A composite material for controlling silver ion sustained release by glucose and its preparation method and application

Abstract

The present invention belongs to the field of antibacterial technology, and specifically relates to a composite material for controlling the sustained release of silver ions by glucose, and a preparation method and application thereof. The composite material is obtained by co-burying AgNPs@UiO‑66 and glucose oxidase in acrylamide hydrogel microspheres. The composite material can be used in antibacterial materials, which can not only enrich nanosilver, but also effectively avoid the formation of aggregated nanosilver and the use of high-dose nanosilver. At the same time, harmless glucose can be used to catalyze glucose oxidase to generate hydrogen peroxide, which greatly improves the antibacterial efficiency, thereby enabling AgNPs@UiO‑66 to slowly release a large amount of Ag on demand. + To achieve the purpose of antibacterial.

Description

Technical Field

[0001] The invention belongs to the technical field of antibacterial, and in particular relates to a composite material for controlling the sustained release of silver ions using glucose, and a preparation method and application thereof. Background Art

[0002] Bacterial wound infection has become a global health threat due to its high morbidity and mortality. The use of antibiotics is the most common and effective method in traditional antibacterial treatment, but bacterial resistance caused by its abuse has become an urgent problem that needs to be solved. Nanosilver (AgNPs) has become a new antibacterial method to replace antibiotics because of its small particles, small specific area, no drug resistance, and broad-spectrum antibacterial properties. The antibacterial principle of nanosilver is to release silver ions (Ag + ) and produce ROS for synergistic antibacterial effect. Under acidic conditions, AgNPs decompose and release Ag in the presence of hydrogen peroxide. + , accompanied by the formation of hydroxyl radicals. Ag + Antibacterial effects have long been reported, with micromolar levels of Ag + It can inhibit electron transfer in the respiratory chain or interfere with membrane permeability to protons and phosphate. In addition, higher concentrations of Ag + It also interacts with cytoplasmic components and nucleic acids. ROS are highly toxic to bacteria due to their strong oxidative properties and have no drug resistance. + It works synergistically with ROS to fight bacteria, greatly improving the antibacterial efficiency.

[0003] However, AgNPs are prone to aggregation, which affects the + The release of Ag, and high doses can lead to + The excessive release of AgNPs may lead to relatively high cytotoxicity. In addition, the high concentration of hydrogen peroxide required in the prior art will indiscriminately damage normal cells and bacteria. + New materials with targeted on-demand antibacterial properties are highly necessary. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide a composite material for controlling the sustained release of silver ions by glucose and a preparation method and application thereof. The specific scheme is as follows:

[0005] A glucose-controlled silver ion sustained-release composite material, wherein the composite material is obtained by co-embedding AgNPs@UiO-66 and glucose oxidase (GOx) in polyacrylamide (PAAm) hydrogel microspheres; and the AgNPs@UiO-66 is obtained by embedding nanosilver in UiO-66.

[0006] The UiO-66 used in the present invention is a zirconium-based metal organic framework (Zr-MOF) with the advantages of good stability, large specific surface area and low toxicity, and can be used as a carrier of metal nanoparticles. AgNPs are embedded in UiO-66, and AgNPs@UiO-66 is equivalent to Ag + The storage reservoir can not only enrich AgNPs, but also effectively avoid the formation of AgNPs aggregation and the use of high doses of AgNPs, which can continuously release Ag. + On this basis, AgNPs@UiO-66 and GOx were co-encapsulated in polyacrylamide hydrogel microspheres. The hydrogel provided a microenvironment in which AgNPs@UiO-66 and GOx could coexist. Polyacrylamide is a three-dimensional network structure similar to the extracellular matrix, which is conducive to the transfer of intermediates between AgNPs@UiO-66 and GOx. Therefore, polyacrylamide hydrogel microspheres can not only encapsulate a large amount of AgNPs@UiO-66 and glucose oxidase, enabling it to use glucose to catalyze glucose oxidase to generate hydrogen peroxide, but also can not only encapsulate a large amount of AgNPs@UiO-66 and glucose oxidase ... + Under the premise of releasing, the transfer of intermediate products is shortened, thereby increasing the Ag content in AgNPs@UiO-66. + The release efficiency of Ag is improved and the controlled release triggered by glucose is realized. On the one hand, it avoids the problem of indiscriminate damage to normal cells and bacteria caused by the use of high-concentration hydrogen peroxide in the prior art. On the other hand, it also truly realizes the Ag + The efficient and controlled release further avoids the high concentration of Ag + Damage to normal cells.

[0007] As a further preferred embodiment, the above-mentioned AgNPs are obtained by dissolving AgNO3 in water, stirring and heating to boiling, then adding 1% sodium citrate, continuing heating for 30 minutes, and cooling to room temperature. Sodium citrate reduction method is one of the commonly used methods for synthesizing precious metal nanocrystals in aqueous phase. Sodium citrate has good biocompatibility. At the same time, since citrate is a weaker ligand, it is easier to exchange ligands with other ligands or biomolecules. The surface of the nanoparticles is stabilized by the coordination of citrate, and the stable conformation of citrate on the surface of silver nanoparticles is confirmed. In addition, the sodium citrate reduction method can also well control the size and morphology of nanosilver, which is conducive to the application of silver nanoparticles in biological systems.

[0008] The present invention also provides a method for preparing the above-mentioned glucose-controlled silver ion sustained-release composite material, comprising the following steps:

[0009] S1: AgNPs@UiO-66 was obtained by solvothermal reaction of nanosilver, 1,4-phthalic acid and zirconium chloride tetrahydrate;

[0010] S2: Glucose oxidase, AgNPs@UiO-66, acrylamide, BIS and PI were prepared into 1 mL enzyme / pregel solution, and then the pregel solution was gelled to obtain AgNPs@UiO-66 / GOx@pAAm.

[0011] As a further preferred implementation, the specific process of S1 above is as follows:

[0012] 1,4-Benzenedicarboxylic acid, nanosilver and zirconium chloride tetrahydrate were dissolved in a solvent, acetic acid was added, ultrasound was applied, and then heated at 80°C-100°C for 15-20 hours to obtain AgNPs@UiO-66.

[0013] As a further preferred embodiment, the solvent is preferably N,N-dimethylformamide.

[0014] As a further preferred embodiment, the ratio of the above 1,4-phthalic acid, nanosilver, zirconium chloride tetrahydrate, N,N-dimethylformamide and acetic acid is 100 mg: 10 mg: 8 mL: 4 mL. Zirconium chloride tetrahydrate provides Zr 4+ UiO-66 is formed by constructing with dicarboxylic acid ligands, and the synthesis of UiO-66 can be adjusted by changing the amount of 1,4-phthalic acid. As the concentration increases, the size of UiO-66 crystals will become larger and larger, and it will also become a single crystal. Acetic acid can systematically adjust the linker site, but the pore surface area of ​​UiO-66 will change with the change of acetic acid concentration. The size of UiO-66 synthesized in this ratio is about 200nm, which is small in size and large in specific surface area, and is more suitable in the present invention.

[0015] As a further preferred embodiment, the heating temperature is 90° C. and the heating time is 18 hours.

[0016] As a further preferred embodiment, the ratio of acrylamide, BIS and PI is 150mg:10mg:10mg. Acrylamide is used as a coagulant, and under the irradiation of ultraviolet light, acrylamide polymerizes into polyacrylamide; BIS and PI are used as cross-linking agents to increase the polymerization rate.

[0017] As a further preferred embodiment, the microfluidics technology is used to promote the gelation of the enzyme / pregel solution.

[0018] The beneficial effects of the present invention are:

[0019] The glucose-controlled silver ion sustained-release composite material proposed in the present invention can be applied to antibacterial materials. It can not only enrich nanosilver, but also effectively avoid the formation of nanosilver aggregation and the use of high-dose nanosilver. At the same time, it can use harmless glucose to catalyze glucose oxidase to generate hydrogen peroxide, which greatly improves the antibacterial efficiency, thereby enabling AgNPs@UiO-66 to slowly release a large amount of Ag on demand. + To achieve the purpose of antibacterial. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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.

[0021] Figure 1 Shown are SEM images of UiO-66 and Ag@UiO-66: (a) UiO-66; (b) Ag@UiO-66;

[0022] Figure 2 Shown are SEM images of AgNPs@UiO-66 / GOx@pAAm: (a) overall view; (b) cross-section; (c) pore details;

[0023] Figure 3 Shown is the absorbance of different components of Silane;

[0024] Figure 4 Shown is a graph of the antimicrobial performance of different components. DETAILED DESCRIPTION

[0025] The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments and drawings to fully understand the purpose, scheme and effects of the present invention.

[0026] Example 1

[0027] A composite material for glucose-controlled silver ion sustained release, wherein the composite material is composed of 20 mg of AgNPs@UiO-66 with a concentration of 20 mg / mL and 2 mg of glucose oxidase (GOx) with a concentration of 2 mg / mL co-encapsulated in polyacrylamide (PAA m) hydrogel microspheres (obtained by gelation of 0.15g acrylamide (15% wt), 0.01g BIS (1% wt), and 0.01g PI (1% wt)); the above-mentioned AgNPs@UiO-66 was obtained by embedding 10mg nanosilver in UiO-66 (zirconium chloride tetrahydrate (30mg, 0.066mmol) was dissolved in 8mL N,N-dimethylformamide, and 4.0mL acetic acid was added to the reaction mixture. After 60s of ultrasonic treatment, the solution was heated at 90°C for 18 hours). The above-mentioned AgNPs were obtained by dissolving 18mg AgNO3 in 100mL water, stirring and heating to boiling, then adding 2mL 1% sodium citrate, continuing heating for 30min, and cooling to room temperature.

[0028] Example 2

[0029] A method for preparing a composite material for glucose-controlled silver ion sustained release, comprising the following steps:

[0030] S1: Dissolve 18 mg of AgNO3 in 100 mL of distilled water, heat to boiling under vigorous stirring, then add 2 mL of 1% sodium citrate to the boiling solution, continue heating for 30 min, and cool to room temperature to obtain the obtained AgNPs, which are stored in a dark bottle;

[0031] S2: 1,4-Benzenedicarboxylic acid (100 mg, 0.60 mmol) was added to a stainless steel autoclave, 10 mg of AgNPS and zirconium chloride tetrahydrate (30 mg, 0.066 mmol) were dissolved in 8 mL of N,N-dimethylformamide, and 4.0 mL of acetic acid was added to the reaction mixture. After 60 s of ultrasonic treatment, the solution was heated at 90 °C for 18 h to obtain AgNPs@UiO-66 particles;

[0032] S3: Weigh 2 mg / mL GOx, 20 mg / mL AgNPs@UiO-66, 0.15 g (15% wt) acrylamide, 0.01 g BIS (1% wt), and 0.01 g PI (1% wt), and prepare 1 mL of enzyme / pregel mixed solution. Then, the enzyme / pregel solution is gelled using microfluidic technology to obtain AgNPs@UiO-66 / GOx@pAAm.

[0033] Example 3

[0034] After adding glucose, Ag +The release experiment records the absorbance of different components of the test silver spirit, which specifically includes the following steps:

[0035] S1: phosphate buffer (PBS) with pH = 7.4 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;

[0036] S2: Use 4 0.5 mL centrifuge tubes, numbered a, b, c, and d;

[0037] S3: The solution in centrifuge tube a is prepared by mixing 10 μL glucose (200 mM) and 40 μL trypsin (0.02%) in 165 μL PBS buffer solution at pH = 7.4;

[0038] S4: The solution in centrifuge tube b was prepared by mixing 5 μL AgNPs (10 mg / mL), 5 μL GOx (200 μg / mL), 10 μL glucose (200 mM) and 40 μL trypsin (0.02%) in 165 μL PBS buffer solution at pH = 7.4;

[0039] S5: The solution in centrifuge tube c was prepared by mixing 5 μL AgNPs@UiO-66(AU) (10 mg / mL), 5 μL GOx (200 μg / mL), 10 μL glucose (200 mM) and 40 μL trypsin (0.02%) in 170 μL PBS buffer solution at pH = 7.4;

[0040] S6: The solution in centrifuge tube d was prepared by mixing 5 μL AgNPs@UiO-66 / GOx@pAAm(AUGP) (10 mg / mL), 5 μL GOx (200 μg / mL), 10 μL glucose (200 mM) and 40 μL trypsin (0.02%) in 160 μL PBS buffer solution at pH = 7.4;

[0041] S7: Place the four centrifuge tubes at 37°C for 60 min in the dark.

[0042] Test results such as Figure 3 Compared with the mixed AgNPs+GOx and AgNPs@UiO-66(AU)+GOx systems, the construction of the AgNPs@UiO-66 / GOx@pAAm(AUGP) system did not affect the glucose-triggered Ag + It can be slowly released from the nanocarrier under the condition of

[0043] Example 4

[0044] Different reaction systems: Blank, UiO-66, pAAm, GOx, AgNPs, Au, Ag + The antibacterial properties of GOx+AU and AUG P were compared, and then cultured on agar plates and the number of colonies was counted, which specifically included the following steps:

[0045] S1: 25 g / L broth medium was used as the solution, the total volume was 2.2 mL, the reaction temperature was 37 °C, and the reaction time was

[0046] 30h;

[0047] S2: Use 9 5 mL glass tubes, numbered a, b, c, d, e, f, g, h, i;

[0048] S3: The solution in glass tube a is prepared as follows: 60 μL AUGP in 2.15 mL containing 1×10 8 (CFU) / mL S.aureus (Staphylococcus aureus) in broth medium solution;

[0049] S4: The solution in glass tube b is prepared by mixing 60 μL of sterile water in 2.15 mL of 1×10 8 (CFU) / mL S. aureus in broth medium solution;

[0050] S5: The solution in glass tube c was prepared by mixing 60 μL of the same concentration of UiO-66 mixed solution with 2.15 mL of 1×10 8 (CFU) / mL S. aureus in broth medium solution;

[0051] S6: The solution in glass tube d was prepared by mixing 60 μL of pAAm mixture with the same concentration in 2.15 mL of 1×10 8 (CFU) / mL S. aureus in broth medium solution;

[0052] S7: The solution in glass tube e was prepared by mixing 60 μL of the same concentration of GOx mixed solution with 2.15 mL of 1×10 8 (CFU) / mL S. aureus in broth medium solution;

[0053] S8: The solution in glass tube f was prepared by mixing 60 μL of the same concentration of AgNPs mixed solution with 2.15 mL of 1×10 8 (CFU) / mL S. aureus in broth medium solution;

[0054] S9: The solution in glass tube g is prepared by: 60 μL of the same concentration of Au mixed solution is added to 2.15 mL containing 1×10 8(CFU) / mL S. aureus in broth medium solution;

[0055] S10: The solution in glass tube h is prepared as follows: 60 μL of the same concentration of Ag + The mixture contained 1×10 8 (CFU) / mL S. aureus in broth medium solution;

[0056] S11: The solution in glass tube i was prepared by mixing 60 μL of GOx+AU mixed solution of the same concentration in 2.15 mL containing 1×10 8 (CFU) / mL S. aureus in broth medium solution;

[0057] S12: 9 glass tubes were placed in a 37°C wet incubator for 30 hours;

[0058] S13: Then culture on an agar plate and count the number of colonies.

[0059] Test results such as Figure 4 As shown in Figure 2, the plate containing AUGP almost achieved complete sterilization, while a large number of bacteria were still found to survive in S. aureus treated with UiO-66, pAAm, and GOx alone, and a small number of bacteria died in S. aureus treated with free AgNPs and Au. The antibacterial activity of the mixture of GOx and AU was slightly higher than that of free Ag. + This is because glucose catalyzes glucose oxidase to generate hydrogen peroxide, triggering the release of Ag. + , while producing gluconic acid to promote the production of highly toxic ROS, AUGP has a higher antibacterial activity because the Ag released in the hydrogel matrix + The proximity of hydrogen protons allows for a higher concentration of cytotoxic agents. Plates containing AUGP almost completely prevented colony formation compared to the control, whereas Ag alone + A large number of colonies were still visible on Luria-Bertani (LB) agar treated with GOx or a mixture of GOx and AU.

[0060] In summary, the present invention proposes a composite material for sustained release of silver ions controlled by glucose, which utilizes the enrichment of drugs by MOF and the protection of enzymes by hydrogel to prepare AgNPs@UiO-66 / GOx@pAAm, and achieves efficient and controllable antibacterial effect. In this antibacterial system, drugs are used under enzyme catalysis, so that macromolecules can slowly release a large amount of small molecules as needed to achieve the purpose of antibacterial.

[0061] 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. A composite antibacterial material for controlling the sustained release of silver ions by glucose, characterized in that: The composite antibacterial material for glucose-controlled silver ion sustained release is obtained by co-embedding AgNPs@UiO-66 and glucose oxidase (GOx) in polyacrylamide hydrogel microspheres; the AgNPs@UiO-66 is obtained by embedding nanosilver in UiO-66; The nano silver is obtained by the following steps: Dissolve AgNO3 in water, stir and heat until boiling, then add 1% sodium citrate, continue heating for 30 minutes, and cool to room temperature to obtain; The preparation method of the composite antibacterial material for controlling the sustained release of silver ions by glucose comprises the following steps: S1: 1,4-phthalic acid, nanosilver and zirconium chloride tetrahydrate were dissolved in a solvent, acetic acid was added, ultrasonication was performed, and then heating was performed at 80℃-100℃ for 15-20 hours to obtain AgNPs@UiO-66; S2: Glucose oxidase, AgNPs@UiO-66, acrylamide, BIS (N,N'-methylenebisacrylamide) and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylacetophenone are prepared into an enzyme / pregel solution, and then the enzyme / pregel solution is gelated to obtain AgNPs@UiO-66 / GOx@pAAm.

2. The composite antibacterial material for glucose-controlled silver ion sustained release according to claim 1, characterized in that: The solvent is N,N-dimethylformamide.

3. The composite antibacterial material for controlled silver ion release by glucose according to claim 2, characterized in that: The ratio of 1,4-phthalic acid, nanosilver, zirconium chloride tetrahydrate, N,N-dimethylformamide and acetic acid is 100 mg: 10 mg: 30 mg: 8 mL: 4 mL.

4. The composite antibacterial material for glucose-controlled silver ion sustained release according to claim 1, characterized in that: In S1, the heating temperature is 90°C and the heating time is 18 hours.

5. The composite antibacterial material for controlled silver ion release by glucose according to claim 1, characterized in that: The usage ratio of acrylamide, BIS and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylacetophenone is 150 mg:10 mg:10 mg.

6. The composite antibacterial material for controlled silver ion sustained release by glucose according to claim 1, characterized in that: Microfluidic technology was used to promote the gelation of enzyme / pregel solution.

7. Use of the composite antibacterial material with glucose-controlled silver ion sustained release as claimed in any one of claims 1 to 6 in the preparation of antibacterial materials.

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