An antibacterial composite material based on hydrogen bond organic framework and its preparation and application

Abstract

The present invention relates to an antibacterial composite material based on a hydrogen-bonded organic framework, its preparation and application, and belongs to the field of material chemistry and biological technology. The antibacterial composite material based on a hydrogen-bonded organic framework is based on a hydrogen-bonded organic framework as the main body, glucose oxidase is encapsulated on the main body, and ultra-small copper sulfide wrapped with chitosan is adsorbed on the surface, and its preparation method is disclosed. The antibacterial composite material disclosed in the present application exerts an antibacterial synergistic effect through the combination of chemodynamic therapy, photothermal therapy, and starvation therapy, and has a good antibacterial effect. Experimental verification shows that the antibacterial composite material disclosed in the present application has obvious antibacterial effects on Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli, and preliminary evaluation shows that it has good biocompatibility and blood compatibility, which lays the foundation for the preparation of corresponding antibacterial drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of material chemistry and biology, and in particular to an antibacterial composite material based on a hydrogen bond organic framework and the preparation and application thereof. Background Art

[0002] Bacterial infections have been a serious health problem for centuries. They can lead to various diseases, including non-healing wounds, pneumonia, and soft tissue infections. Antibiotics are widely used as a traditional treatment in clinical practice. However, improper or overuse of antibiotics has led to the emergence of multidrug-resistant strains, posing a serious threat to human health. The World Health Organization predicts that by 2050, the global death toll from drug-resistant bacteria will reach 10 million, making drug-resistant infections the most serious health crisis. Therefore, there is an urgent need to explore alternative treatments to combat bacterial infections.

[0003] Faced with the complex bacterial microenvironment, chemokinetic antibacterial has been widely studied in recent years. Chemokinetic antibacterial uses the slightly acidic environment of bacteria to exert the Fenton or Fenton-like effect of metals to produce highly toxic hydroxyl radicals and then kill bacteria. However, a single treatment mode is often ineffective. By combining with other therapies such as photothermal therapy, photodynamic therapy, and starvation therapy to exert synergistic effects, an antibacterial effect of 1+1>2 can be achieved. CN 114984241 A discloses a chitosan / manganese dioxide / glucose oxidase nanocomposite material, which consists of chitosan nanospheres, and manganese dioxide and glucose oxidase loaded on the surface of chitosan nanospheres. It can achieve the dual antibacterial and anti-tumor effects of starvation therapy and chemokinetic antibacterial. Since the Fenton-like effect of manganese dioxide is weak and the glucose oxidase is exposed to the outside, the enzyme activity is easily affected, which in turn affects the catalytic activity of the material.

[0004] While chemokinetics and photothermal therapy hold significant promise as alternatives to antibiotics, they face numerous limitations. Insufficient endogenous hydrogen peroxide in the bacterial microenvironment and low acidity significantly reduce the antibacterial efficacy of chemokinetics. While photothermal therapy exhibits excellent antibacterial efficacy, its high temperatures often damage normal cells, compromising therapeutic efficacy.

[0005] Based on the above background, the present application discloses an antibacterial composite material based on a hydrogen-bonded organic framework, which can utilize the bacterial microenvironment to provide hydrogen peroxide, increase acidity, and achieve multi-component synergistic antibacterial effects in synergistic manner with mild light and heat. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an antibacterial composite material based on a hydrogen bond organic framework and its preparation and application.

[0007] In order to solve the above problems, the technical solution adopted by the present invention is:

[0008] Technical Topic 1

[0009] An antibacterial composite material based on a hydrogen-bonded organic framework, wherein the composite material is based on the hydrogen-bonded organic framework, glucose oxidase is encapsulated in the framework, and copper sulfide wrapped with chitosan is adsorbed on the surface;

[0010] The building block of the hydrogen bond organic framework is 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene.

[0011] As a further improvement of the present invention, the loading amount of glucose oxidase in the composite material is 2.35%wt-9.97%wt.

[0012] Technical Topic 2

[0013] A method for preparing an antibacterial composite material based on a hydrogen-bonded organic framework as described in the first technical topic comprises the following steps:

[0014] S1: 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene was dissolved in solvent I as solution A, and glucose oxidase was dispersed in water as solution B;

[0015] S2: Rapidly add solution B prepared in S1 into solution A, stir for 5-10 minutes, place in the dark for 20-30 minutes, centrifuge, and wash with water to obtain GOx@HOF;

[0016] S3: Add copper chloride and sodium citrate to water in sequence, stir evenly, then add sodium sulfide, stir for 10-12 hours, centrifuge, wash with water, and dry to obtain copper sulfide;

[0017] S4: The copper sulfide obtained in S3 was dispersed in the chitosan solution and stirred at 1000-1200 rpm for 2-4 h, centrifuged, washed with water, and dried to obtain CuS@CS;

[0018] S5: GOx@HOF obtained in S2 and CuS@CS obtained in S4 were added to water, ultrasonically dispersed uniformly, stirred at 1200-1500 rpm for 10-14 h, centrifuged, and washed with water to obtain GOx@HOF-CuS@CS.

[0019] As a further improvement of the present invention, the mass ratio of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene to glucose oxidase in S2 is 1:0.1-0.7;

[0020] The molar ratio of copper chloride: sodium citrate: sodium sulfide in S3 is 0.9-1.1:0.6-0.9:1;

[0021] The mass ratio of copper sulfide to chitosan in S4 is 1:3-8;

[0022] The mass ratio of GOx@HOF to CuS@CS in the S5 is 1:1.5-2.5.

[0023] As a further improvement of the present invention, solvent I is selected from DMF, DMAc, NMP, and DMSO; the volume ratio of solution A to solution B in S2 is 1:8-10; and the mass volume ratio of GOx@HOF to water in S5 is 4 mg:5-20 mL.

[0024] As a further improvement of the present invention, the mass volume ratio of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene to solvent I in solution A is 5 mg:400-800 μL;

[0025] As a further improvement of the present invention, the centrifugal speed in S2 is 12000-13000 rpm, and the centrifugal time is 10-15 min;

[0026] In S3, the centrifugal speed is 13000-15000 rpm and the centrifugation time is 20-30 min;

[0027] In S4, the centrifugal speed is 13000-15000 rpm and the centrifugation time is 20-30 min;

[0028] The centrifugal speed in S5 is 11000-13000 rpm, and the centrifugal time is 10-15 min.

[0029] As a further improvement of the present invention, the concentration of sodium citrate in S3 is 0.1-0.4 mg / mL.

[0030] As a further improvement of the present invention, the chitosan solution in S4 is prepared by dissolving chitosan in a 0.5-1.5% glacial acetic acid solution, and the concentration of chitosan is 5-50 mg / mL.

[0031] As a further improvement of the present invention, the concentration of chitosan is 5-8 mg / mL.

[0032] As a further improvement of the present invention, the mass ratio of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene to glucose oxidase in S2 is 1:0.5;

[0033] The molar ratio of copper chloride: sodium citrate: sodium sulfide in S3 is 1.05:0.78:1

[0034] The mass ratio of copper sulfide to chitosan in S4 is 1:5;

[0035] The mass ratio of GOx@HOF to CuS@CS in S5 is 1:2.

[0036] Technical Theme 3

[0037] An application of an antibacterial composite material based on a hydrogen bond organic framework as described in Technical Topic 1 in the preparation of antibacterial drugs.

[0038] The beneficial effects of adopting the above technical solution are:

[0039] The invention discloses an antibacterial composite material based on a hydrogen bond organic framework, which has glucose oxidase activity, can consume glucose, exert starvation antibacterial effects, produce gluconic acid, regulate the bacterial microenvironment, and provide a substrate for chemical kinetics.

[0040] The loaded ultrasmall copper sulfide has excellent chemical kinetics and photothermal properties. The ultrasmall copper sulfide is modified by chitosan with antibacterial activity and loaded onto the surface of the hydrogen-bonded organic framework, thereby achieving multi-component synergistic antibacterial properties of the antibacterial composite material based on the hydrogen-bonded organic framework and having broad-spectrum antibacterial activity.

[0041] The GOx@HOF-CuS@CS prepared in this example encapsulates glucose oxidase, demonstrating stable catalytic performance. Under 808nm near-infrared light irradiation, the antibacterial composite exhibited significant antibacterial activity against both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Preliminary evaluation also demonstrated good biocompatibility and hemocompatibility, paving the way for the preparation of corresponding antibacterial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 TEM images of materials prepared in a specific embodiment of the present invention, wherein A is CuS@CS prepared in Example 1, B is GOx@HOF prepared in Example 1, C is GOx@HOF-CuS@CS prepared in Example 1, and D is HOF prepared in Comparative Example 1;

[0043] Figure 2 1 is a structural comparison diagram of GOx@HOF in Example 1 of the present invention and HOF in Comparative Example 1, wherein A is an infrared spectrum; B is an X-ray powder diffraction spectrum;

[0044] Figure 3 This is a graph showing potential changes of the materials prepared in Example 1 and Comparative Example 1 of the present invention;

[0045] Figure 4 The UV absorption graphs of the materials prepared in Example 1 and Comparative Example 1 of the present invention and chitosan (CS);

[0046] Figure 5 UV absorption spectra of different groups of materials after reaction with TMB, where Gs represents glucose, laser is 808nm, 1W / cm 2 , 5min;

[0047] Figure 6This is the enzyme activity diagram of GOx@HOF-CuS@CS prepared in Example 1 of the present invention;

[0048] Figure 7 This is the enzyme activity diagram of GOx@HOF-CuS@CS and GOx prepared in Example 1 of the present invention under different treatment conditions, Acetone is acetone, Trypsin is trypsin, and Urea is urea;

[0049] Figure 8 The activity of S. aureus bacteria after treatment with GOx@HOF+Gs, GOx@HOF-CuS@CS+Gs, and GOx@HOF-CuS@CS+Gs+Laser prepared in Example 1 of the present invention, where Gs represents glucose, and the laser is 808 nm and 1 W / cm 2 , 5min;

[0050] Figure 9 The bacterial activity of E.coil after treatment with GOx@HOF+Gs, GOx@HOF-CuS@CS+Gs, and GOx@HOF-CuS@CS+Gs+Laser prepared in Example 1 of the present invention, where Gs represents glucose, and the laser is 808 nm, 1 W / cm 2 , 5min;

[0051] Figure 10 The flat plate and data statistics of S. aureus after GOx@HOF, GOx@HOF-CuS@CS, and GOx@HOF-CuS@CS+Laser treatment prepared in Example 1 of the present invention, wherein the laser is 808nm, 1W / cm 2 , 5min;

[0052] Figure 11 The flat plate and data statistics of E.coil after GOx@HOF, GOx@HOF-CuS@CS, and GOx@HOF-CuS@CS+Laser treatment prepared in Example 1 of the present invention, wherein the laser is 808nm, 1W / cm 2 , 5min;

[0053] Figure 12 This is a diagram showing the safety test of HUVECs cells using GOx@HOF-CuS@CS prepared in Example 1 of the present invention;

[0054] Figure 13 This is the hemolysis condition of GOx@HOF-CuS@CS prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0055] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0056] Chitosan was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., C766421, CAS: 9012-76-4.

[0057] Example 1

[0058] S1: Weigh 5 mg of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene (H4TBAPy) into a 2 mL centrifuge tube, add 500 μL of DMF, and ultrasonicate in an ultrasonic cleaner for 30 min to obtain an H4TBAPy solution. Weigh 2.5 mg of GOx into a 5 mL centrifuge tube, add 4.5 mL of purified water, and disperse evenly to obtain a GOx solution for later use.

[0059] S2: The GOx solution was quickly added to the H4TBAPy solution, stirred at 800 rpm for 5 min in the dark, and aged for 25 min. After the reaction was completed, the material was washed by multiple centrifugation at 13,000 rpm for 10 min with purified water until the supernatant was colorless to obtain a GOx@HOF stock solution (1 mg / mL). The enzyme content in GOx@HOF was determined to be 23.04% wt by BCA assay and was set aside.

[0060] S3: Place 100 mL of purified water in a flask, add 17.75 mg of copper chloride dihydrate, followed by 20 mg of sodium citrate, and stir for 10 minutes. Finally, add 24 mg of sodium sulfide nonahydrate and stir at 1000 rpm for 10 hours. After the reaction is complete, wash the material by centrifugation at 15,000 rpm for 20 minutes multiple times with purified water until the supernatant is colorless. Dry in a vacuum oven to obtain CuS, which is then set aside.

[0061] S4: Dissolve chitosan in 1% glacial acetic acid solution, stir 5 mg CuS in 5 mL of 5 mg / mL chitosan solution at 1200 rpm for 3 h. After the reaction is completed, use purified water to centrifuge and wash the material at 15000 rpm for 20 min, dry it in a vacuum drying oven, and set aside.

[0062] S5: 2 mL of GOx@HOF stock solution (1 mg / mL) and 4 mg of CuS@CS were added to 4 mL of purified water, ultrasonically dispersed, and stirred at 1200 rpm for 12 h. After the reaction, the material was washed with purified water by multiple centrifugation at 11000 rpm for 15 min to obtain GOx@HOF-CuS@CS. The enzyme content was determined to be 7.78% wt by BCA assay.

[0063] Example 2:

[0064] S1: Weigh 5 mg of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene (H4TBAPy) into a 2 mL centrifuge tube, add 500 μL of DMF, and ultrasonicate in an ultrasonic cleaner for 30 min to obtain an H4TBAPy solution. Weigh 2.5 mg of GOx into a 5 mL centrifuge tube, add 4.5 mL of purified water, and disperse evenly to obtain a GOx solution for later use.

[0065] S2: The GOx solution was quickly added to the H4TBAPy solution, stirred at 800 rpm for 10 min in the dark, and aged for 20 min. After the reaction was completed, the material was washed by multiple centrifugation at 12,000 rpm for 15 min with purified water until the supernatant was colorless to obtain a GOx@HOF stock solution (1 mg / mL). The enzyme content in GOx@HOF was determined to be 25.16% wt by BCA assay and was set aside.

[0066] S3: Place 100 mL of purified water in a flask, add 15 mg of copper chloride dihydrate, then 20 mg of sodium citrate, and stir at 1000 rpm for 10 minutes. Finally, add 20 mg of sodium sulfide nonahydrate and stir for 10 hours. After the reaction is complete, wash the material with purified water by centrifugation at 13,000 rpm for 30 minutes multiple times until the supernatant is colorless. Dry the resulting CuS in a vacuum oven to obtain CuS and set aside.

[0067] S4: Dissolve chitosan in 1% glacial acetic acid solution, stir 5 mg CuS in 5 mL of 5 mg / mL chitosan solution at 1200 rpm for 3 h. After the reaction is completed, use purified water to centrifuge and wash the material at 13000 rpm for 30 min, dry it in a vacuum drying oven, and set aside.

[0068] S5: 2 mL of GOx@HOF stock solution (1 mg / mL) and 3.5 mg of CuS@CS were added to 4 mL of purified water and ultrasonically dispersed. The mixture was stirred at 1200 rpm for 12 h. After the reaction, the material was washed by centrifugation multiple times at 11000 rpm for 15 min with purified water to obtain GOx@HOF-CuS@CS. The enzyme content was determined to be 9.15% wt by BCA assay.

[0069] Example 3:

[0070] S1: Weigh 5 mg of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene (H4TBAPy) into a 2 mL centrifuge tube, add 500 μL of DMF, and ultrasonicate in an ultrasonic cleaner for 30 min to obtain an H4TBAPy solution. Weigh 3.0 mg of GOx into a 5 mL centrifuge tube, add 4.5 mL of purified water, and disperse evenly to obtain a GOx solution for later use.

[0071] S2: The GOx solution was quickly added to the H4TBAPy solution, stirred at 800 rpm for 5 min in the dark, and aged for 25 min. After the reaction was completed, the material was washed by multiple centrifugation at 13,000 rpm for 10 min with purified water until the supernatant was colorless to obtain a GOx@HOF stock solution (1 mg / mL). The enzyme content in GOx@HOF was determined to be 29.97% wt by BCA assay and was set aside.

[0072] S3: Place 100 mL of purified water in a flask, add 17.75 mg of copper chloride dihydrate, followed by 20 mg of sodium citrate, and stir at 1000 rpm for 10 minutes. Finally, add 24 mg of sodium sulfide nonahydrate and stir for 10 hours. After the reaction is complete, wash the material by centrifugation at 15,000 rpm for 20 minutes multiple times with purified water until the supernatant is colorless. Dry in a vacuum oven to obtain CuS, which is then set aside.

[0073] S4: Chitosan was dissolved in 1% glacial acetic acid solution. 5 mg CuS was stirred in 5 mL of 5 mg / mL chitosan solution at 1200 rpm for 3 h. After the reaction was completed, the material was centrifuged and washed with purified water at 15,000 rpm for 20 min and dried in a vacuum drying oven for subsequent use.

[0074] S5: 1 mL of GOx@HOF stock solution (1 mg / mL) and 2 mg of CuS@CS were added to 4 mL of purified water, ultrasonically dispersed, and stirred at 1200 rpm for 12 h. After the reaction, the material was washed by centrifugation multiple times in purified water at 11000 rpm for 15 min to obtain GOx@HOF-CuS@CS. The enzyme content was determined by the BCA method to be 9.97% wt.

[0075] Example 4:

[0076] S1: Weigh 5 mg of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene (H4TBAPy) into a 2 mL centrifuge tube, add 500 μL of DMF, and ultrasonicate in an ultrasonic cleaner for 30 min to obtain an H4TBAPy solution. Weigh 0.5 mg of GOx into a 5 mL centrifuge tube, add 4.0 mL of purified water, and disperse evenly to obtain a GOx solution for later use.

[0077] S2: The GOx solution was quickly added to the H4TBAPy solution, stirred at 800 rpm for 5 min in the dark, and aged for 25 min. After the reaction was completed, the material was washed by multiple centrifugation at 13,000 rpm for 10 min with purified water until the supernatant was colorless to obtain a GOx@HOF stock solution (1 mg / mL). The enzyme content in GOx@HOF was determined to be 5.72% wt by BCA assay and was set aside.

[0078] S3: Place 100 mL of purified water in a flask, add 15.30 mg of copper chloride dihydrate, followed by 15.5 mg of sodium citrate, and stir at 1000 rpm for 10 minutes. Finally, add 24 mg of sodium sulfide nonahydrate and stir for 10 hours. After the reaction is complete, wash the material by centrifugation at 15,000 rpm for 20 minutes multiple times with purified water until the supernatant is colorless. Dry in a vacuum oven to obtain CuS, which is then set aside.

[0079] S4: Chitosan was dissolved in 0.5% glacial acetic acid solution. 5 mg CuS was stirred in 3 mL of 5 mg / mL chitosan solution at 1000 rpm for 4 h. After the reaction was completed, the material was centrifuged and washed with purified water at 15000 rpm for 20 min. The material was then dried in a vacuum drying oven for subsequent use.

[0080] S5: 2 mL of GOx@HOF stock solution (1 mg / mL) and 3 mg of CuS@CS were added to 4 mL of purified water, ultrasonically dispersed, and stirred at 1200 rpm for 14 h. After the reaction, the material was washed with purified water by centrifugation multiple times at 13000 rpm for 10 min to obtain GOx@HOF-CuS@CS. The enzyme content was determined to be 2.35% wt by BCA assay.

[0081] Example 5:

[0082] S1: Weigh 5 mg of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene (H4TBAPy) into a 2 mL centrifuge tube, add 500 μL of DMF, and ultrasonicate in an ultrasonic cleaner for 30 min to obtain an H4TBAPy solution. Weigh 3.5 mg of GOx into a 5 mL centrifuge tube, add 5.0 mL of purified water, and disperse evenly to obtain a GOx solution for later use.

[0083] S2: The GOx solution was quickly added to the H4TBAPy solution, stirred at 800 rpm for 5 min in the dark, and aged for 30 min. After the reaction was completed, the material was washed by multiple centrifugation at 13,000 rpm for 10 min with purified water until the supernatant was colorless to obtain a GOx@HOF stock solution (1 mg / mL). The enzyme content in GOx@HOF was determined to be 31.24% wt by BCA assay and was set aside.

[0084] S3: Place 100 mL of purified water in a flask, add 37.6 mg of copper chloride dihydrate, followed by 40 mg of sodium citrate, and stir at 1000 rpm for 10 minutes. Finally, add 48 mg of sodium sulfide nonahydrate and stir for 12 hours. After the reaction is complete, wash the material by centrifugation at 15,000 rpm for 20 minutes multiple times with purified water until the supernatant is colorless. Dry in a vacuum oven to obtain CuS, which is then set aside.

[0085] S4: Chitosan was dissolved in 1.5% glacial acetic acid solution. 5 mg CuS was stirred in 5 mL of 8 mg / mL chitosan solution at 1200 rpm for 2 h. After the reaction was completed, the material was centrifuged and washed with purified water at 15,000 rpm for 20 min and dried in a vacuum drying oven for subsequent use.

[0086] S5: 2 mL of GOx@HOF stock solution (1 mg / mL) and 5 mg of CuS@CS were added to 4 mL of purified water, ultrasonically dispersed, and stirred at 1500 rpm for 10 h. After the reaction, the material was washed by centrifugation multiple times at 11000 rpm for 15 min with purified water to obtain GOx@HOF-CuS@CS. The enzyme content was determined to be 9.25% wt by BCA assay.

[0087] Comparative Example 1

[0088] 5 mg of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene was dissolved in 500 μL of DMF, 4.5 mL of purified water was added, and the mixture was stirred at room temperature for 8 min. The mixture was allowed to stand in the dark for 25 min, centrifuged, and washed with water to obtain HOF.

[0089] Effect Example 1 Structure Verification

[0090] The CuS@CS, GOx@HOF, GOx@HOF-CuS@CS obtained in Example 1 of the present application and the HOF obtained in Comparative Example 1 were observed using a transmission electron microscope. The results are shown in the attached figure. Figure 1 As shown in A, 1B, 1C, and 1D, it is proved that GOx@HOF-CuS@CS was successfully prepared, and the results of observation showed that Figure 1As shown in A and 1D, the hydrogen-bonded organic framework encapsulates glucose oxidase, and the morphology of the material does not change significantly.

[0091] The GOx@HOF prepared in Example 1 and the HOF obtained in Comparative Example 1 were subjected to infrared detection and X-ray powder diffraction. The results are as follows: Figure 2 As described above, it is proved that after the hydrogen bond organic framework encapsulates glucose oxidase, the structure of the organic framework does not change.

[0092] The zeta potential of HOF prepared in Comparative Example 1, GOx@HOF prepared in Example 1, copper sulfide prepared in Example 1, CuS@CS prepared in Example 1 and GOx@HOF-CuS@CS prepared in Example 1 were measured. The results are shown in the attached figure. Figure 3 As shown in Figure 3, the potential of GOx@HOF-CuS@CS is -16.36 mV. The large absolute value of the negative potential enables GOx@HOF-CuS@CS to be stably dispersed in aqueous solution.

[0093] Ultraviolet absorption tests were conducted on chitosan, GOx@HOF prepared in Example 1, copper sulfide prepared in Example 1, HOF prepared in Comparative Example 1, CuS@CS prepared in Example 1, and GOx@HOF-CuS@CS prepared in Example 1. The results are as follows: Figure 4 As shown in the figure, the UV absorption peak of HOF highly overlaps with that of GOx@HOF, further proving that the encapsulation of glucose oxidase by the hydrogen-bonded organic framework has little effect on the structure of the hydrogen-bonded organic framework. The successful preparation of GOx@HOF-CuS@CS was verified by comparing the UV absorption peaks of GOx@HOF, CuS@CS, and GOx@HOF-CuS@CS.

[0094] Effect Example 2

[0095] In order to verify the chemical kinetics of the antibacterial composite material, this application used 3,3',5,5'-tetramethylbenzidine (TMB) as a probe to evaluate the catalytic activity of GOx@HOF-CuS@CS prepared in Example 1 by comparing different groups.

[0096] Five groups of experiments were set up, and the contents of the components in the reaction system (the solvent was 0.01 mol / L PBS solution) are shown in Table 1:

[0097]

[0098] After the samples were prepared, they were incubated at 37°C for 2 h. The fifth group was illuminated with 808 nm near-infrared light (1.0 W·cm -2) for 5 min, and the absorption spectrum at 400-800 nm was detected using UV-vis. The chemical kinetics of the antibacterial composite material was compared based on the absorption value at 652 nm.

[0099] The results are as follows Figure 5 As shown, the GOx@HOF-CuS@CS material disclosed in this application has a photodynamic effect, and photothermal therapy can promote the photodynamic effect.

[0100] Effect Example 3 Enzyme Stability Test

[0101] In order to verify the enzyme stability of the antibacterial composite material, the present application verifies the enzyme stability at different times and under different conditions by using titanium sulfate test solution.

[0102] The GOx@HOF-CuS@CS solution prepared by the method described in Example 1 was prepared into a 1 mg / mL solution and placed at 4°C for 14 days. Samples were taken at the same time every day. A GOx@HOF-CuS@CS solution with a concentration of 50 μg / mL was incubated with 10 mM glucose at 37°C for 2 h. Subsequently, 200 μL of the sample was taken and 200 μL of titanium sulfate detection solution (6 μg / mL) was added. The enzyme stability was evaluated by ultraviolet detection of H2O2 production at 415 nm. The results are shown in the attached figure. Figure 6 shown.

[0103] Free GOx and GOx@HOF-CuS@CS were prepared into 1. a 1 mg / mL aqueous solution (60°C); 2. a 1 mg / mL acetone solution; 3. a 1 mg / mL aqueous solution containing 6 M urea; and 4. a 1 mg / mL aqueous solution containing 5 mM pancreatin. After 30 minutes of treatment, 20 μL of the sample described in 1-4 was added to 400 μL of PBS containing 10 mM glucose and reacted at 37°C for 2 hours. Subsequently, 200 μL of the sample was sampled and 200 μL of titanium sulfate detection solution (6 μg / mL) was added. The enzyme stability was evaluated by UV detection of H2O2 production. The results are shown in the attached figure. Figure 7 shown.

[0104] The results show that the antibacterial composite material of the present application has excellent enzyme stability.

[0105] Effect Example 4 Bacteria Test

[0106] In order to verify the antibacterial effect of the antibacterial composite material, the growth inhibition ability of materials with different concentrations on Staphylococcus aureus (S. aureus) and Escherichia coli (E. coil) was detected by OD value.

[0107] Staphylococcus aureus and Escherichia coli on agar medium were transferred to liquid Luria-Bertani (LB) medium, which was shaken at 200 rpm at 37°C for 8 h. The bacteria were then collected by centrifugation and diluted to 1 × 10 using LB medium containing 10 mM glucose in a 48-well plate. 8 CFU / mL. 1 μL of PBS was added as a negative control group; 1 μL of GOx@HOF solution prepared in Example 1 with different concentrations, and 1 μL of GOx@HOF-CuS@CS solution prepared in Example 1 with different concentrations, so that the concentrations of the corresponding materials in the 48-well plate were 10, 25, 50, 75, and 100 μg∙mL, respectively. -1 The culture medium containing GOx@HOF-CuS@CS was divided into two groups. One group was irradiated with 808 nm near-infrared light (1.0 W·cm -2 ) irradiated for 5 minutes, and the plate was incubated at 37°C and a bacterial shaker at 200 rpm. After 4 hours, the absorbance at 600 nm was measured to assess the bacterial concentration. The results are shown in the attached figure. Figure 8 , Attachment Figure 9 shown.

[0108] The results showed that under 808nm near-infrared light irradiation, the bacterial activity of both Staphylococcus aureus and Escherichia coli was significantly reduced at a concentration of 100μg / mL of GOx@HOF-CuS@CS. This demonstrates that the antibacterial composite material can inhibit bacterial growth and exhibits excellent broad-spectrum antibacterial activity.

[0109] Effect Example 5: Bacterial Plate Test

[0110] In order to further verify the antibacterial effect of the antibacterial composite material, the bactericidal ability of different groups against Staphylococcus aureus and Escherichia coli was verified by the plate count method. The GOx@HOF and GOx@HOF-CuS@CS used were prepared in Example 1.

[0111] Staphylococcus aureus and Escherichia coli stored in frozen glycerol were transferred to Luria-Bertani (LB) liquid medium and shaken at 200 rpm at 37°C for 10 h. The bacteria were then collected by centrifugation and diluted to 1×10 using 1. LB medium containing 100 μg / mL GOx@HOF and 10 mM glucose; 2. LB medium containing 100 μg / mL GOx@HOF-CuS@CS and 10 mM glucose; and 3. LB medium containing 10 mM glucose. 8CFU / mL, and after dilution, the cells were incubated at 37°C and 200 rpm for 4 h. The culture medium containing GOx@HOF-CuS@CS was divided into two groups. One group was illuminated with 808 nm near-infrared light (1.0 W·cm -2 ) irradiated for 5 min. After incubation, the bacterial suspension was diluted with LB medium to 1×10 5 Then 100 μL of the diluted bacterial suspension was spread on the solid culture medium through glass beads and incubated at 37°C for 12 hours before counting the number of colonies. The results are shown in the attached figure. Figure 10 and attached Figure 11 The results showed that the PBS, GOx@HOF, and GOx@HOF-CuS@CS groups all had obvious colonies, and under 808nm near-infrared light irradiation, the antibacterial composite material had a sterilization rate of more than 99% against Staphylococcus aureus and Escherichia coli.

[0112] Effect Example 6: Biocompatibility Evaluation

[0113] The GOx@HOF-CuS@CS used in this effect example was prepared in Example 1.

[0114] To evaluate the biocompatibility of normal cells with GOx@HOF-CuS@CS, human umbilical vein endothelial cells (HUVECs) were seeded in 96-well plates (8000 cells / well) and cultured with DMEM medium. The cells were incubated at 37°C under normoxic conditions (O2 21%) for 24 h. The original medium was discarded and the HUVECs containing GOx@HOF-CuS@CS at concentrations of 0, 5, 10, 25, 50, 75, and 100 μg∙mL were added. -1 The cells were incubated with DMEM medium for 24 h and the cell survival rate was detected by standard CCK-8 method. Figure 12 As shown, it is demonstrated that GOx@HOF-CuS@CS has good biocompatibility.

[0115] 100 μL of heparin was mixed with 900 μL of saline, and 20 μL of the heparin solution was added to a centrifuge tube. Blood was collected from the canthus using a capillary tube soaked in heparin solution and dripped into a centrifuge tube containing heparin for anticoagulation. The cells were centrifuged at 3500 rpm for 10 min, the upper plasma layer was removed, and the blood cells were washed with pH 7.4 PBS (10 mM) until the supernatant was free of foam. 760 μL of GOx@HOF-CuS@CS containing different concentrations (10, 25, 50, 100, 200, 400, and 500 μg∙mL) was added. -140 μL of red blood cells were added to PBS (100 μL / min); 40 μL of red blood cells were added to 760 μL of PBS and deionized water, respectively, as negative and positive controls. All the above samples were incubated at room temperature for 30 min, centrifuged, and photographed, and the supernatant was collected. The ultraviolet absorbance at 570 nm was measured using a UV-Vis spectrophotometer. The hemolysis results are shown in the attached figure. Figure 13 As shown, the hemolysis rate (Hemolysis) is calculated as follows:

[0116]

[0117] Among them A sample is the absorbance value of the sample, A negative and A positive The absorbance values ​​of negative and positive samples are shown respectively. The results show that the antibacterial composite material has preliminary biocompatibility.

[0118] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibacterial composite material based on a hydrogen bond organic framework, characterized in that: The composite material is mainly composed of a hydrogen bond organic framework, glucose oxidase is encapsulated in the main body, and copper sulfide wrapped with chitosan is adsorbed on the surface; The building block of the hydrogen bond organic framework is 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene; The preparation method of the composite material comprises the following steps: S1: 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene was dissolved in solvent I as solution A, and glucose oxidase was dispersed in water as solution B; S2: Rapidly add solution B prepared in S1 into solution A, stir for 5-10 minutes, place in the dark for 20-30 minutes, centrifuge, and wash with water to obtain GOx@HOF; S3: Add copper chloride and sodium citrate to water in sequence, stir evenly, then add sodium sulfide, stir for 10-12 hours, centrifuge, wash with water, and dry to obtain copper sulfide; S4: The copper sulfide obtained in S3 was dispersed in the chitosan solution and stirred at 1000-1200 rpm for 2-4 h, centrifuged, washed with water, and dried to obtain CuS@CS; S5: GOx@HOF obtained in S2 and CuS@CS obtained in S4 were added to water, ultrasonically dispersed, stirred at 1200-1500 rpm for 10-14 h, centrifuged, and washed with water to obtain GOx@HOF-CuS@CS; The mass ratio of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene to glucose oxidase in S2 is 1:0.1-0.7; The molar ratio of copper chloride: sodium citrate: sodium sulfide in S3 is 0.9-1.1:0.6-0.9:1; The mass ratio of copper sulfide to chitosan in S4 is 1:3-8; The mass ratio of GOx@HOF to CuS@CS in S5 is 1:1.5-2.5; The solvent I is selected from one of N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, and dimethyl sulfoxide; the volume ratio of solution A to solution B in S2 is 1:8-10; the mass volume ratio of GOx@HOF to water in S5 is 4 mg:5-20 mL.

2. The antibacterial composite material based on hydrogen bond organic framework according to claim 1, characterized in that: The loading amount of glucose oxidase in the composite material is 2.35%wt-9.97%wt.

3. The antibacterial composite material based on hydrogen bond organic framework according to claim 1, characterized in that: In S2, the centrifugal speed is 12000-13000 rpm and the centrifugation time is 10-15 min; In S3, the centrifugal speed is 13000-15000 rpm and the centrifugation time is 20-30 min; In S4, the centrifugal speed is 13000-15000 rpm and the centrifugation time is 20-30 min; The centrifugal speed in S5 is 11000-13000 rpm, and the centrifugal time is 10-15 min.

4. The antibacterial composite material based on hydrogen bond organic framework according to claim 1, characterized in that The concentration of sodium citrate in the S3 is 0.1-0.4 mg / mL.

5. The antibacterial composite material based on hydrogen bond organic framework according to claim 1, characterized in that: The chitosan solution in S4 is prepared by dissolving chitosan in 0.5-1.5% glacial acetic acid solution, and the concentration of chitosan is 5-50 mg / mL.

6. The antibacterial composite material based on hydrogen bond organic framework according to claim 1, characterized in that: The mass ratio of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene to glucose oxidase in S2 is 1:0.5; The molar ratio of copper chloride: sodium citrate: sodium sulfide in S3 is 1.05:0.78:1 The mass ratio of copper sulfide to chitosan in S4 is 1:5; The mass ratio of GOx@HOF to CuS@CS in S5 is 1:

2.

7. Use of the antibacterial composite material based on hydrogen-bonded organic framework according to any one of claims 1 to 6 in the preparation of antibacterial drugs.

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