An antibacterial material with on-demand activated cascade reaction and antibiotic synergy, and preparation method and application thereof

Abstract

The present invention belongs to the field of antibacterial technology, and specifically relates to an antibacterial material that is activated on demand and coordinated with an antibiotic in a cascade reaction, and a preparation method and application thereof. The antibacterial material is obtained by embedding antibiotics in Fe-ZIF-8, and then co-embedded with glucose oxidase in polyacrylamide hydrogel microspheres. The antibacterial material can use ATP secreted by bacteria to break the pH limit of Fe-ZIF-8, so that it also has relatively excellent catalytic activity under physiological conditions, and can smoothly cascade with glucose oxidase to produce ROS. After ROS destroys the cell wall of drug-resistant cells and oxidizes the proteins in the body, the antibiotics further act to avoid the repair of the bacterial cell wall, thereby achieving the purpose of completely killing bacteria on demand and reducing bacterial resistance.

Description

Technical Field

[0001] The present invention belongs to the field of antibacterial technology, and specifically relates to an antibacterial material with an on-demand activated cascade reaction and antibiotic synergy, and a preparation method and application thereof. Background Art

[0002] Bacterial infection is one of the main causes of human illness and death, posing a major threat to human safety. In clinical treatment, antibiotics are still the most frequently used and effective drugs. However, the frequent use of antibiotics will lead to an increase in highly harmful multidrug-resistant bacteria, putting public safety in a more severe predicament.

[0003] Reactive oxygen species (ROS), as a byproduct of cellular energy metabolism, participate in a variety of intracellular biochemical reactions. Studies have shown that ROS can destroy bacterial biofilms, oxidize their proteins and degrade DNA due to their oxidative properties, while avoiding the problem of drug resistance while killing bacteria, and play an important role in the treatment of bacterial infections. At present, ROS are mainly produced by the cascade reaction of natural enzymes and nanozymes. Compared with natural enzymes, nanozymes are more efficient in the research process due to their advantages such as easy synthesis, high stability and durability. In addition, nanozymes can also be modified to change their own properties to improve their overall catalytic activity and biocompatibility. Therefore, nanozymes show great application potential in different fields such as analysis, biomedicine and environmental chemistry. However, the extremely short half-life and extremely narrow diffusion distance of ROS are still a major challenge in antibacterial applications, and the excessive nonspecific ROS generated during the treatment process will cause serious damage to healthy tissues around the infected site. Therefore, it is urgent to develop an antibacterial material that can avoid drug resistance and have good antibacterial properties. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide an antibacterial material that can be activated on demand and can cooperate with antibiotics in a cascade reaction and a preparation method and application thereof, and specifically adopts the following technical solutions:

[0005] An antibacterial material with an on-demand activated cascade reaction and antibiotic synergy. The antibacterial material (AFeGp) is obtained by encapsulating antibiotics in Fe-ZIF-8 and then co-embedded with glucose oxidase (GOx) in polyacrylamide hydrogel microspheres.

[0006] The present invention selects Fe-ZIF-8 with excellent catalytic activity as a simulated enzyme. Compared with other nanozymes, Fe-ZIF-8 can show excellent enzyme activity under neutral conditions through the activation of ATP. At the same time, Fe-ZIF-8 also has the function of a carrier and has good stability under normal circumstances. Encapsulating antibiotics in Fe-ZIF-8 can avoid premature triggering of antibiotics. On this basis, antibiotics @Fe-ZIF-8 and glucose oxidase are co-embedded in polyacrylamide hydrogel microspheres. When bacteria are present, ATP secreted by bacteria can be used to activate the AFeGp (AMP@Fe-ZIF-8 / GOx@pAAm) antibacterial system. On the one hand, ATP can break the pH limit of Fe-ZIF-8, so that it also has relatively excellent catalytic activity under physiological conditions, thereby solving the problem that Fe-ZIF-8 can only show catalytic activity in a narrow pH range, so that Fe-ZIF-8 can smoothly cascade with glucose oxidase to produce ROS. In addition, this antibacterial system does not react in the absence of ATP, and produces ROS on demand, avoiding the damage to normal cells caused by excessive ROS under normal circumstances. On the other hand, due to the coordination between ATP and metal ions, Fe-ZIF-8 is responsive to ATP and can respond to release the antibiotics in the presence of ATP. After ROS destroys the cell wall of drug-resistant cells and oxidizes the proteins in their bodies, the antibiotics further act to avoid the repair of bacterial cell walls. The synergistic effect of the two can effectively kill multi-drug resistant bacteria in the case of low ROS content and low concentration of antibiotics. In addition, the hydrogel microspheres provide a microenvironment where antibiotics @Fe-ZIF-8 and glucose oxidase can coexist. The polyacrylamide in them is a three-dimensional grid structure similar to the extracellular matrix (ECM), which is conducive to the transfer of product intermediates of glucose oxidase and Fe-ZIF-8, improves the reaction efficiency of the cascade reaction, and ultimately achieves the purpose of killing bacteria on demand and reducing bacterial resistance through the dual functions of ATP.

[0007] As a further preferred embodiment, the antibiotic is AMP (ampicillin sodium). AMP, as an antibiotic drug, can be loaded in Fe-ZIF-8. In the presence of ATP secreted by bacteria, AMP@Fe-ZIF-8 can be decomposed and release AMP in response, thereby acting on bacteria.

[0008] The present invention also provides a method for preparing the above-mentioned on-demand activated cascade reaction and antibiotic-synergistic antibacterial material, comprising the following steps:

[0009] AMP, 2-methylimidazole and ferrous sulfate heptahydrate are mixed, and zinc acetate dihydrate is added and allowed to react for 20 min-50 min to obtain AMP@Fe-ZIF-8; AMP@Fe-ZIF-8, glucose oxidase, acrylamide, BIS and PI (2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone) are configured into an enzyme / pregel solution, and then the enzyme / pregel solution is gelated to obtain the antibacterial material.

[0010] As a further preferred embodiment, the ratio of the above AMP, 2-methylimidazole, ferrous sulfate heptahydrate and zinc acetate dihydrate is (18-22) mg: (5-9) mL: (0.3-0.5) mL: (1.5-1.7) mL. 2-methylimidazole and zinc acetate dihydrate can form a MOF framework by combining them in a fixed ratio to produce a carrier that can be used as an antibiotic AMP; after the addition of ferrous sulfate heptahydrate, MOF can have peroxidase-like activity, and at the same time, an appropriate content can maintain the original morphology of MOF.

[0011] As a further preferred embodiment, the ratio of AMP, 2-methylimidazole, ferrous sulfate heptahydrate and zinc acetate dihydrate is: 20 mg: 7 mL: 0.4 mL: 1.6 mL. Fe-ZIF-8 is a ZIF-8 synthesized by adding Fe 2+ The structure formed by ZIF-8 is mainly composed of organic ligand-2-methylimidazole and metal ion-Zn 2+ Coordination formation, changing the concentration of the corresponding reactants, the morphology of ZIF-8 will also change accordingly. The protonation of excess 2-methylimidazole and the solvent is the basis for the formation of Fe-ZIF-8 crystal nuclei. At the same time, excessive 2-methylimidazole will adsorb on the surface of the crystal nucleus to slow down the growth of the crystal nucleus. Therefore, with the increase of 2-methylimidazole concentration, the particle size of Fe-ZIF-8 will gradually decrease; zinc acetate dihydrate is the main metal ion source in the synthesis process, which is crucial to the formation of the material structure. If its concentration is too low or too high, it may be difficult to form a three-dimensional metal organic framework structure. For ferrous sulfate heptahydrate, if the concentration is too high, the color and morphology of the product will change. If the concentration is too low, it is difficult to give the MOF framework obvious enzyme-like activity. Therefore, this ratio has formed a stable three-dimensional framework structure and has excellent enzyme-like activity.

[0012] As a further preferred embodiment, the above-mentioned static reaction time is 30 min

[0013] As a further preferred embodiment, the static reaction further includes centrifugation and washing three times.

[0014] As a further preferred embodiment, the ratio of the above-mentioned AMP@Fe-ZIF-8, glucose oxidase, acrylamide, BIS and PI (2-hydroxy-4'-(2-hydroxyethoxy)-2-methylacetophenone) is 5 mg: 2 mg: 150 mg: 10 mg: 10 mg.

[0015] As a further preferred embodiment, the microfluidic UV polymerization technology is used to promote the gelation of the enzyme / pregel solution.

[0016] The beneficial effects of the present invention are:

[0017] The antibacterial material proposed by the present invention can be used in the preparation of medical materials. It can use ATP secreted by bacteria to break the pH limit of Fe-ZIF-8, so that it also has relatively excellent catalytic activity under physiological conditions, and can smoothly cascade with glucose oxidase to produce ROS. In addition, due to the coordination effect between ATP and metal ions, Fe-ZIF-8 is responsive to ATP. When ATP is present, it can respond to release the antibiotics therein. After ROS destroys the cell wall of the drug-resistant cell and oxidizes the protein in the body, the antibiotics further act to avoid the repair of the bacterial cell wall, thereby achieving the purpose of coordinated antibacterial on demand and reducing bacterial resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 Shown are scanning electron microscopy images of RBITC@Fe-ZIF-8 with and without ATP;

[0020] Figure 2 Shown is the comparison of biocatalytic activities of different reaction systems in the presence of glucose;

[0021] Figure 3 Shown is a comparison of the antibacterial properties of different reaction systems. DETAILED DESCRIPTION

[0022] The concept, specific structure and technical effects of the present invention will be clearly and completely described below in combination with the embodiments and drawings to fully understand the purpose, scheme and effect of the present invention.

[0023] Example 1

[0024] An antibacterial material with an on-demand activated cascade reaction and antibiotic synergy. The antibacterial material is obtained by encapsulating antibiotics in Fe-ZIF-8 and then co-embedded with glucose oxidase in polyacrylamide hydrogel microspheres. The antibiotic is AMP (ampicillin sodium).

[0025] Example 2

[0026] A method for preparing an antibacterial material that is activated on demand and synergistic with an antibiotic cascade reaction, specifically comprising the following steps:

[0027] S1: 20 mg of ampicillin sodium (AMP) was stirred in 7 mL of 2 M 2-methylimidazole solution, and then 0.4 mL of 200 mM ferrous sulfate heptahydrate solution was added and stirred, followed by 1.6 mL of 200 mM zinc acetate dihydrate and allowed to react for 30 min. The mixture was centrifuged and washed three times to obtain AMP@Fe-ZIF-8.

[0028] S2: 5 mg 5 mg / mL AMP@Fe-ZIF-8, 2 mg 2 mg / mL glucose oxidase, 150 mg acrylamide (5%wt), 10 mg BIS (1%wt) and 10 mg PI (2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone) (1%wt) were dissolved in 1 ml ultrapure water to prepare an enzyme / pregel solution, and then microfluidic UV technology was used to promote the gelation of the enzyme / pregel solution to obtain AFeGp.

[0029] Example 3

[0030] RBITC (rhodamine B isothiocyanate)@Fe-ZIF-8 was prepared according to the following steps, and its scanning electron micrographs in the presence / absence of ATP were compared, and its maximum emission peak was recorded using a fluorescence spectrophotometer to detect the response release ability of Fe-ZIF-8 under the action of ATP, which specifically included the following steps:

[0031] S1: 2 mg RBITC was stirred in 7 mL 2 M 2-methylimidazole solution, and then 0.4 mL 200 mM ferrous sulfate heptahydrate solution was added and stirred, followed by 1.6 mL 200 mM zinc acetate dihydrate, and the mixture was allowed to react for 30 min. The mixture was centrifuged and washed three times to obtain RBITC@Fe-ZIF-8.

[0032] S2: HEPES (hydroxyethylpiperazine ethanesulfonic acid) with pH = 7 was used as the buffer solution, with a total volume of 200 μL, a reaction temperature of 37 °C, and a reaction time of 30 min;

[0033] S3: Use three 0.5 mL centrifuge tubes, numbered a, b, and c;

[0034] S4: The solution in centrifuge tube a was prepared by mixing 5 μL ATP (200 mM) and 10 μL RBITC@Fe-ZIF-8 (200 μg / mL) in 185 μL HEPES buffer solution at pH = 7;

[0035] S5: The solution in centrifuge tube b was prepared as follows: 10 μL RBITC@Fe-ZIF-8 (200 μg / mL) was mixed in 190 μL HEPES buffer solution at pH = 7;

[0036] S6: The solution in centrifuge tube c was prepared as follows: 10 μL Fe-ZIF-8 (200 μg / mL) was mixed in 190 μL HEPES buffer solution at pH = 7;

[0037] S7: Place the three centrifuge tubes at 37°C for 30 min in the dark;

[0038] S8: Place the solution in a scanning electron microscope to observe the morphological changes, and use a fluorescence spectrophotometer to measure the relative fluorescence intensity of the solution.

[0039] Test results such as Figure 1 The scanning electron microscopy results are shown in Figure 1 a and 1 b, in the presence of ATP, the morphology of RBITC@Fe-ZIF-8 changed significantly and it was difficult to maintain the original configuration, indicating that Fe-ZIF-8 has structural responsiveness to ATP. Figure 1 c. When ATP is present, RBITC@Fe-ZIF-8 can release the embedded fluorescent substance RBITC, while there is no fluorescence peak in the absence of ATP, indicating that Fe-ZIF-8 has the ability to respond to ATP and release.

[0040] Example 4

[0041] The ROS activity experiment after the addition of glucose was used to record the intensity corresponding to the maximum absorption peak by a UV spectrophotometer to detect the biocatalytic activity of different catalytic systems with or without ATP, which specifically includes the following steps:

[0042] S1: 5 mg 5 mg / mL Fe-ZIF-8 or 5 mg 5 mg / mL AMP@Fe-ZIF-8, 150 mg acrylamide (15%wt), 10 mg BIS (1%wt) and 10 mg PI (2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone) (1%wt) were prepared into enzyme / pregel solution, and then microfluidic UV technology was used to promote the gelation of enzyme / pregel solution to obtain Fep or AFep;

[0043] S2: HEPES with pH = 7 was used as the buffer solution, the total volume was 200 μL, the reaction temperature was 37 °C, and the reaction time was 60 min;

[0044] S3: Use 8 0.5 mL centrifuge tubes, numbered a, b, c, d, e, f, g, h;

[0045] S4: The solution in centrifuge tube a was prepared by mixing 10 μL Fe-ZIF-8, 10 μL TMB (20 mM) and 10 μL glucose (200 mM) in 170 μL HEPES buffer solution at pH = 7; accordingly, 5 μL ATP (200 mM) was added to centrifuge tube b.

[0046] S5: The solution in centrifuge tube c was prepared by mixing 10 μL GOx, 10 μL Fep, 10 μL TMB (20 mM) and 10 μL glucose (20 mM) in 160 μL HEPES buffer solution at pH = 7; accordingly, 5 μL ATP (200 mM) was added to centrifuge tube d.

[0047] S6: The solution in centrifuge tube e was prepared as follows: 10 μL GOx, 10 μL AFep, 10 μL TMB (20 mM) and 10 μL glucose (200 mM) were mixed in 160 μL HEPES buffer solution at pH = 7; accordingly, 5 μL ATP (200 mM) was added to the solution in centrifuge tube f.

[0048] S7: The solution in centrifuge tube g was prepared by mixing 10 μL AFeGp, 10 μL TMB (20 mM) and 10 μL glucose (200 mM) in 170 μL HEPES buffer solution at pH = 7; accordingly, 5 μL ATP (200 mM) was added to centrifuge tube h.

[0049] S8: The content of Fe-ZIF-8 or GOx in all centrifuge tubes was kept consistent;

[0050] S9: Place 8 centrifuge tubes at a reaction temperature of 37°C for 60 min in the dark, and use a UV spectrophotometer to measure the absorbance of the solutions.

[0051] Test results such as Figure 2 As shown in the figure, in the absence of ATP, each component has no color development effect, and the absorbance value is almost 0; after adding ATP, since the substrate is glucose, Fe-ZIF-8 still does not develop color, while GOx + Fep, GOx + AFep and AFeGp all have obvious ultraviolet absorption, and the ultraviolet absorption of AFeGp is ​​significantly higher. This shows that the microenvironment provided by the hydrogel microspheres is conducive to the transfer of product intermediates between glucose oxidase and Fe-ZIF-8, thereby improving the efficiency of the cascade reaction. At the same time, there is almost no difference in the absorption intensity of GOx + Fep and GOx + AFep, indicating that the encapsulation of AMP has almost no effect on the enzyme-like activity of Fe-ZIF-8.

[0052] Example 5

[0053] The antibacterial properties of different reaction systems: Blank, AMP, GOx + Fe-ZIF-8, FeGp, GOx + AMP@Fe-ZIF-8 and AFeGp were compared, and then cultured on agar plates and the number of colonies was counted, which specifically included the following steps:

[0054] S1: 25 g / L broth medium was used as the solution, the total volume was 2 mL, the reaction temperature was 37 °C, and the reaction time was 30 h;

[0055] S2: Use 6 5 mL glass tubes, numbered a, b, c, d, e, and f;

[0056] S3: The solution in glass tube a was prepared by dissolving 60 μL AFeGp in 2 mL containing 1×10 8 (CFU) / mL MRSA (methicillin-resistant Staphylococcus aureus) in broth solution;

[0057] S4: The solution in glass tube b was prepared by mixing 60 μL of the same concentration of GOx + AMP@Fe-ZIF-8 mixed solution in 2 mL containing 1×10 8 (CFU) / mL MRSA in broth solution;

[0058] S5: The solution in glass tube c was prepared by mixing 60 μL of FeGp mixture with the same concentration in 2 mL of 1×10 8(CFU) / mL MRSA in broth solution;

[0059] S6: The solution in glass tube d was prepared by mixing 60 μL of the same concentration of GOx + Fe-ZIF-8 mixed solution with 2 mL of 1×10 8 (CFU) / mL MRSA in broth solution;

[0060] S7: The solution in glass tube e was prepared by mixing 60 μL of the same concentration of AMP mixed solution with 2 mL of 1×10 8 (CFU) / mL MRSA in broth solution;

[0061] S8: The solution in glass tube f is prepared as follows: 2 mL contains 1×10 8 (CFU) / mL broth solution of MRSA;

[0062] S9: Place the six glass tubes in a 37°C humidified incubator for 30 h;

[0063] S10: Then culture on an agar plate and count the number of colonies.

[0064] Test results such as Figure 3 As shown in the figure, the plate containing AFeGp almost completely sterilized after 30 h, while a large number of active bacteria still existed on the MRSA treated with Blank, AMP, GOx + Fe-ZIF-8, FeGp and GOx + AMP@Fe-ZIF-8 mixture. Therefore, AFeGp has higher antibacterial activity because, on the one hand, ATP breaks the pH limit of Fe-ZIF-8, allowing Fe-ZIF-8 to smoothly produce ROS in the glucose oxidase cascade. On the other hand, due to the responsiveness of Fe-ZIF-8 to ATP, antibiotics are released in response. After ROS destroys the cell wall of drug-resistant cells and oxidizes the proteins in their bodies, antibiotics further act to avoid the repair of bacterial cell walls. Compared with the control group, the plate containing AFeGp almost completely prevented colony formation after 30 h, while a large number of colonies still existed on the plates treated with Blank, AMP, GOx + Fe-ZIF-8, FeGp and GOx + AMP@Fe-ZIF-8 mixture.

[0065] In summary, the present invention proposes a method for preparing a synergistic antibacterial system, and uses the protective effect of hydrogel on the spatial conformation of enzymes and the loading of antibiotics by nano-MOFs with enzyme-like activity to prepare AFeGp synergistic antibacterial materials, thereby achieving efficient and controllable antibacterial effects. In this antibacterial system, through the activation and response release of ATP, AFeGp activates the activity of nanoenzymes to produce ROS to destroy the cell walls of drug-resistant cells, oxidizes the proteins in the cells, and then further avoids the repair of bacterial cell walls through the response-released antibiotics, thereby achieving the on-demand synergistic bactericidal effect of reactive oxygen and antibiotic drug molecules.

[0066] Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Mentioning "embodiment" in this article means that the specific features, structures or characteristics described in conjunction with the embodiment may be included in at least one embodiment of the present embodiment application. The phrase appearing in various positions in the specification is not necessarily the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. It can be understood explicitly and implicitly by those skilled in the art that the embodiments described herein can be combined with other embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application. Although the embodiments of the present invention have been shown and described, it can be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

Claims

1. An antibacterial material with an on-demand activated cascade reaction and antibiotic synergy, characterized in that: The antibacterial material is obtained by embedding antibiotics in Fe-ZIF-8 and then co-embedding with glucose oxidase in polyacrylamide hydrogel microspheres; The antibiotic is AMP (ampicillin sodium); The method for preparing the antibacterial material comprises the following steps: AMP, 2-methylimidazole and ferrous sulfate heptahydrate are mixed, and zinc acetate dihydrate is added and allowed to react for 20 min-50 min to obtain AMP@Fe-ZIF-8; AMP@Fe-ZIF-8, glucose oxidase, acrylamide, BIS (N,N'-methylenebisacrylamide) and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylacetophenone are configured into an enzyme / pregel solution, and then the enzyme / pregel solution is gelated to obtain the antibacterial material.

2. The antibacterial material according to claim 1, characterized in that: The ratio of AMP, 2-methylimidazole, ferrous sulfate heptahydrate and zinc acetate dihydrate is (18-22) mg: (5-9) mL: (0.3-0.5) mL: (1.5-1.7) mL.

3. The antibacterial material according to claim 2, characterized in that: The ratio of AMP, 2-methylimidazole, ferrous sulfate heptahydrate, and zinc acetate dihydrate is 20 mg: 7 mL: 0.4 mL: 1.6 mL.

4. The antibacterial material according to claim 1, characterized in that: The static reaction time is 30 min.

5. The preparation method according to claim 1, characterized in that: The static reaction further includes centrifugation and washing three times.

6. The antibacterial material according to claim 1, characterized in that: The ratio of the amount of AMP@Fe-ZIF-8, glucose oxidase, acrylamide, BIS and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylacetophenone is 5 mg: 2 mg: 150 mg: 10 mg: 10 mg.

7. The antibacterial material according to claim 1, characterized in that: Microfluidic UV polymerization technology was used to promote the gelation of the enzyme / pregel solution.

8. Use of the antibacterial material according to any one of claims 1 to 7 in the preparation of medical materials.

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