A sugar-grafted metal-covalent organic framework artificial enzyme for rapid capture and clearance of bacteria, and its preparation method and application

Abstract

The present invention discloses a sugar-grafted metal-covalent organic framework artificial enzyme for rapidly capturing and clearing bacteria, and its preparation method and application, belonging to the technical field of biomedicine. The artificial enzyme comprises a covalent organic framework polymer with a porous structure; the covalent organic framework polymer is a covalent organic framework polymer containing porphyrin, and the porphyrin coordinates with a metal through a four-coordination center, so that the metal is anchored in the covalent organic framework polymer; sugars are grafted onto the covalent organic framework polymer. The present invention first prepares a covalent organic framework material, then performs glycosylation modification to obtain an ester, then coordinates with copper, and finally hydrolyzes the ester to obtain the sugar-grafted metal-covalent organic framework artificial enzyme. This artificial enzyme not only promotes the effects of photothermal therapy (PTT) and photodynamic therapy (PDT), but also has a strong adsorption effect on bacteria and inhibits the synthesis of bacterial cell membranes; it can capture and clear bacteria at extremely low concentrations and in an extremely short time.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a sugar-grafted metal-covalent organic framework artificial enzyme for rapidly capturing and clearing bacteria, and a preparation method and application thereof. Background Art

[0002] Infectious diseases caused by pathogenic bacteria cause irreversible damage to human health, and the prevalence of antibiotic resistance genes due to the overuse of antibiotics. To address this issue, a variety of novel bacterial inactivation methods have been developed, including enzyme therapy, photodynamic therapy, ion sterilization therapy, photothermal therapy (PTT), and other technologies. Among them, phototherapy and enzyme therapy are two important treatment means. Taking peroxidase (POD) as an example, it can be activated by overexpressed H2O2 in acidic IME, becoming a promising in-situ bacterial inactivation treatment method. However, although the level of H2O2 induced by infection is increased compared with normal tissues, its level is still far from sufficient, which poses a major challenge to enzyme therapy that relies on the generation of H2O2-dependent hydroxyl radicals (•OH).

[0003] Currently, due to the limited efficacy of a single treatment mode, the dilemma of bacterial drug resistance has not been effectively alleviated. How to effectively eliminate bacteria while avoiding the development of bacterial drug resistance remains a major challenge for clinicians and researchers. Bacterial attachment plays a key role in the infection process, and they can attach to the infection site through various forces such as Coulomb force, van der Waals force, and hydrogen bond. Subsequently, these bacteria aggregate, lose their flagella, and begin to secrete extracellular matrix (ECM), resulting in irreversible adhesion, especially in the immune microenvironment (IME). Therefore, to effectively solve this problem, there is an urgent need to develop bacterial-targeted intelligent materials that can interfere with the interaction between bacteria and the infection site. These materials can not only eradicate and capture pathogenic bacteria but also effectively reshape IME to overcome the bacterial threat. Currently, achieving functional transformation through reasonable material design is the focus and challenge of research, and such design can hinder the action of bacteria and ultimately delay the formation of biofilms.

[0004] Covalent organic frameworks (COFs) are a class of emerging multifunctional materials formed by covalently linking organic building units. Their diverse chemical bond types and structural building units provide endless research possibilities for COFs and exhibit great potential in various applications. COFs have unique chemical stability and tunability and can serve as an integrated structure and function to achieve precise control of material properties. It is expected to play an increasingly important role in biomedical research and clinical practice. These unique properties enable COFs to separate and aggregate bacterial cells, minimizing adverse damage. However, further research is still needed to address the challenges in the production of functional-targeted COFs through rational structural design and post-modification to achieve functional integration and maximize their therapeutic effects. Carbohydrates are ubiquitous in organisms and are one of the three key substances in the life process, involved in physiological processes such as bacterial infection, cell signaling, and inflammation. Notably, the process of bacterial infection is mainly promoted through specific sugar-protein binding interactions, and the adhesion of bacteria to cells depends on the recognition of sugar molecules on the surface of host cells. Glycobiology has become an important research direction in the biomedical field. However, if the specific recognition of sugar-protein binding between sugar and bacteria is used to capture bacteria, the bacteria will metabolize by phagocytosing the sugar after binding to the sugar, which instead promotes the reproduction of bacteria and fails to achieve the capture and clearance of bacteria. Therefore, it is necessary to design a material that contains both sugar and covalent organic frameworks, which can not only achieve the capture and clearance of bacteria but also achieve the rapid capture and clearance of bacteria. Summary of the Invention

[0005] In view of the above-mentioned prior art, the object of the present invention is to provide a sugar-grafted metal-covalent organic framework artificial enzyme for rapidly capturing and clearing bacteria, and its preparation method and application. The present invention first prepares a covalent organic framework polymer, then grafts sugar onto the covalent organic framework polymer, and then anchors copper into the covalent organic framework polymer, thereby obtaining an artificial enzyme that can rapidly capture and clear bacteria, which can achieve the capture and clearance of bacteria at extremely low concentrations and in an extremely short time.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, there is provided a sugar-grafted metal-covalent organic framework artificial enzyme for rapidly capturing and clearing bacteria, wherein the sugar-grafted metal-covalent organic framework artificial enzyme comprises a covalent organic framework polymer having a porous structure; the covalent organic framework polymer is a covalent organic framework polymer containing porphyrin, and the porphyrin coordinates with the metal through a four-coordination center, so that the metal is anchored in the covalent organic framework polymer; sugar is grafted onto the covalent organic framework polymer; the sugar is a monosaccharide; and the metal is copper.

[0008] Preferably, the covalent organic framework polymer is obtained by copolymerizing 2,6-bis(2-propynyloxy)naphthalene-1,5-dicarbaldehyde and tetraaminophenyl porphyrin.

[0009] Preferably, the monosaccharide is mannose.

[0010] In a second aspect of the present invention, a method for preparing a sugar-grafted metal-covalent organic framework artificial enzyme is provided, comprising the following steps:

[0011] (1) Add tetraaminophenyl porphyrin and 2,6-bis(2-propynyloxy)naphthalene-1,5-dicarbaldehyde into an organic solvent, and heat and react to obtain a dark purple powder PN-COF;

[0012] (2) Add copper iodide, PN-COF, N,N-diisopropylethylamine and azide-modified sugar into a mixed solution of THF, H2O and tert-butanol to obtain a suspension. The suspension is stirred overnight at room temperature under a protective atmosphere. After the reaction is completed, filter the solid and wash and dry it to obtain a brown solid COF-sugar;

[0013] (3) Add COF-sugar and copper acetate into methanol, and heat and reflux to react. After the reaction is completed, cool and filter to obtain a purple-black powder Cu-COF-sugar;

[0014] (4) Add a methanol solution to Cu-COF-sugar and ultrasonically disperse it evenly; then add a methanol solution containing sodium methoxide, and let it stand overnight at room temperature under a protective atmosphere. Filter, wash and dry to obtain a brown solid SCu-COF-sugar, which is the sugar-grafted metal-covalent organic framework artificial enzyme.

[0015] Preferably, in step (1), the molar ratio of tetraaminophenyl porphyrin to 2,6-bis(2-propynyloxy)naphthalene-1,5-dicarbaldehyde is 1:2; the organic solvent is a mixture of 1,4-dioxane, mesitylene and acetic acid in a volume ratio of 6:3:2; the temperature of the heating reaction is 120 °C, and the time of the heating reaction is 5 days.

[0016] Preferably, in step (2), the volume ratio of THF, H2O and tert-butanol is 3:1:1; the addition amounts of copper iodide, PN-COF, N,N-diisopropylethylamine and azide-modified sugar are in a ratio of 27.69 mg:100 mg:87 µL:10 mg.

[0017] Preferably, the azide-modified sugar is azide-modified mannose.

[0018] Preferably, in step (3), the mass ratio of COF-sugar to copper acetate is 5:1; the temperature of the heating reflux reaction is 65 °C, and the time of the heating reflux reaction is 5 days.

[0019] Preferably, in step (4), the addition ratio of the Cu-COF-sugar, methanol, and the methanol solution containing sodium methoxide is 25:4:1; the concentration of the methanol solution containing sodium methoxide is 1 M.

[0020] In the third aspect of the present invention, there is provided the use of sugar-grafted metal-covalent organic framework artificial enzyme (SCu-COF-Man) in the preparation of antibacterial drugs or drugs for removing biofilms. The concentration of the sugar-grafted metal-covalent organic framework artificial enzyme is 75 μg / mL, and it can capture and remove bacteria within 1-2 h.

[0021] The beneficial effects of the present invention are as follows:

[0022] (1) The sugar-grafted metal-covalent organic framework artificial enzyme (SCu-COF-Man) prepared by the present invention has a light-responsive property and can be used as an intelligent platform for efficient sterilization and wound healing. During the entire antibacterial process, sugar molecules not only promote the effects of photothermal therapy (PTT) and photodynamic therapy (PDT), but also have a strong adsorption effect on bacteria and inhibit the synthesis of bacterial cell membranes. At the same time, the addition of metal ions also provides catalase activity, generating a large amount of toxic ROS to provide bactericidal power; and SCu-COF-Man has excellent biosafety at therapeutic doses, greatly expanding the application prospects. This work provides an innovative strategy for the development of COF structures with novel sugar modifications.

[0023] (2) The SCu-COF-Man of the present invention can not only capture and remove bacteria, but also rapidly capture and remove bacteria. It can rapidly capture and remove bacteria within 1-2 h at an extremely low concentration (75 μg / mL). Compared with the porous polymer artificial enzyme that adsorbs bacteria by charge, the sterilization speed is faster and the drug concentration is extremely low. It provides a new idea for rapidly capturing bacteria and sterilizing them.

[0024] (3) The present invention utilizes the targeting ability of sugar to pathogens and the multi-hydroxyl groups contained in sugar to mediate directional functionalization, realizes spatial regulation through stereochemical diversity, and ensures physiological tolerance through natural biocompatibility. Description of the Drawings

[0025] Figure 1:Physical characteristics of polymers: (a) Infrared spectra of TAPP, NA, PN-COF, and SCu-COF-Man; (b) Infrared spectra of S-COF-Gal, S-COF-Glc, and S-COF-Man; (c) Solid-state NMR of SCu-COF-Man; (d) Zeta potential changes after PN-COF, S-COF-Man, S-COF-Glc, S-COF-Gal, and SCu-COF-Man; (e) Thermogravimetric curve before modification; (f) Thermogravimetric curve after modification; (g) Low-temperature N2 adsorption isotherm; (h) Pore size distribution curve of SCu-COF-Man;

[0026] Figure 2 :(a) Total elemental XPS of SCu-COF-Man; (b) High-resolution XPS of N 1s; (c) High-resolution XPS spectrum of O 1s; (d) High-resolution XPS spectrum of Cu 2p;

[0027] Figure 3 :XRD of SCu-COF-Man;

[0028] Figure 4 :(a) Scanning electron microscopy of SCu-COF-Man at 1000 nm; (b) Scanning electron microscopy of SCu-COF-Man at 500 nm; (c) Scanning electron microscopy of SCu-COF-Man at 200 nm; (d) Transmission electron microscopy of SCu-COF-Man at 200 nm; (e) Transmission electron microscopy of SCu-COF-Man at 50 nm; (f) Transmission electron microscopy of SCu-COF-Man at 5 nm; (g) Transmission electron microscopy of SCu-COF-Man at 0.5 nm; (h) HADDF-STEM image of SCu-COF-Man; (i) C element mapping of SCu-COF-Man; (j) Cu element mapping of SCu-COF-Man; (k) N element mapping of SCu-COF-Man; (l) O element mapping of SCu-COF-Man;

[0029] Figure 5 :EDS of SCu-COF-Man;

[0030] Figure 6: (a) Comparison of bacteria captured by S-COF-Man, S-COF-Glc, S-COF-Gal, and PN-COF at the same time; (b) Kinetic curves of Escherichia coli captured by S-COF-Man, S-COF-COF-Glc, S-COF-Gal, and PN-COF; (c) Kinetic curves of Staphylococcus aureus captured by S-COF-Man, S-COF-Glc, S-COF-Gal, and PN-COF; (d) Initial color of REG; (e) Color of REG after bacteria rupture; (f) Color changes of Escherichia coli treated with different glycopolymers; (g) Color changes of Staphylococcus aureus treated with different glycopolymers;

[0031] Figure 7 : (a) UV spectra of DPBF after laser irradiation; (b) UV spectra of MB after laser irradiation; (c) Fluorescence spectra of DHR123 after laser irradiation;

[0032] Figure 8 : (a) UV-visible spectra of SCu-COF-Man and DPBF added under laser irradiation; (b) UV-visible spectra of SCu-COF-Man and MB added under laser irradiation; (c) Fluorescence spectra of SCu-COF-Man and DHR123 added under laser irradiation; (d) Decay curves of SCu-COF-Man and DPBF added under laser irradiation; (e) Decay curves of SCu-COF-Man and MB added under laser irradiation; (f) Decay curves of SCu-COF-Man and DHR123 under laser irradiation;

[0033] Figure 9 : (a) UV-visible spectra of SCu-COF-Man catalyzing hydrogen peroxide at different pH (150 μg / mL); (b) Different concentrations at the same pH (pH = 5.5); (c) Influence of laser on the catalytic effect; (d) Elimination curves of GSH at different concentrations (0, 50, 75, 100 μg / mL); (e) Elimination curves of GSH at the same concentration (75 μg / mL) and different incubation times (0, 5, 10, 15 min); (f) Histogram of Staphylococcus aureus inhibition percentage; (g) Histogram of Escherichia coli inhibition percentage; (h) Generation curves of •OH incubated at different times;

[0034] Figure 10 : (a) Antibacterial plates of different sugar cluster materials at the same concentration (190 μg / mL); (b) Histogram of bacterial viability;

[0035] Figure 11: (a) Antibacterial plate images of SCu-COF-Man at different concentrations (0 - 75 μg / mL); (b) Bacterial viability histogram;

[0036] Figure 12 : (a) Colony images of Staphylococcus aureus on the plate; (b) Bacterial viability histogram of Staphylococcus aureus; (c) Colony images of Escherichia coli on the plate; (d) Bacterial viability histogram of Escherichia coli;

[0037] Figure 13 : Biofilm experiment of SCu-COF-Man;

[0038] Figure 14 : (a) Inverted fluorescence images of Staphylococcus aureus and Escherichia coli treated by in vitro antibacterial test; (b) TEM of Staphylococcus aureus and Escherichia coli treated by in vitro antibacterial test;

[0039] Figure 15 : (a) Hemolysis test of SCu-COF-Man; (b) MTT experiment of SCu-COF-Man; (c) MTT experiment of SCu-COF-Man in the presence of laser irradiation; (d) MTT experiment of SCu-COF-Man in the presence of hydrogen peroxide;

[0040] Figure 16 : (a) Changes in the back wounds of mice in different groups on days 1 - 9; (b) Percentage of wound healing during treatment; (c) Changes in the body weight of mice;

[0041] Figure 17 : HE and Masson staining of mouse back wound sections on day 9;

[0042] Figure 18 : (a) Changes in white blood cells of mice on day 9; (b) Changes in red blood cells of mice on day 9; (c) Changes in hemoglobin of mice on day 9; (d) Changes in mean corpuscular hemoglobin of mice on day 9; (e) Mean corpuscular volume of mice on day 9; (f) Changes in hematocrit of mice on day 9; (g) H&E staining of the heart, liver, spleen, lungs, and kidneys of mice in different groups;

[0043] Figure 19 : Comparative experiment on the rapid bactericidal effects of the PBS group, Cu-COF group, HLV-Cu group, and SCu-COF-Man group;

[0044] Figure 20 : Synthesis schematic diagram. Detailed implementation methods

[0045] It should be noted that the following detailed description is illustrative and aims to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0046] As introduced in the background art section, bacteria and sugars can achieve the capture of sugars by specific sugar-protein binding, and the adhesion of bacteria to cells depends on the recognition of sugar molecules on the surface of host cells. If this property can be used to reversely capture bacteria with sugars, it will be helpful for antibacterial. However, after bacteria bind to sugars, they will metabolize the sugars by phagocytosis, which instead promotes the reproduction of bacteria and cannot achieve the capture and clearance of bacteria with sugars.

[0047] Based on this, the object of the present invention is to provide a sugar-grafted metal-covalent organic framework artificial enzyme for rapid capture and clearance of bacteria, and its preparation method and application. The present invention designs a bacteria capture and inactivation platform based on COFs, and prepares a sugar-grafted metal-covalent organic framework artificial enzyme for rapid capture and clearance of bacteria through step-by-step functional modification (metal embedding and sugar modification). The surface sugar modification of the two-dimensional (2D) layered substrate endows COFs with selective bacteria recognition ability, further promoting the interaction between the polymer matrix and bacteria. The embedding of copper enables COFs to have dual enzyme activities of mimicking peroxidase (POD) and glutathione (GSH) oxidase. The ability to mimic POD enables COFs to generate highly toxic hydroxyl radicals (•OH) using endogenous H2O2, specifically killing bacteria in the pathological weak acidic IME, while the GSH oxidase activity promotes the consumption of GSH, providing conditions for the generation of H2O2 and realizing a self-cascade enzyme reaction. The abnormally high GSH level induced by anaerobic glycolysis in infected tissues will scavenge the generated reactive oxygen species (ROS), reducing the ROS content and thus affecting the treatment efficiency. The GSH oxidase activity promotes the consumption of GSH, which can avoid the above problems. It is of great significance to consume GSH to enhance the synergistic treatment effect.

[0048] The modification of different saccharides endows COFs with the ability to specifically recognize bacterial cell surface proteins (called lectins). These proteins present on the bacterial surface help bacteria bind to specific saccharides on the surface of foreign cells through their interaction with carbohydrates. For example, mannose binds to Escherichia coli, etc. By utilizing the antibacterial principle of the present invention, the variety of sugar copolymers is expanded. The copolymers of the present invention can be grafted with various monosaccharides or polysaccharides, and more drugs with different functions can be developed by using the functions of different saccharides. Among multivalent ligands, sugar copolymers have broad prospects in bacterial adhesion research. The sugar moiety of the sugar copolymer recognizes and captures bacteria, enhancing the adhesion affinity of the nanomaterial for bacteria, thereby inhibiting infection. At the same time, after double modification, the photothermal and photodynamic capabilities of COFs are also greatly improved. Through the significantly enhanced affinity, a superimposed effect can be achieved, effectively avoiding the loss of ROS and heat caused by laser irradiation, and thus being used for bacterial filtration and sterilization.

[0049] In addition, in the research of the present invention, it is also found that in addition to using the specific binding of sugar to bacteria, SCu-COF-Man also binds to negatively charged bacteria through the charges it carries. Compared with other porous polymers that only adsorb bacteria by charge and sterilize, the SCu-COF-Man of the present invention has both an adsorption effect and a capture effect on bacteria, so it can bind to bacteria faster and has a better capture effect. After the sugar contained in the SCu-COF-Man of the present invention is captured by bacteria, since the sugar and COFs are grafted through triazole bonds, and the triazole bonds are very strong and bacteria cannot break them, so bacteria cannot phagocytose the sugar after capturing it, but instead the sugar captures the bacteria. In the structure of the present invention, after bacteria phagocytose the sugar in SCu-COF-Man, the sugar and the Cu-COF part act together to reduce the probability of the generated reactive oxygen species leaking into the environment. The reactive oxygen species act directly on bacteria to quickly sterilize; and copper consumes GSH to catalyze the production of hydrogen peroxide, and then the generated hydroxyl radicals (•OH) can also act directly on bacteria, reducing the probability of leaking into the environment. The above multiple effects all act directly on bacteria, thus achieving rapid sterilization at extremely low concentrations.

[0050] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0051] LB broth, agar, and bacterial viability detection kits were purchased from Shanghai Beten Biotechnology Co., Ltd.;

[0052] 4-triformylbenzene was purchased from Anhui Zesheng Technology Co., Ltd., and propionic acid was purchased from Shanghai Macklin Biochemical Technology Co., Ltd.

[0053] Unless otherwise specified, the control group in the present invention refers to the PBS group, and the pH of PBS is 7.4.

[0054] In the attached drawings of the specification, L represents laser, and S represents SCu-COF-Man; for example, S+L represents the SCu-COF-Man + laser group.

[0055] In the present invention, azide-modified sugars such as azide-modified mannose, azide-modified glucose, and azide-modified galactose are all commercially available products, and can also be prepared by the following method:

[0056] The acetylated monosaccharide (acetylated mannose, acetylated glucose or acetylated galactose) and 2-[2-(chloroethoxy)ethoxy]ethanol (1.1 g) were added to a 100 mL round-bottom flask in a molar ratio of 1:1, 2 mL of boron trifluoride diethyl ether and 50 mL of dichloromethane were added, and the mixture was stirred overnight at 33 °C, quenched with water, washed and dried, and the product was collected and purified by column chromatography. The mobile phase was DCM:EA (dichloromethane:ethyl acetate) = 9:1 (v / v). After rotary evaporation, a colorless oily liquid was obtained. Take 5 g of the above product, add 974 mg of KN3 (potassium azide) and 923 mg of BU4NI (tetrabutylammonium iodide). Add about 30 mL of DMF and react overnight at room temperature. After the reaction, the obtained product was purified by silica gel column chromatography. The mobile phase was PE:EA (petroleum ether:ethyl acetate) = 1:1 (v / v). The obtained liquid was dried by rotary evaporation to obtain a yellow oily substance, which was the azide-modified sugar (azide-modified mannose, azide-modified glucose or azide-modified galactose).

[0057] Among them, the brand of acetylated mannose is Aladdin Chemistry (CAS No.: 4163-65-9); acetylated glucose was purchased from Tianjin Xiensi Biochemical Technology Co., Ltd. (CAS No.: 604-69-3); the brand of acetylated galactose is Aladdin Chemistry (CAS No.: 4163-60-4).

[0058] The test materials used in the examples of the present invention are all conventional test materials in the art and can be obtained through commercial channels.

[0059] Example 1: Preparation of SCu-COF-Man

[0060] (1) Synthesis of 2,6-bis(2-propynyloxy)naphthalene-1,5-dicarbaldehyde

[0061] 2,6-Dihydroxynaphthalene-1,5-dicarbaldehyde (0.58 g) and K2CO3 (1.87 g) were refluxed in THF (60 mL) for 30 minutes. After turning off the heating and cooling to room temperature, propargyl bromide (1.3 mL) was added, and the mixture was heated to reflux for 72 hours. After the reaction was completed, cold water was added, and the mixture was extracted with dichloromethane, washed three times with NaOH solution (5%, 80 mL) and NaCl solution (5%, 100 mL), dried over anhydrous sodium bicarbonate, filtered, and the solvent was removed under pressure. Finally, it was purified by column chromatography (DCM: petroleum ether = 1:1) to obtain 2,6-bis(2-propynyloxy)naphthalene-1,5-dicarbaldehyde as a pale yellow powder. The synthetic route is as follows:

[0062] 。

[0063] (2) Synthesis of TAPP

[0064] Under a nitrogen atmosphere, p-nitrobenzaldehyde (11 g, 73 mmol) and acetic anhydride (12 mL, 127 mmol) were dissolved in 400 mL of propionic acid. After heating to 120 °C, pyrrole (5 mL, 73 mmol) was added dropwise, and the reaction solution was stirred at 140 °C for 2 hours. After cooling to room temperature, the mixture was placed in the refrigerator overnight, and then filtered and washed with methanol (300 mL) and deionized water (300 mL). Then, the obtained black solid was recrystallized from pyridine and then washed with methanol / acetone (1:1) to obtain a purple product (TNPP, yield 20.5%). TNPP (1.8 g, 2.26 mmol) was dissolved in concentrated hydrochloric acid (30 mL), and then a concentrated hydrochloric acid solution (50 mL) of SnCl2 (7.0 g, 29 mmol) was added dropwise. The reaction solution was stirred at room temperature for 2 hours and then heated to 80 °C under a nitrogen atmosphere for 0.5 hour. After the reaction was completed, the mixture was cooled to 0 °C and neutralized with ammonia water. The purple product (TAPP, yield 80%) was obtained by filtration and Soxhlet extraction with chloroform.

[0065] (3) Synthesis of PN-COF

[0066] 1,4-Dioxane (12 mL), mesitylene and acetic acid with a concentration of 6 M were mixed in a volume ratio of 6:3:2 to obtain a mixed solution. TAPP (50 mg) and 2,6-bis(2-propynyloxy)naphthalene-1,5-dicarbaldehyde (85 mg) were added to the mixed solution. The above mixture was transferred to a Schlenk tube, ultrasonically dissolved, and after three freeze-thaw cycles in a liquid nitrogen environment, the reaction was transferred to an environment at 120 °C for reaction for 5 days. After the reaction was completed, the mixture was cooled to room temperature and washed successively with methanol, tetrahydrofuran and dichloromethane to obtain a dark purple powder, which is PN-COF. The synthetic route is as follows:

[0067] .

[0068] (4)Glycosylation modification

[0069] Mix THF, H2O and tert-butanol t-BuOH in a volume ratio of 3:1:1 to obtain a mixed solution. Add 27.69 mg of copper iodide and 100 mg of PN-COF to 13 mL of the mixed solution, and then purify with argon for 5 minutes. Then add N,N-diisopropylethylamine (DIPEA) (87 µL, 0.50 mmol), purify the mixture with argon, and finally add about 10 mg of azide-modified mannose (Man) to obtain a suspension. The suspension is stirred overnight at room temperature under an argon atmosphere. After the reaction, filter the solid, and then wash it successively with water, acetonitrile and tetrahydrofuran and further dry it to obtain a brown solid, which is COF-Man.

[0070] (5)Ionization modification

[0071] Take about 100 mg of COF-Man, add about 20 mg of copper acetate, heat it at 65 °C in 30 mL of methanol and reflux for 5 days, cool and filter to obtain a purple-black powder, which is Cu-COF-Man.

[0072] (6)Hydrolysis

[0073] Add about 20 mL of methanol solution to Cu-COF-Man and disperse it evenly by ultrasonic treatment; then add about 5 mL of methanol solution containing sodium methoxide (1 M), protect it with argon and let it stand overnight at room temperature. Filter and wash with methanol to obtain a brown solid, which is SCu-COF-Man. The synthesis schematic diagram is shown in Figure 20 .

[0074] Comparative Example 1

[0075] The difference from Example 1 is that the azide-modified mannose (Man) is replaced with an equal amount of azide-modified glucose (Glc), and finally SCu-COF-Glc is obtained.

[0076] Comparative Example 2

[0077] The difference from Example 1 is that the azide-modified mannose (Man) is replaced with an equal amount of azide-modified galactose (Gal), and finally SCu-COF-Gal is obtained.

[0078] Comparative Example 3

[0079] (1)Prepare PN-COF according to the method of step (3) of Example 1.

[0080] (2) Take about 100 mg of PN-COF, add about 20 mg of copper acetate, heat it at 65 °C in 20 mL of methanol and reflux for 5 days, cool and filter to obtain a purple-black powder, which is Cu-COF.

[0081] Comparative Example 4

[0082] Multifunctional cationic covalent organic framework material prepared according to the examples in CN116789980B: HLV-Cu polymer.

[0083] Example 2: Characterization and property study

[0084] (1) Figure 1 Shows the synthetic route of SCu-COF-Man. Through Figure 1 (a) Fourier transform infrared spectroscopy was used to preliminarily explore the bonding and chemical structure of SCu-COF-Man, using the reaction monomers and unmodified samples as controls. The results showed that the fine vibration of -NH in TAPP (~3400 cm -1 ) and the -C=O of NA (1700 cm -1 ) disappeared, while the C=N of NA (~1580 cm -1 ) appeared, indicating the successful construction of PN-COF. The alkyne vibration of NA (~2210 cm -1 ) was also shown on PN-COF. With the insertion of metal ions and the modification of sugar molecules, its characteristics disappeared, and instead, the vibration peak of the triazole ring 2260 cm -1 bond generated by the click reaction appeared. These characteristics also appeared in Figure 1 (b) The infrared spectra of SCu-COF-Man, SCu-COF-Glc and SCu-COF-Gal, and the characteristics of sugar (3500 cm -1 , 1550 cm -1 ) also appeared. The characteristic carbon resonance peaks obtained by solid-state 13C CP / MAS NMR further verified the formation of imine and triazole bonds. As Figure 1 (c) shown, 13C-NMR showed a broad peak signal covering saturated and unsaturated carbons, and strong carbon peaks were shown at 65, 122, 133, 148 and 155 ppm respectively. The unsaturated carbon signals at 122, 148 and 155 ppm were attributed to imine bonds and triazole ring bonds, and the remaining peaks were attributed to aromatic carbons in porphyrin and naphthalene units and unreacted alkynyl carbons respectively. The saturated carbon signals were attributed to sugar units. As Figure 1As shown in (d), the Zeta potential of COF was determined to be -3.11 eV, and it changed to -13.06, -12.3, and -13.26 eV after being modified by Man, Glc, and Gal molecules, respectively. It is worth noting that the Zeta potential of SCu-COF-Man increased to 2.91 eV after being modified by metal ions, enabling it to adhere well to the bacterial surface.

[0085] (2)Thermogravimetric analysis (TGA) showed that the synthesized SCu-COF-Man had excellent thermal stability. Figure 1 In (f), the residual weight of SCu-COF-Man exceeded 50% at 800 °C, and its thermal stability was enhanced compared with the Figure 1 thermogravimetric results of PN-COF in (e), indicating that the participation of Cu 2+ increased the overall stability of the carbon skeleton. The porosity of SCu-COF-Man was estimated by low-temperature nitrogen (N2) adsorption-desorption measurements. As Figure 1 shown in (g) and Figure 1 (h), SCu-COF-Man exhibited a type-IV isotherm, and a typical H3-type hysteresis loop appeared in the later stage of the curve, indicating its mesoporous-dominated layered structure porosity. The specific surface area of SCu-COF-Man calculated based on the Brunauer Emmett Teller model was 23.2 m 2 g -1 , and the cumulative pore volume was 0.068 cm 3 g -1 . The pore size distribution (PSD) curve of SCu-COF-Man further revealed its mesoporous-dominated porosity, with the main peak located at 2.4 nm and the secondary peaks located at 13.8 and 21.7 nm, respectively. In addition, X-ray photoelectron spectroscopy (XPS) was used to study the elemental composition of SCu-COF-Man. As Figure 2 shown in (a), the survey spectrum revealed the coexistence of Cu, N, C, and O on the surface of SCu-COF-Man, with atomic ratios of 45.83%, 2.25%, 9.98%, and 41.92%, respectively. Figure 2 (b) shows the fine spectrum of N 1s, where the obvious peak signals were attributed to C=N, N=N (398.4 eV), porphyrin-N (399.8 eV), and Cu-N, N=N-N (400.5 eV), respectively, while the other peak signals may be attributed to π-π* conjugation. Figure 2 The high-resolution O 1s spectrum in (c) was divided into three peaks at 532.7, 531.4, and 530.7 eV, which were attributed to C=O, aromatic-O ether, O-C, and O-H bonds, respectively. Figure 2(d)The Cu 2p spectrum of SCu-COF-Man was fitted with five peaks located at 934.8, 941.7, 944.4, 954.8, and 963.1 eV. The signals at 963.1, 941.7, and 944.4 eV were attributed to satellite peaks, while the remaining three peaks were attributed to Cu 2+ 2p 2 / 3 and Cu 2+ 2p 1 / 3 , indicating that it was produced by CuO. The growth of CuO during the polymerization process could also be verified by powder X-ray diffraction (PXRD). The PXRD pattern of SCu-COF-Man showed distinct peaks at 32.78, 35.50, and 38.52, corresponding to the (1,1,0), (0,0,1), and (1,1,1) crystal planes of the crystalline CuO structure ( Figure 3 ). Through Figure 4 (a)~ Figure 4 (c) field emission scanning electron microscopy and Figure 4 (d)~ Figure 4 (g) transmission electron microscopy, the microscopic morphology of SCu-COF-Man was studied. The SEM images showed that SCu-COF-Man exhibited a typical layered structure, and the surface topological structure was characterized by interconnected macropores and cracks. The TEM results showed that there were a large number of tiny pores in the layered stacked structure. High-resolution TEM (HR-TEM) showed that micropores were simultaneously present on the polymer matrix, visible through light and dark contrast. At the same time, HR-TEM also showed widespread lattice fringes with a fringe spacing of 0.23 nm, which might be due to the presence of CuO, consistent with the XRD analysis. This unique structure provided multiple adsorption sites for bacteria of different shapes and sizes, thus promoting the adsorption process. This aggregation could be attributed to the complex hydrogen bond interactions within the sugar clusters. In addition, consistent with the XPS results, energy-dispersive X-ray spectroscopy ( Figure 5 ) obtained by high-angle annular dark-field scanning TEM (HAADF-TEM) and the corresponding elemental mapping revealed the uniform distribution of N, Cu, and O elements in the C-element-dominated structural framework.

[0086] (3) Since the positively charged Cu in SCu-COF-Man would also adsorb negatively charged bacteria, in order to investigate the capture of bacteria by the sugar in SCu-COF-Man, S-COF-Man without copper was prepared. S-COF-Man, S-COF-Glc, and S-COF-Gal were prepared by omitting step (5) ionization modification according to the methods of Example 1 and Comparative Examples 1~2, and PN-COF was prepared according to the method of step (3) of Example 1. Their binding abilities to Escherichia coli and Staphylococcus aureus were investigated. Figure 6 (a) depicts the four materials at a concentration of 10 8The capture efficiency after 1 hour of interaction between Escherichia coli or Staphylococcus aureus at CFU / mL. Compared with S-COF-Glc, PN-COF, and S-COF-Gal, S-COF-Man showed good capture efficiency for both Escherichia coli and Staphylococcus aureus. Figure 6 (b)~ Figure 6 (c)The kinetics of bacterial capture was determined by measuring the change in the optical density of the bacterial solution at 600 nm over time. It can be seen that the optical density of S-COF-Glc and S-COF-Gal decreased slowly, while the optical density of PN-COF did not change significantly. In contrast, S-COF-Man captured far more bacteria than the other three materials within the specified time, with a decrease of approximately 50% within 40 minutes.

[0087] To more intuitively display the results, S-COF-Man was pretreated with BSA and equilibrated with Escherichia coli and Staphylococcus aureus (both at a concentration of 10 8 CFU / mL). After 1 hour, the supernatant was treated with β-glucuronidase (REG) at room temperature for 2 hours. Since resorcinol was released by the β-glucuronidase present in the supernatant, the solution showed a faint pink color. As Figure 6 (d)~ Figure 6 shown in (e), when the initially blank Escherichia coli and Staphylococcus aureus were treated with REG, the solution showed an orange-yellow color and only turned pink after the addition of cell lysate. This indicates that both strains were positive for REG. In the bacterial solutions equilibrated with S-COF-Man and S-COF-Glc, a pink color was observed when the supernatant was treated with REG. However, according to Figure 6 (f)~ Figure 6 (g), no obvious color change was observed when the supernatant of Escherichia coli and Staphylococcus aureus cells containing S-COF-Gal was treated with REG. These results indicate that the release of β-glucuronidase only occurs when the sample captures bacteria resulting in bacterial cell lysis. It can be seen that after removing copper from SCu-COF-Man, S-COF-Man has better bacterial capture ability than S-COF-Glc, S-COF-Gal, and PN-COF.

[0088] Therefore, in addition to using sugars to capture bacteria, SCu-COF-Man also combines surface properties with charge effects, making SCu-COF-Man a potential bacterial adsorbent. That is, in addition to bacteria engulfing the sugars on the surface of SCu-COF-Man to achieve bacterial capture, the tiny pores and cracks on the surface of SCu-COF-Man provide abundant adsorption sites (positively charged copper), and the positively charged copper can also adsorb bacteria by binding to negatively charged bacteria. The charge effect promotes the adsorption process between negatively charged bacteria and the positively charged surface of SCu-COF-Man through electrostatic interaction, while hydrogen bonds and van der Waals forces stabilize the adsorption of bacteria on the surface of SCu-COF-Man. Therefore, SCu-COF-Man can capture bacteria through the sugars and copper it contains together, thus enabling faster capture of bacteria.

[0089] (4)Using 1,3-diphenylisobenzofuran (DPBF), methylene blue (MB), and dihydro-rhodamine 123 (DHR123) as chemical probes, the singlet oxygen ( 1 O2), hydroxyl radical (•OH), and superoxide anion (O2 •- ) were respectively studied, and the ability of SCu-COF-Man to generate photoinduced reactive oxygen species (ROS) was finely evaluated. As Figure 8 shown in Figure 7 (d), in sharp contrast to the minimal attenuation Figure 8 (a) observed for the pure DPBF probe, the ultraviolet-visible light absorption in the presence of SCu-COF-Man showed an obvious downward trend, reaching 100% within 1 minute, 1 (a) demonstrating a significant Figure 7 O2 generation ability. Similar to DPBF, Figure 8 (b) the ultraviolet-visible spectrum of the pure MB probe only showed a slight decrease in absorbance after different irradiation times. However, Figure 8 (b) after adding SCu-COF-Man, a significant decrease in absorbance was observed, indicating the generation of •OH. Figure 8 The attenuation rate in Figure 7 (e) intuitively shows the change in curve attenuation before and after the introduction of SCu-COF-Man. In addition, combining •- (c) and Figure 8 (c), it can be seen that compared with the DHR123 solution exposed to laser conditions alone, the DHR123 solution treated with SCu-COF-Man showed a significant increase, indicating a significant •-Generating ability. In addition, the EPR measurement results provide strong evidence for the formation of the above-mentioned ability. In summary, SCu-COF-Man, as a photosensitive material with a multifunctional antibacterial platform, shows great potential. In addition, verifying that the modification of Cu 2+ introduces peroxidase-like activity into SCu-COF-Man. By using 3,3’,5,5’-tetramethylbenzidine (TMB) as a visual indicator of color change, the decomposition of H2O2 is monitored to study this activity. Figure 9 Figure (a) shows that SCu-COF-Man has pH-dependent enzyme activity, and the ultraviolet absorption curve shows an initial increase followed by a decrease, with the peak appearing at pH = 3.5. It is worth noting that SCu-COF-Man also maintains considerable catalytic activity in the bacterial infection environment (pH = 5.5). Similarly, Figure 9 Figure (b) shows the relationship between the enzyme activity of PN-COF and its concentration, and the activity increases with the increase in concentration (under the condition of constant pH = 5.5). Interestingly, compared with before irradiation, the introduction of laser significantly enhances the enzyme activity after irradiation, as shown in Figure 9 Figure (c). This indicates that the photothermal effect further enhances the enzyme activity. Combining with the special bacterial capture ability, a synergistic effect is produced, resulting in severe oxidative damage to bacteria.

[0090] Glutathione (GSH) produced by cells can combine with ROS and scavenge ROS, thus reducing the bactericidal effect; therefore, the GSH scavenging ability of SCu-COF-Man was tested. As can be seen from Figure 9 Figure (d), at the same incubation time (5 minutes), with the increase in concentration, GSH is depleted when the concentration of SCu-COF-Man is 100 μg / mL. Figure 9 In Figure (e), at 75 μg / mL of SCu-COF-Man, GSH is completely eliminated after 15 minutes of incubation. This indicates that SCu-COF-Man has amazing glutathione peroxidase (GPx)-like activity. The above results can be more intuitively observed in Figure 9 Figure (f) and Figure 9 Figure (g).

[0091] To verify whether the consumption of GSH enhances the generation of •OH, the change in •OH concentration at different incubation times after adding GSH was detected. As shown in Figure 9 Figure (h), in the presence of GSH, the generation of •OH initially increases with the increase in incubation time. This indicates that the elimination of GSH can continue to generate •OH by supplementing H2O2, thereby producing a bactericidal effect.

[0092] Test Example 1: Antibacterial Test

[0093] The in vitro antibacterial ability of the prepared SCu-COF-Man in the examples was evaluated by the bacterial plate counting method. PN-COF and the prepared SCu-COF-Glc and SCu-COF-Gal in Comparative Examples 1-2 were used as controls, and Escherichia coli and Staphylococcus aureus were used as model strains.

[0094] First, the above materials were prepared with PBS to a concentration of 190 μg / mL. They were respectively denoted as the SCu-COF-Man group, the SCu-COF-Glc group, the SCu-COF-Gal group, and the PN-COF group. The total volume of the mixed solution in each group was 1 mL, and the bacterial solution concentration was 100 μL / mL (the number of colonies was 10 8 CFU / mL -1 ). Each group was irradiated with 638 nm infrared laser, the power density was 1.5 W / cm -2 , and the irradiation duration was 10 min. Finally, the mixed solution of each group was placed in a shaker and incubated at 37 °C for 2 h. 100 μL of the mixed solution from each group was transferred to a solid medium, the morphology was observed, and the number of colonies was calculated. And it was compared with the control group (PBS group) to evaluate the antibacterial effect.

[0095] SCu-COF-Man showed the most significant bactericidal effect after incubation at room temperature for 2 h under the condition of pH = 7.35 in PBS ( Figure 10 ).

[0096] Secondly, the synergistic antibacterial potential of SCu-COF-Man at different concentrations was studied ( Figure 11 ). The preparation solution was PBS with pH = 5.5, the H2O2 concentration was 10 μL / mL, and after laser irradiation for 10 minutes, it was spread on the plate for observation.

[0097] It can be seen that after 2 h of incubation, as the concentration of SCu-COF-Man increased (0 to 75 μg / mL), the bacterial survival rate gradually decreased. At 75 μg / mL, the survival rates of Staphylococcus aureus and Escherichia coli were only 0.28 ± 0.12% and 4.01 ± 1.11% respectively.

[0098] Finally, to emphasize the synergistic antibacterial advantage of SCu-COF-Man, Staphylococcus aureus and Escherichia coli were respectively subjected to different treatments. The experimental groups were as follows: (I) control group (PBS), (II) H2O2 group, (III) SCu-COF-Man group, (IV) SCu-COF-Man + H2O2 group, (V) PBS + laser group, (VI) H2O2 + laser group, (VII) SCu-COF-Man + laser group, and (VIII) SCu-COF-Man + H2O2 + laser group. The concentrations of SCu-COF-Man and H2O2 were set at 75 μg / mL and 10 μL / mL respectively. As Figure 12 shown, similar to the control groups (I and V) with PBS, the H2O2 groups (II and VI) showed negligible bactericidal effects whether or not they received laser irradiation (laser irradiation for 10 minutes), indicating that the concentration of H2O2 in this experiment was not sufficient to cause bacterial damage. In contrast, laser irradiation led to significant changes in the bacterial survival rates in the remaining groups (SCu-COF-Man and SCu-COF-Man + H2O2). Specifically, due to the significant change in affinity, after treatment with SCu-COF-Man, the bacterial survival rate decreased significantly, affecting the normal metabolism of bacteria and resulting in damage to the cell structure. In addition, after only exposure to laser, the bacterial mortality rates in the SCu-COF-Man-treated groups increased from 61.02 ± 6.8% and 51.32 ± 6.6%. It is worth noting that the SCu-COF-Man + H2O2 + laser group (VIII) combined the synergistic effects of enzyme activity, photoactivity, and glyco-cluster molecular affinity, demonstrating the most significant bactericidal performance, with the survival rates of Staphylococcus aureus and Escherichia coli being 1.20 ± 0.34% and 0.33 ± 0.47% respectively.

[0099] Experimental Example 2: Biological Evaluation

[0100] (1) To visually evaluate the anti-biofilm activity of SCu-COF-Man, a biofilm test of Staphylococcus aureus was designed, and its ability was quantified using crystal violet staining and enzyme markers. The experimental groups were the same as in Experimental Example 1. Bacterial solution was added to a 96-well plate, 150 μL per well, and incubated on a shaker for 2 days. First, 100 μL was aspirated out, then 50 μL of the material was added, irradiated with light, after irradiation, 100 μL of PBS was added, 150 μL was aspirated out, 100 μL of methanol was added to fix for half an hour, then 100 μL was aspirated out, 150 μL of PBS was added, 150 μL was aspirated out, then 10 - 50 μL of crystal violet was added for 15 - 20 minutes, washed with PBS until clear, 100 μL of glacial acetic acid was added for 30 minutes, aspirated onto a blank plate and photographed. The results were observed to be consistent with the in vitro antibacterial results. The SCu-COF-Man + L + H2O2 group showed a lighter color compared with other groups (Figure 13 ). Generally speaking, these results highlight the excellent antibacterial ability of SCu-COF-Man, which can simultaneously achieve bactericidal and elimination of organisms.

[0101] (2) To visually observe the damage caused to bacteria by different treatment groups, bacterial live / dead staining and transmission electron microscopy (TEM) techniques were carried out. Bacterial live / dead staining used a SYTO-9 and PI double staining system to label live bacteria and dead bacteria respectively. Grouped according to Test Example 1 into 8 groups (the total volume of the mixed solution in each group was 1 mL), 400 μL of the solution was taken from each group, and 20 μL of SYTO-9 (1.0×10 -3 M) and 20 μL of PI (1.5×10 -3 M) were added thereto, and incubated for 15 min in the dark at 37°C. After that, the excess SYTO-9 and PI were removed by centrifugation. Then the bacteria in each group of solution were resuspended with 50 μL of PBS, and the images were observed through an inverted fluorescence microscope.

[0102] As Figure 14 shown in (a), consistent with the plate counting results, the bacteria in the control group and the H2O2 group in the fluorescence microscope images mainly showed green staining whether or not they were irradiated with laser. On the contrary, obvious red fluorescence of different degrees was detected in the remaining groups. Specifically, the proportion of the red area was in the order of: SCu-COF-Man group < SCu-COF-Man + H2O2 group < SCu-COF-Man + laser group < SCu-COF-Man + H2O2 + laser group. This result indicates that the SCu-COF-Man + H2O2 + laser group contains the most dead bacteria and almost no green area. In contrast, the SCu-COF-Man + laser group and the SCu-COF-Man + H2O2 group showed similar percentages of the red area, but were significantly higher than the SCu-COF-Man group.

[0103] (3) TEM technology was used to evaluate the changes in the state and morphology of bacteria under different treatments. Grouped according to the grouping of Test Example 1 into 8 groups. The solutions of each group were added to 2.5 wt% glutaraldehyde solution and fixed at 4°C for 24 h, then washed 3 times with PBS, embedded with agar and sealed. The bacteria were dehydrated by treating them with ethanol solutions of 30 wt%, 50 wt%, 70 wt%, 90 wt%, 95 wt% and 100 wt% concentrations in sequence for 10 min, and then treated with acetone for 3 h for gradient osmotic embedding. Finally, the bacteria were negatively stained and fixed on a nickel grid for observation through a transmission electron microscope.

[0104] As Figure 14As shown in (b), after treatment with the control group and H2O2, whether irradiated with laser or not, the surface of the bacteria remained intact, Staphylococcus aureus remained spherical, and Escherichia coli remained rod-shaped. However, in the presence of SCu-COF-Man, the surface of the bacteria was wrapped by the material, resulting in significant damage to the bacteria, which was attributed to the excellent adhesion of the sugar molecules, shortening the action distance. This damage was further exacerbated by laser irradiation and the addition of H2O2, intuitively reflecting the superiority of the synergistic bactericidal effect.

[0105] (4) As a basic prerequisite for the practical application of biomedical technology, the in vitro biocompatibility of SCu-COF-Man was evaluated. The blood used in the experiment was taken from 5-week-old female KM mice (purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd.). The obtained fresh blood was centrifuged (10,000 rpm, 20 min), and then the supernatant was removed to collect red blood cells. Subsequently, PBS buffer was added for washing, and the volume ratio of the added PBS buffer to red blood cells was approximately 1:1. After centrifugation, the supernatant was removed, and this was repeated 4 times. Then the red blood cells and PBS buffer were mixed at a volume ratio of 3:11, and the mixed red blood cells and SCu-COF-Man (concentration 50 - 200 μg / mL) were mixed at a ratio of 1:9 (v / v) and incubated at 37 °C for 4 h. 100 μL of the supernatant from each tube was added to a 96-well plate, and the absorbance was measured using an enzyme-linked immunosorbent assay (ELISA) reader. Distilled water was used as the positive control, and the PBS group was used as the negative control. The calculation formula is as follows:

[0106] Hemolysis rate (%) = (A - An) / (Ap - An) × 100%;

[0107] A: Absorbance obtained from the supernatant after adding iso -CMP-1 to red blood cells;

[0108] An: Absorbance obtained from the supernatant after adding PBS to red blood cells (negative control);

[0109] Ap: Absorbance obtained from the supernatant after adding distilled water to red blood cells (positive control).

[0110] As can be seen from Figure 15 (a), SCu-COF-Man showed a concentration-dependent response when co-incubated with blood, and the hemolysis percentage increased with increasing concentration. It should be noted that within the experimental range (50 - 200 μg / mL), the hemolysis rate remained below 2%, far lower than the recommended threshold of 4%.

[0111] (5) In a 96-well plate, NIH / 3T3 cells (from the Cell Bank of the Chinese Academy of Sciences) were seeded at 5 × 10 3Inoculate at a density of individual cells, with 100 μL of cells in each well. The edges of the plate are sealed with 100 μL of PBS to prevent excessive evaporation. After incubating for 24 h, the control group is treated with PBS, and the experimental groups are treated with different concentrations of SCu-COF-Man (concentrations of 50, 100, 150, 200, 300 μg / mL). After standing for 24 h, the supernatant is discarded. Then, 10 μL of MTT (tetramethylthiazolyl blue, 5 mg / mL) is added to each well. After culturing in the incubator for 4 h, the supernatant is aspirated, and 100 μL of dimethyl sulfoxide is added. After standing for about 10 min, the absorbance is measured at 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Each experiment is repeated 3 times.

[0112] As Figure 15 shown in (b)~ Figure 15 (d), even when the concentration of SCu-COF-Man is as high as 300 μg / mL, the cell viability still remains at 85%. In contrast, the cell viability after laser irradiation (1.2 W cm -2 , 10 minutes) slightly decreases but still reaches 80%. In the presence of hydrogen peroxide, the cell survival rate is not as good as the former, although sufficient cell safety is maintained at the therapeutic dose. These findings together reveal the excellent biocompatibility of SCu-COF-Man.

[0113] Test Example 3: Animal Experiment

[0114] Female KM mice at 5 weeks of age and weighing about 25 g (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.) are used to establish a wound trauma model. The mice are randomly divided into 8 groups: control group (PBS), control (PBS)+laser group, H2O2 group, H2O2+laser group, SCu-COF-Ma group, SCu-COF-Man+laser group, SCu-COF-Man+H2O2 group, and SCu-COF-Man+H2O2+laser group, with 6 mice in each group. Normal-fed mice are used as the blank group (6 mice) for comparison with the above other groups. Before formal infection, the weight of each group of mice is measured. Except for the blank group, the mice in other groups are anesthetized, the hair on the back is shaved off, and disinfected with 75 wt% ethanol to form a wound with a radius of about 5 mm. Staphylococcus aureus (1×10 6 CFU / mL) is added to the wound. After infecting for 24 h, the groups are divided and the mice in each group are treated according to the grouping.

[0115] Figure 16(a) shows the photographs of the back wounds on the 1st, 3rd, 6th, and 9th days. On the 1st day, the back of the mice was punctured and infected with a bacterial droplet. Over time, the wounds of the mice treated with different treatments showed different degrees of contraction. Compared with the minimum healing progress of the wounds in the control group, control group + laser, H2O2, and H2O2 + laser groups, the SCu-COF-Man-related groups showed a higher degree of wound shrinkage on the 9th day, especially the SCu-COF-Man + H2O2 + laser group. The phototherapy group (SCu-COF-Man + laser), enzyme group (SCu-COF-Man + H2O2), and synergistic group (SCu-COF-Man + H2O2 + laser) demonstrated the most significant wound healing, achieving wound shrinkages of 36.22 ± 4.48%, 32.47 ± 4.20%, and 10.60 ± 2.34% respectively. As Figure 16 shown in the wound area histogram in (b), Figure 16 it can be seen from (c) the change in the body weight of the mice, indicating that during the treatment period, except for the steady increase in body weight in the blank group, there was no significant fluctuation in the body weight of other groups. In addition, Figure 17 shows the cross-section of the back wounds after different treatments on the 9th day. In the absence of SCu-COF-Man, regardless of whether light treatment was received, the degree of skin healing remained basically unchanged. However, in the presence of SCu-COF-Man, the laser treatment group showed a better degree of scab formation than the non-laser group, and the synergistic group even showed signs of new capillary and skin regeneration. Finally, Figure 18 (a)~ Figure 18 (f) shows the blood indexes and histological test results of the mice treated in different subgroups. The parameters shown in the routine blood test were all within the normal range and there was no statistically significant difference compared with the control group. At the same time, Figure 18 the histological analysis of the major organs of the mice in (g) showed that there were no abnormalities in the heart, liver, spleen, lungs, and kidneys. The above results indicate that SCu-COF-Man can also maintain good safety in vivo and has no significant impact on the organs.

[0116] Test Example 4: Bactericidal Rate Test

[0117] The antibacterial rate was evaluated by the bacterial plate counting method, using Escherichia coli and Staphylococcus aureus as model strains.

[0118] The test was divided into 4 groups: PBS as the control group, SCu-COF-Man group, Cu-COF group prepared in Comparative Example 3, and HLV-Cu group prepared in Comparative Example 4. The above materials were prepared with PBS as the solution to a concentration of 75 μg / mL, the total volume of each group of the mixture was 1 mL, and the concentration of the bacterial solution was 100 μL / mL (the number of colonies was 10 8 CFU / mL -1). Each group was irradiated with 638 nm infrared laser at a power density of 1.5 W cm -2 , and the irradiation duration was 10 min. Finally, the mixture of each group was placed in a shaker and incubated at 37 °C for 1 h. 100 μL of the mixture of each group was transferred to a solid medium to observe the morphology and calculate the number of colonies. And it was compared with the control group (PBS group) to evaluate the antibacterial effect.

[0119] As can be seen from Figure 19 , compared with PBS, about 50% of the bacteria in the Cu-COF group and the HLV-Cu group were still alive after 1 h of incubation, while more than 90% of the bacteria in the SCu-COF-Man group were killed, and the bactericidal speed was faster than that of other groups.

[0120] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A sugar-grafted metal-covalent organic framework artificial enzyme for rapid capture and clearance of bacteria, characterized in that The sugar-grafted metal-covalent organic framework artificial enzyme includes a covalent organic framework polymer with a porous structure; the covalent organic framework polymer is a covalent organic framework polymer containing porphyrin, and the porphyrin coordinates with the metal through a four-coordination center, so that the metal is anchored in the covalent organic framework polymer; sugar is grafted onto the covalent organic framework polymer; the sugar is a monosaccharide; the metal is copper; The covalent organic framework polymer is copolymerized from 2,6-bis(2-propynyloxy)naphthalene-1,5-dicarbaldehyde and tetraaminophenyl porphyrin; The monosaccharide is mannose.

2. The preparation method of the sugar-grafted metal-covalent organic framework artificial enzyme according to claim 1, characterized in that, It includes the following steps: (1) Add tetraaminophenyl porphyrin and 2,6-bis(2-propynyloxy)naphthalene-1,5-dicarbaldehyde into an organic solvent, and heat and react to obtain a dark purple powder PN-COF; (2) Add copper iodide, PN-COF, N,N-diisopropylethylamine and azide-modified sugar into a mixed solution of THF, H2O and tert-butanol to obtain a suspension. The suspension is stirred at room temperature overnight under a protective atmosphere. After the reaction is completed, filter the solid, wash and dry it to obtain a brown solid COF-sugar; (3) Add COF-sugar and copper acetate into methanol, heat and reflux the reaction. After the reaction is completed, cool and filter to obtain a purple-black powder Cu-COF-sugar; (4) Add a methanol solution to Cu-COF-sugar, and disperse it evenly by ultrasonic treatment; then add a methanol solution containing sodium methoxide, and let it stand overnight at room temperature under a protective atmosphere. Filter, wash and dry it to obtain a brown solid SCu-COF-sugar, which is the sugar-grafted metal-covalent organic framework artificial enzyme.

3. The preparation method according to claim 2, wherein In step (1), the molar ratio of tetraaminophenyl porphyrin to 2,6-bis(2-propynyloxy)naphthalene-1,5-dicarbaldehyde is 1:2; the organic solvent is a mixture of 1,4-dioxane, mesitylene and acetic acid in a volume ratio of 6:3:2; the temperature of the heating reaction is 120 °C, and the time of the heating reaction is 5 days.

4. The preparation method according to claim 2, characterized in that, In step (2), the volume ratio of THF, H2O and tert-butanol is 3:1:1; the addition amounts of copper iodide, PN-COF, N,N-diisopropylethylamine and azide-modified sugar are in a ratio of 27.69 mg:100 mg:87 μL:10 mg.

5. The preparation method according to claim 4, characterized in that, The azide-modified sugar is azide-modified mannose.

6. The preparation method according to claim 2, wherein In step (3), the mass ratio of COF-sugar to copper acetate is 5:1; the temperature of the heating reflux reaction is 65 °C, and the time of the heating reflux reaction is 5 days.

7. The preparation method according to claim 2, characterized in that, In step (4), the addition amounts of Cu-COF-sugar, methanol and the methanol solution containing sodium methoxide are in a ratio of 25:4:1; the concentration of the methanol solution containing sodium methoxide is 1 M.

8. Use of the sugar-grafted metal-covalent organic framework artificial enzyme according to claim 1 in the preparation of antibacterial drugs or drugs for removing biofilms, characterized in that, The concentration of the sugar-grafted metal-covalent organic framework artificial enzyme is 75 μg / mL, and it can capture and remove bacteria within 1-2 h.

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