pH 7.4) to a concentration of 6 wt% to obtain an oxidized fucoidan solution; carboxymethyl chitosan was prepared with PBS buffer (0.01 mol / L, pH 7.4) to a concentration of 6 wt% to obtain a carboxymethyl chitosan solution; 0.5 mL of the oxidized fucoidan solution and 0.5 mL of the carboxymethyl chitosan solution were mixed evenly, 1 mg of zinc peroxide / copper nanoparticles and 1 mg of L-arginine were added, and the mixture was stirred at room temperature for 16 s until it became a gel, thereby obtaining a pH-responsive dual synergistic therapy hydrogel (ZCLA@OFC).

[0040] Comparative Example 1: This comparative example provides a method for preparing a hydrogel, which is carried out according to the following steps: S1: Dissolve 1g of fucoidan in 100mL of deionized water, add 1.7g of sodium periodate, react at room temperature in the dark for 6h, then add 10mL of ethylene glycol, continue to react at room temperature in the dark for 1h, transfer the resulting reaction solution to a dialysis bag with a molecular weight cutoff of 8000-14000g / mol, dialyze with deionized water for 2 days, and freeze-dry after dialysis to obtain oxidized fucoidan (OFU).

[0041] S2: The oxidized fucoidan was prepared with PBS buffer (0.01 mol / L, pH 7.4) to a concentration of 6 wt% to obtain an oxidized fucoidan solution; carboxymethyl chitosan was prepared with PBS buffer (0.01 mol / L, pH 7.4) to a concentration of 6 wt% to obtain a carboxymethyl chitosan solution; 0.5 mL of the oxidized fucoidan solution and 0.5 mL of the carboxymethyl chitosan solution were mixed evenly, and stirred at room temperature for 12 s until it became a gel, to obtain a hydrogel (OFC).

[0042] Comparative Example 2: This comparative example provides a method for preparing a hydrogel, which is carried out according to the following steps: S1: Dissolve 1g of fucoidan in 100mL of deionized water, add 1.7g of sodium periodate, react at room temperature in the dark for 6h, then add 10mL of ethylene glycol, continue to react at room temperature in the dark for 1h, transfer the resulting reaction solution to a dialysis bag with a molecular weight cutoff of 8000-14000g / mol, dialyze with deionized water for 2 days, and freeze-dry after dialysis to obtain oxidized fucoidan (OFU).

[0043] S2: Add 0.566g zinc acetate to 60mL anhydrous ethanol, ultrasonically treat until completely dissolved, then add 1.4g polyvinyl pyrrolidone, ultrasonically treat again until completely dissolved, add 4mL NH3·H2O under stirring, stir and react at room temperature for 10min, then add 800μL H2O2 under stirring, stir and react at room temperature, continue to stir and react for 1h after the solution becomes milky turbid, centrifuge, collect the precipitate, wash the collected precipitate with anhydrous ethanol by centrifugation 3 times to obtain zinc peroxide; add 100mg zinc peroxide to 60mL anhydrous ethanol, then add 100mg copper chloride, stir and react at room temperature for 1h, centrifuge, collect the precipitate, wash the collected precipitate with anhydrous ethanol by centrifugation 3 times, then dry it in vacuo at 60℃ for 12h, grind it into powder to obtain zinc peroxide / copper nanoparticles (ZnO2-Cu).

[0044] S3: The oxidized fucoidan was prepared with PBS buffer (0.01 mol / L, pH 7.4) to a concentration of 6 wt% to obtain an oxidized fucoidan solution; carboxymethyl chitosan was prepared with PBS buffer (0.01 mol / L, pH 7.4) to a concentration of 6 wt% to obtain a carboxymethyl chitosan solution; 0.5 mL of the oxidized fucoidan solution and 0.5 mL of the carboxymethyl chitosan solution were mixed evenly, 1 mg of zinc peroxide / copper nanoparticles were added, and the mixture was stirred at room temperature for 12 s until it became a gel, to obtain a hydrogel (ZC@OFC).

[0045] Figure 1 The infrared characterization images of oxidized fucoidan before and after cross-linking with carboxymethyl chitosan prove the aldehyde formation of fucoidan and the cross-linking through imino bonds. Figure 2 X-ray diffraction pattern of zinc peroxide / copper nanoparticles. Figure 3 This is a scanning electron micrograph of zinc peroxide / copper nanoparticles.

[0046] Depend on Figure 4 It can be seen that when the rhodamine-stained ZCLA@OFC hydrogel and the unstained ZCLA@OFC hydrogel were cut in half and placed together, the two hydrogels of different colors recovered into a complete hydrogel within 5 minutes; the rhodamine-stained ZCLA@OFC hydrogel was placed in a syringe and injected with a syringe with a needle diameter of 0.41 mm to produce hydrogel filaments. This proves that the ZCLA@OFC hydrogel has good self-healing and injectability, which is beneficial for gastric injection applications.

[0047] The ZCLA@OFC hydrogel was fixed on different materials (skin, polypropylene, plastic, metal) by Figure 5 It can be seen that ZCLA@OFC hydrogel has good adhesion, which is conducive to adhering to gastric mucus to increase the drug delivery concentration at the infection site.

[0048] Figure 6 This is the actual picture of ZCLA@OFC hydrogel before and after gelation.

[0049] Figure 7 The swelling rate and degradation rate of ZCLA@OFC hydrogel in simulated gastric fluid (pH 1.5) and PBS buffer (0.01 mol / L, pH 7.4) are shown. It proves that ZCLA@OFC hydrogel has high swelling capacity and good biodegradability under gastric acid conditions.

[0050] Figure 8 The storage modulus (G′) and loss modulus (G″) of the hydrogel were obtained by using a rheometer in strain scanning mode. As can be seen from the figure, compared with OFC hydrogel, the mechanical properties of ZCLA@OFC hydrogel are improved and it still has self-healing properties.

[0051] KI was dissolved in simulated gastric fluid (pH 1.5) or PBS buffer (0.01 mol / L, pH 7.4) to prepare a 0.5 M working solution. Then, ZCLA@OFC hydrogel was incubated with 5 mL of the working solution. 200 μL of the mixed solution was added to a cuvette containing 1.8 mL of ultrapure water at regular intervals. The test was performed under a UV spectrophotometer to observe the changes in the characteristic peak at 350 nm and record the H2O2 production performance of ZCLA@OFC hydrogel. The results are shown in Fig. 9 .

[0052] TMB was dissolved in DMSO, and simulated gastric fluid (pH 1.5) or PBS buffer (0.01 mol / L, pH 7.4) was added to prepare a 40 μg / mL working solution. ZCLA@OFC hydrogel was then incubated with 5 mL of the working solution. 200 μL of the mixed solution was added to a cuvette containing 1.8 mL of ultrapure water at regular intervals. The test was performed under a UV spectrophotometer to observe the changes in the characteristic peak at 450 nm and record the OH production of ZCLA@OFC hydrogel. - The performance of Fig.10 .

[0053] The ZCLA@OFC hydrogel was placed in simulated gastric fluid (pH 1.5) or PBS buffer (0.01 mol / L, pH 7.4), and 200 μL of liquid was taken at regular intervals to test the NO concentration according to the instructions of the NO detection kit. The results are shown in Fig.11 .

[0054] The pre-cultured Helicobacter pylori was diluted with simulated gastric fluid (pH 1.5) or PBS buffer (0.01 mol / L, pH 7.4) to an OD 600 = 0.1 bacterial solution, then take 1mL of hydrogel and 1mL of Helicobacter pylori bacterial solution and culture them in a microaerobic environment (oxygen concentration is 10%) in a three-gas incubator for 2h, then take 100μl and apply it on the Columbia blood agar plate prepared in advance, continue to culture in the three-gas incubator for 3-5 days to observe the colony results, the results are shown in Fig.12 At pH = 7.4, each hydrogel had no antibacterial effect on Helicobacter pylori. At pH = 1.5, with the addition of zinc peroxide / copper nanoparticles and L-arginine, Helicobacter pylori was almost completely killed. This demonstrated the pH-responsive dual combined antibacterial function.

[0055] Take 100 μL of Helicobacter pylori diluted bacterial solution and add 10 mL of liquid culture medium to culture at 37°C in a microaerobic environment (oxygen concentration is 10%) for 48 to 72 h. Take 200 μL of the bacterial solution at the gas-liquid interface after culture and add it to a 48-well plate. Add 200 μL of liquid culture medium to each well and dilute the concentration of each well to 1×10 6 cfu / mL, incubated at 37℃ in a microaerobic environment (oxygen concentration is 10%) for 48h. After 3-5 days of culture, the upper culture medium was aspirated and added with 200μL hydrogel and 200μL simulated gastric juice with pH=1.5, incubated for 2h, then the bacteria were resuspended in phosphate buffer and dropped on the glass slide, fixed with glutaraldehyde fixative for 2h, the glass slide was taken out, rinsed with phosphate buffer, and then treated with gradient ethanol until completely dehydrated, sprayed with gold, and the biofilm was observed with a scanning electron microscope. The results are shown in Fig.13 .from Fig.13It can be seen that after the hydrogel and biofilm were co-cultured in a simulated gastric acid environment of pH = 1.5, the Helicobacter pylori biofilm was greatly destroyed, and the degree of destruction gradually increased with the addition of zinc peroxide / copper nanoparticles and L-arginine.

[0056] The above are all preferred implementations of the present invention and do not limit the protection scope of the present invention. All other implementation examples obtained by ordinary professionals in this technical field after reading the present invention without making any creative work are within the protection scope of the present invention.

Claims

1. A method for preparing a pH-responsive dual synergistic therapy hydrogel, characterized in that: The following steps are involved: S1: Dissolve fucoidan in deionized water, add sodium periodate, react at room temperature in the dark for 6 hours, then add ethylene glycol, continue to react at room temperature in the dark for 1 hour to quench excess sodium periodate, dialyze the resulting reaction solution in deionized water for 2 days, and freeze-dry to obtain oxidized fucoidan; S2: Add zinc acetate to anhydrous ethanol, ultrasonically treat until completely dissolved, add polyvinyl pyrrolidone, treat again until completely dissolved, then add NH3·H2O under stirring, stir at room temperature for 10 min, then add H2O2 under stirring, stir at room temperature, continue stirring for 1 h after the solution becomes milky white turbidity, centrifuge and wash to collect the precipitate, and obtain zinc peroxide; Disperse zinc peroxide in anhydrous ethanol, add copper chloride, stir and react at room temperature for 1 hour, collect the precipitate by centrifugation and washing, and vacuum dry to obtain zinc peroxide / copper nanoparticles; S3: Oxidized fucoidan and carboxymethyl chitosan were respectively prepared into solutions with PBS buffer; the oxidized fucoidan solution and the carboxymethyl chitosan solution were mixed, zinc peroxide / copper nanoparticles and arginine were added, and stirred at room temperature until they were in a gel state, thereby obtaining a pH-responsive dual synergistic therapy hydrogel.

2. The preparation method according to claim 1, characterized in that: In step S1, the mass ratio of fucoidan to sodium periodate is 1:1.

7.

3. The preparation method according to claim 1, characterized in that: In step S2, the mass ratio of zinc acetate to polyvinyl pyrrolidone is 0.566:1.

4.

4. The preparation method according to claim 1, characterized in that: In step S2, the mass ratio of zinc peroxide to cupric chloride is 1:

1.

5. The preparation method according to claim 1, characterized in that: In step S3, the concentration of PBS buffer is 0.01 M and the pH value is 7.

4.

6. The preparation method according to claim 1, characterized in that: In step S3, the concentrations of the oxidized fucoidan solution and the carboxymethyl chitosan solution are both 6 wt %.

7. The preparation method according to claim 1, characterized in that: In step S3, the mass ratio of oxidized fucoidan, carboxymethyl chitosan, zinc peroxide / copper nanoparticles and arginine in the hydrogel is 0.03:0.03:1:

1.

8. A pH-responsive dual synergistic therapy hydrogel prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the dual synergistic therapy hydrogel as claimed in claim 8 in the preparation of anti-Helicobacter pylori drugs.

10. The use according to claim 9, characterized in that: Anti-Helicobacter pylori drugs include drugs that kill Helicobacter pylori and drugs that destroy Helicobacter pylori biofilms.

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