Sugar-grafted metal-covalent organic framework artificial enzyme capable of rapidly capturing and removing bacteria as well as preparation method and application of sugar-grafted metal-covalent organic framework artificial enzyme

Abstract

The invention discloses a sugar grafted metal-covalent organic framework artificial enzyme capable of rapidly capturing and removing bacteria as well as a preparation method and application of the sugar grafted metal-covalent organic framework artificial enzyme, and belongs to the technical field of biological medicines. The artificial enzyme comprises a covalent organic framework polymer with a porous structure; the covalent organic framework polymer is a porphyrin-containing covalent organic framework polymer, and porphyrin is coordinated with metal through a four-coordination center, so that the metal is anchored in the covalent organic framework polymer; the covalent organic framework polymer is grafted with sugar. The preparation method comprises the following steps: firstly, preparing a covalent organic framework material, then carrying out glycosyl modification to obtain ester, then carrying out coordination with copper, and finally, carrying out ester hydrolysis to obtain the sugar grafted metal-covalent organic framework artificial enzyme. The artificial enzyme not only promotes photothermal therapy (PTT) and photodynamic therapy (PDT) effects, but also has a strong adsorption effect on bacteria and inhibits synthesis of bacterial cell membranes; the bacteria can be captured and cleared within extremely low concentration and extremely short time.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a sugar-grafted metal-covalent organic framework artificial enzyme capable of rapidly capturing and removing 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 overuse of antibiotics has led to the prevalence of antibiotic resistance genes. To address this problem, a variety of new 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 methods. Taking peroxidase (POD) as an example, it can be activated by overexpressed H2O2 in acidic IME and become a promising in situ bacterial inactivation treatment method. However, although the infection-induced H2O2 level is increased compared with normal tissues, its level is still far from sufficient, which poses a major challenge to enzyme therapy that relies on H2O2-dependent hydroxyl radical (•OH) generation.

[0003] At present, the dilemma of bacterial resistance has not been effectively alleviated due to the limited efficacy of single treatment modes. How to effectively eliminate bacteria while avoiding the development of bacterial 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 multiple forces such as Coulomb force, van der Waals force and hydrogen bonding. Subsequently, these bacteria aggregate, lose flagella, and begin to secrete extracellular matrix (ECM), resulting in irreversible adhesion, especially in the immune microenvironment (IME). Therefore, in order to effectively solve this problem, it is urgent to develop bacteria-targeted smart materials that can interfere with the interaction between bacteria and infection sites. These materials can not only eradicate and capture pathogens, but also effectively reshape IME and overcome bacterial threats. At present, the functional transformation through rational material design is the focus and challenge of research, which can hinder the action of bacteria and ultimately delay the formation of biofilm.

[0004] Covalent organic frameworks (COFs) are an emerging class of multifunctional materials that are made of organic building blocks connected by covalent bonds. The diverse chemical bond types and structural building blocks provide COFs with endless research possibilities and show great potential in various applications. COFs have unique chemical stability and tunability, and can be used as an integrator of structure and function to achieve precise control of material properties. They are expected to play an increasingly important role in biomedical research and clinical practice. These unique properties enable COFs to separate and aggregate bacterial cells and minimize 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. Sugars are ubiquitous in organisms and are one of the three key substances that constitute life processes. They are involved in physiological processes such as bacterial infection, cell signaling, and inflammation. It is worth noting that the bacterial infection process is mainly promoted by specific sugar-protein binding interactions, and bacterial adhesion 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 and bacterial sugar-protein binding is used to capture bacteria, the bacteria will metabolize by engulfing the sugar after binding with the sugar, which will promote the reproduction of bacteria and fail to capture and eliminate the bacteria. Therefore, it is necessary to design a material that contains both sugar and covalent organic framework, which can not only capture and eliminate bacteria, but also quickly capture and eliminate bacteria. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a sugar-grafted metal-covalent organic framework artificial enzyme that can quickly capture and remove 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 quickly capture and remove bacteria, and can achieve the capture and removal 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 solution: The first aspect of the present invention provides a sugar-grafted metal-covalent organic framework artificial enzyme that can quickly capture and remove bacteria. 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 is coordinated with the metal through a four-coordination center, so that the metal is anchored in the covalent organic framework polymer; sugar is grafted on the covalent organic framework polymer; the sugar is a monosaccharide; and the metal is copper.

[0007] Preferably, the covalent organic framework polymer is obtained by copolymerization of 2,6-bis(2-propynyloxy)naphthalene-1,5-dicarboxaldehyde and tetraaminophenylporphyrin.

[0008] Preferably, the monosaccharide is mannose.

[0009] The second aspect of the present invention provides a method for preparing a sugar-grafted metal-covalent organic framework artificial enzyme, comprising the following steps: (1) Tetraaminophenylporphyrin and 2,6-bis(2-propynyloxy)naphthalene-1,5-dicarboxaldehyde are added to an organic solvent and heated to react to obtain a dark purple powder PN-COF; (2) adding cuprous iodide, PN-COF, N,N-diisopropylethylamine and azide-modified sugar to a mixture of THF, H2O and tert-butanol to obtain a suspension, stirring the suspension at room temperature overnight under a protective atmosphere, and after the reaction, filtering the solid, washing and drying it to obtain a brown solid COF-sugar; (3) COF-sugar and copper acetate are added to methanol, heated under reflux for reaction, and after the reaction is completed, cooled and filtered to obtain purple-black powder Cu-COF-sugar; (4) Adding methanol solution to Cu-COF-sugar and dispersing it evenly by ultrasonication; then adding methanol solution containing sodium methoxide, standing overnight at room temperature in a protective atmosphere, filtering, washing, and drying to obtain brown solid SCu-COF-sugar, which is the sugar-grafted metal-covalent organic framework artificial enzyme.

[0010] Preferably, in step (1), the molar ratio of tetraaminophenylporphyrin 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 heating reaction time is 5 days.

[0011] Preferably, in step (2), the volume ratio of THF, H2O and tert-butanol is 3:1:1; the added amount ratio of cuprous iodide, PN-COF, N,N-diisopropylethylamine and azide-modified sugar is 27.69 mg: 100 mg: 87 µL: 10 mg.

[0012] Preferably, the azide-modified sugar is azide-modified mannose.

[0013] Preferably, in step (3), the mass ratio of the COF-sugar to copper acetate is 5:1; the temperature of the heating reflux reaction is 65° C., and the heating reflux reaction time is 5 days.

[0014] Preferably, in step (4), the ratio of the added amounts of the Cu-COF-sugar, methanol and the methanol solution containing sodium methoxide is 25:4:1; and the concentration of the methanol solution containing sodium methoxide is 1M.

[0015] The third aspect of the present invention provides the use of a sugar-grafted metal-covalent organic framework artificial enzyme (SCu-COF-Man) in the preparation of antibacterial drugs or biofilm removal drugs, wherein the sugar-grafted metal-covalent organic framework artificial enzyme has a concentration of 75 μg / mL and can capture and remove bacteria within 1 to 2 hours.

[0016] Beneficial effects of the present invention: (1) The sugar-grafted metal-covalent organic framework artificial enzyme (SCu-COF-Man) prepared by the present invention is photoresponsive and can be used as an intelligent platform for efficient sterilization and wound healing. The sugar molecules not only promote the photothermal therapy (PTT) and photodynamic therapy (PDT) effects in the entire antibacterial process, 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 extraordinary biosafety at therapeutic doses, greatly expanding its application prospects. This work provides an innovative strategy for the development of COF structures with new sugar modifications.

[0017] (2) The SCu-COF-Man of the present invention can not only capture and remove bacteria, but also quickly capture and remove bacteria. It can quickly capture and remove bacteria within 1-2 hours at an extremely low concentration (75 μg / mL). Compared with porous polymer artificial enzymes that use electric charges to adsorb bacteria, the sterilization speed is faster and the drug concentration is extremely low. This provides a new idea for quickly capturing bacteria and sterilizing them.

[0018] (3) The present invention utilizes the targeting ability of sugars to pathogens and the polyhydroxyl groups contained in sugars to mediate directional functionalization, stereochemical diversity to achieve spatial regulation, and natural biocompatibility to ensure physiological tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1:Physical characteristics of polymers: (a) IR spectra of TAPP, NA, PN-COF and SCu-COF-Man; (b) IR spectra of S-COF-Gal, S-COF-Glc and S-COF-Man; (c) S-S NMR of SCu-COF-Man; (d) Zeta potential changes of PN-COF, S-COF-Man, S-COF-Glc, S-COF-Gal and SCu-COF-Man; (e) Thermogravimetric curves before modification; (f) Thermogravimetric curves after modification; (g) Low-temperature N2 absorption isotherm; (h) Pore size distribution curve of SCu-COF-Man; Figure 2 :(a)Total element XPS of SCu-COF-Man;(b)High-resolution XPS of N 1s;(c)High-resolution XPS of O 1s;(d)High-resolution XPS of Cu 2p; Figure 3 : XRD of SCu-COF-Man; Figure 4 :(a)Scanning electron microscopy of SCu-COF-Man at 1000nm;(b)Scanning electron microscopy of SCu-COF-Man at 500nm;(c)Scanning electron microscopy of SCu-COF-Man at 200nm;(d)Transmission electron microscopy of SCu-COF-Man at 200nm;(e)Transmission electron microscopy of SCu-COF-Man at 50nm;(f)Transmission electron microscopy of SCu-COF-Man at 5nm;(g)Transmission electron microscopy of SCu-COF-Man at 0.5nm;(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; Figure 5 :EDS of SCu-COF-Man; 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)The kinetic curves of Escherichia coli captured by S-COF-Man, S-COF-COF-Glc, S-COF-Gal and PN-COF;(c)The kinetic curves of Staphylococcus aureus captured by S-COF-Man, S-COF-Glc, S-COF-Gal and PN-COF;(d)The initial color of REG;(e)The color of REG after bacterial rupture;(f)The color change of Escherichia coli after treatment with different glycosyl polymers;(g)The color change of Staphylococcus aureus after treatment with different glycosyl polymers; Figure 7 :(a)UV spectrum of DPBF after laser irradiation;(b)UV spectrum of MB after laser irradiation;(c)Fluorescence spectrum of DHR123 after laser irradiation; Figure 8 :(a)UV-visible spectra after adding SCu-COF-Man and DPBF under laser irradiation;(b)UV-visible spectra after adding SCu-COF-Man and MB under laser irradiation;(c)Fluorescence spectra after adding SCu-COF-Man and DHR123 under laser irradiation;(d)Attenuation curve after adding SCu-COF-Man and DPBF under laser irradiation;(e)Attenuation curve after adding SCu-COF-Man and MB under laser irradiation;(f)Attenuation curve of SCu-COF-Man and DHR123 under laser irradiation; Fig. 9 :(a) UV-visible spectra of hydrogen peroxide catalyzed by SCu-COF-Man at different pH (150 μg / mL); (b) different concentrations at the same pH (pH=5.5); (c) the effect of laser on the catalytic effect with or without laser; (d) GSH elimination curves at different concentrations (0, 50, 75, 100 μg / mL); (e) GSH elimination curves at the same concentration (75 μg / mL) and different incubation times (0, 5, 10, 15 min); (f) Staphylococcus aureus inhibition percentage histogram; (g) Escherichia coli inhibition percentage histogram; (h) •OH production curves after incubation for different time periods; Fig.10 :(a)Antibacterial plate image of different sugar cluster materials at the same concentration (190 μg / mL);(b)Bacterial activity histogram; Fig.11 :(a)Antibacterial plate image of SCu-COF-Man at different concentrations (0-75 μg / mL);(b)Bacterial activity histogram; Fig.12 :(a) Plate colony image of Staphylococcus aureus;(b) Bacterial activity histogram of Staphylococcus aureus;(c) Plate colony image of Escherichia coli;(d) Bacterial activity histogram of Escherichia coli; Fig.13 :SCu-COF-Man biofilm experiment; Fig.14 : (a) Inverted fluorescence image 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; Fig.15:(a) SCu-COF-Man hemolysis test;(b) SCu-COF-Man MTT test;(c) SCu-COF-Man MTT test under laser irradiation;(d) SCu-COF-Man MTT test in the presence of hydrogen peroxide; Fig.16 :(a)Changes in back wounds of mice in different groups from 1 to 9 days;(b)Percentage of wound healing during treatment;(c)Changes in mouse body weight; Fig.17 : HE and Masson staining of the back wound sections of mice on day 9; Fig.18 :(a)Changes in leukocytes of mice on day 9;(b)Changes in erythrocytes 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)Changes in mean corpuscular volume of mice on day 9;(f)Changes in hematocrit of mice on day 9;(g)H&E staining of heart, liver, spleen, lung and kidney of mice in different groups; Fig.19 : Comparative experiment on rapid bactericidal effect among PBS group, Cu-COF group, HLV-Cu group and SCu-COF-Man group; Fig. 20 : Synthesis schematic diagram. DETAILED DESCRIPTION

[0020] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0021] As introduced in the background technology section, bacteria and sugar can be combined with specific sugar-proteins to achieve sugar capture by bacteria, and bacterial adhesion to cells depends on the recognition of sugar molecules on the surface of host cells. If this characteristic can be used to reversely capture bacteria using sugar, it will help with antibacterial. However, after bacteria combine with sugar, they will metabolize by engulfing sugar, which will promote the reproduction of bacteria instead, and it is impossible to use sugar to capture and remove bacteria.

[0022] Based on this, the purpose of the present invention is to provide a sugar-grafted metal-covalent organic framework artificial enzyme that can quickly capture and remove bacteria, and a preparation method and application thereof. The present invention designs a COFs-based bacterial capture and inactivation platform, and prepares a sugar-grafted metal-covalent organic framework artificial enzyme that can quickly capture and remove bacteria through step-by-step functional modification (metal embedding and sugar modification). The surface sugar modification of the two-dimensional (2D) layered substrate gives COFs selective bacterial recognition ability, further promoting the interaction between the polymer matrix and bacteria. The embedding of copper enables COFs to have dual enzyme activities that simulate peroxidase (POD) and glutathione (GSH) oxidase. The ability to simulate POD enables COFs to use endogenous H2O2 to generate highly toxic hydroxyl radicals (•OH), which specifically kill bacteria in pathological weakly acidic IME, while the GSH oxidase activity promotes the consumption of GSH, provides conditions for the generation of H2O2, and realizes a self-cascade enzyme reaction. Abnormally high GSH levels induced by anaerobic glycolysis in infected tissues will remove the generated reactive oxygen species (ROS), reduce the ROS content and thus affect the treatment efficiency. GSH oxidase activity promotes the consumption of GSH to avoid the above problems. It is of great significance to consume GSH to enhance the synergistic therapeutic effect.

[0023] Modification with different sugars gives COFs the ability to specifically recognize bacterial cell surface proteins (called lectins). These proteins present on the surface of bacteria help bacteria bind to specific sugars on the surface of foreign cells by interacting with carbohydrates. For example, mannose binds to Escherichia coli, etc. By utilizing the antibacterial principle of the present invention, the types of sugar copolymers have been expanded. The copolymers of the present invention can be grafted with various monosaccharides or polysaccharides, and more drugs with different effects can be developed by utilizing the effects of different sugars. Among multivalent ligands, sugar copolymers have broad prospects in bacterial adhesion research. The sugar part of the sugar copolymer recognizes and captures bacteria, enhancing the adhesion affinity of the nanomaterial to bacteria, thereby inhibiting infection. At the same time, after double modification, the photothermal and photodynamic capabilities of COFs have also been greatly improved. Through significantly enhanced affinity, a superposition effect can be achieved, effectively avoiding ROS and heat loss caused by laser irradiation, thereby being used for bacterial filtration and sterilization.

[0024] In addition, the present invention 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 charge it carries. Compared with other porous polymers that only absorb bacteria and kill bacteria through charge, the SCu-COF-Man of the present invention has both adsorption and capture effects on bacteria, so it can bind to bacteria faster and have a better capture effect. After the sugar contained in the SCu-COF-Man of the present invention is captured by bacteria, because the sugar and COFs are grafted through a triazole bond, the triazole bond is very strong and the bacteria cannot break it. Therefore, after the bacteria capture the sugar, they cannot engulf it, and the sugar captures the bacteria in reverse. In the structure of the present invention, after the bacteria engulf the sugar in the SCu-COF-Man, the sugar and the Cu-COF part work together to reduce the probability of the generated active oxygen leaking into the environment, and the active oxygen directly acts on the bacteria to quickly kill bacteria; and copper consumes GSH to catalyze the production of hydrogen peroxide, and the hydroxyl radicals (•OH) produced can also directly act on the bacteria to reduce the probability of leakage into the environment. The above multiple effects all act directly on bacteria, thereby achieving rapid sterilization at extremely low concentrations.

[0025] 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.

[0026] LB broth, agar, and bacterial viability detection kit were purchased from Shanghai Beteng Biotechnology Co., Ltd.; 4-Tribenzaldehyde was purchased from Anhui Zesheng Technology Co., Ltd., and propionic acid was purchased from Shanghai McLaren Biochemical Technology Co., Ltd.

[0027] If not otherwise specified, the control group in the present invention refers to the PBS group, and the pH of PBS is 7.4.

[0028] In the drawings of the specification, L represents laser, and S represents SCu-COF-Man; for example, S+L represents SCu-COF-Man+laser combination.

[0029] The azide-modified sugars in the present invention, such as azide-modified mannose, azide-modified glucose, and azide-modified galactose, are commercially available and can also be prepared by the following method: 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 at a molar ratio of 1:1, and 2 mL of boron trifluoride ether and 50 mL of dichloromethane were added. The mixture was stirred overnight at 33 °C, quenched with water, washed and dried, and the product was collected and purified by column. The mobile phase was DCM:EA (dichloromethane:ethyl acetate) = 9:1 (v / v), and the mixture was spin-dried to obtain a colorless oily liquid. 5 g of the above product was taken, and 974 mg of KN3 (potassium azide) and 923 mg of BU4NI (tetrabutylammonium iodide) were added. About 30 mL of DMF was added and the reaction was allowed to proceed at room temperature overnight. After the reaction, the obtained product was purified by silica gel column with the mobile phase of PE:EA (petroleum ether:ethyl acetate) = 1:1 (v / v). The obtained liquid was cyclone dried to obtain a yellow oily substance, which was the azide-modified sugar (azido-modified mannose, azide-modified glucose or azide-modified galactose).

[0030] Among them, the brand of acetylated mannose is Anage Chemical (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 Anage Chemical (CAS No.: 4163-60-4).

[0031] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0032] Example 1: Preparation of SCu-COF-Man (1) Synthesis of 2,6-bis(2-propynyloxy)naphthalene-1,5-dicarboxaldehyde 2,6-dihydroxynaphthalene-1,5-dicarboxaldehyde (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 under reflux for 72 hours. After the reaction was completed, cold water was added, the mixture was extracted with dichloromethane, washed three times with NaOH solution (5%, 80 mL) and NaCl solution (5%, 100 mL), dried with anhydrous sodium bicarbonate, filtered and the solvent was removed under pressure, and finally purified by column chromatography (DCM: petroleum ether = 1:1) to obtain a light yellow powder 2,6-bis (2-propynyloxy) naphthalene-1,5-dicarboxaldehyde. The synthetic route is as follows: .

[0033] (2) Synthesis of TAPP Under 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 a refrigerator overnight, then filtered and washed with methanol (300 mL) and deionized water (300 mL). Next, 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 nitrogen atmosphere for 0.5 hours. After the reaction was completed, the mixture was cooled to 0°C and neutralized with aqueous ammonia. The purple product (TAPP, yield 80%) was obtained by filtration and Soxhlet extraction with chloroform.

[0034] (3) Synthesis of PN-COF 1,4-dioxane (12 mL), mesitylene and 6M acetic acid 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-dicarboxaldehyde (85 mg) were added to the mixed solution, and the above mixture was transferred to a Schlenk tube, dissolved by ultrasound, and subjected to three freeze-thaw cycles in a liquid nitrogen environment. The reaction was then transferred to a 120°C environment for 5 days. After the reaction was completed, the mixture was cooled to room temperature and washed with methanol, tetrahydrofuran and dichloromethane in sequence to obtain a dark purple powder, which was PN-COF. The synthesis route is as follows: .

[0035] (4) Glycosylation modification THF, H2O and t-BuOH were mixed in a volume ratio of 3:1:1 to obtain a mixed solution. 27.69 mg of cuprous iodide and 100 mg of PN-COF were added to 13 mL of the mixed solution, followed by purging with argon for 5 minutes. N,N-diisopropylethylamine (DIPEA) (87 µL, 0.50 mmol) was then added, the mixture was purged with argon, and finally about 10 mg of azide-modified mannose (Man) was added to obtain a suspension. The suspension was stirred at room temperature overnight under an argon atmosphere. After the reaction was completed, the solid was filtered, then washed with water, acetonitrile and tetrahydrofuran in sequence and further dried to obtain a brown solid, which was COF-Man.

[0036] (5) Ionization modification Take about 100 mg of COF-Man, add about 20 mg of copper acetate, heat to 65°C and reflux in 30 mL of methanol for 5 days, cool and filter to obtain a purple-black powder, which is Cu-COF-Man.

[0037] (6) Hydrolysis Add about 20 mL of methanol solution to Cu-COF-Man and disperse it evenly by ultrasonication; then add about 5 mL of methanol solution containing sodium methoxide (1M), protect with argon and let stand overnight at room temperature. Filter and wash with methanol to obtain a brown solid, which is SCu-COF-Man. Synthesis diagram is shown in Fig. 20 .

[0038] Comparative Example 1 The difference from Example 1 is that the azide-modified mannose (Man) is replaced with an equal amount of azide-modified glucose (Glc), and SCu-COF-Glc is finally obtained.

[0039] Comparative Example 2 The difference from Example 1 is that the azide-modified mannose (Man) is replaced with an equal amount of azide-modified galactose (Gal), and SCu-COF-Gal is finally obtained.

[0040] Comparative Example 3 (1) PN-COF was prepared according to the method of step (3) of Example 1.

[0041] (2) Take about 100 mg of PN-COF, add about 20 mg of copper acetate, heat to 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.

[0042] Comparative Example 4 A multifunctional cationic covalent organic framework material prepared according to the example in CN116789980B: HLV-Cu polymer.

[0043] Example 2: Characterization and performance studies (1) Figure 1 The synthetic route of SCu-COF-Man is shown. Figure 1 (a) Fourier transform infrared spectroscopy was used to preliminarily explore the bonding and chemical structure of SCu-COF-Man, using the reaction monomer and unmodified samples as controls. The results showed that the fine vibration of -NH of TAPP (~3400 cm -1 ) and -C=O of NA (1700 cm -1 ) disappears, 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 ) is also shown on PN-COF. With the insertion of metal ions and the modification of sugar molecules, its features disappear and are replaced by the triazole ring generated by the click reaction at 2260 cm -1 These features also appear in Figure 1 (b) IR spectra of SCu-COF-Man, SCu-COF-Glc, and SCu-COF-Gal, and the characteristic of sugars (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. Figure 1 As shown in (c), 13C-NMR shows broad peak signals covering saturated and unsaturated carbons, showing strong carbon peaks at 65, 122, 133, 148 and 155 ppm, respectively. The unsaturated carbon signals at 122, 148 and 155 ppm are attributed to the imine bond and triazole ring bond, and the remaining peaks are attributed to the aromatic carbon in the porphyrin and naphthalene units and the incompletely reacted alkynyl carbon. The saturated carbon signal is attributed to the sugar unit. Figure 1 As shown in (d), the Zeta potential of COF was measured to be -3.11 eV, which became -13.06, -12.3, and -13.26 eV after modification with Man, Glc, and Gal molecules, respectively. It is worth noting that the Zeta potential of SCu-COF-Man increased to 2.91 eV after metal ion modification, which enabled it to adhere well to the bacterial surface.

[0044] (2) Thermogravimetric analysis (TGA) showed that the synthesized SCu-COF-Man had excellent thermal stability. Figure 1 (f) The residual weight of SCu-COF-Man at 800℃ exceeds 50%, and compared with Figure 1 (e) Thermogravimetric results of PN-COF show that the thermal stability is enhanced, indicating that Cu 2+ The participation of increases the overall stability of the carbon framework. The porosity of SCu-COF-Man was estimated by low-temperature nitrogen (N2) adsorption-desorption measurements. Figure 1 (g) and Figure 1 As shown in (h), SCu-COF-Man exhibits a type IV isotherm, and a typical H3 type hysteresis loop appears in the later stage of the curve, indicating that its layered structure porosity is mainly mesoporous. The specific surface area of ​​SCu-COF-Man calculated based on the Brunauer EmmettTeller model is 23.2 m 2 g -1 The cumulative pore volume is 0.068 cm 3 g -1The pore size distribution (PSD) curve of SCu-COF-Man further revealed that its porosity was mainly mesopores, with the main peak at 2.4 nm and the secondary peaks at 13.8 and 21.7 nm, respectively. In addition, the elemental composition of SCu-COF-Man was studied using X-ray photoelectron spectroscopy (XPS). Figure 2 As shown in (a), the survey spectrum reveals that Cu, N, C and O coexist on the surface of SCu-COF-Man, with atomic proportions of 45.83%, 2.25%, 9.98% and 41.92%, respectively. Figure 2 (b) shows the detailed spectrum of N 1s, in which the obvious peak signals are attributed to C=N, N=N (398.4 eV), porphyrin-N (399.8 eV) and Cu-N, N=NN (400.5 eV), while the other peak signals may be attributed to π-π* conjugation. Figure 2 The high-resolution O 1s spectrum of (c) is divided into three peaks at 532.7, 531.4, and 530.7 eV, which are attributed to C=O, aromatic-O ether, OC, and OH bonds, respectively. Figure 2 (d) The Cu 2p spectrum of SCu-COF-Man is 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 are attributed to satellite peaks, while the remaining three peaks are attributed to Cu 2+ 2p 2 / 3 and Cu 2+ 2p 1 / 3 , indicating that it is produced by CuO. The growth of CuO during the polymerization process can also be verified by powder X-ray diffraction (PXRD). The PXRD spectrum of SCu-COF-Man shows obvious 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, respectively ( Figure 3 ).pass Figure 4 (a) ~ Figure 4 (c) Field emission scanning electron microscopy and Figure 4 (d) ~ Figure 4(g) Transmission electron microscopy was used to investigate the micromorphology of SCu-COF-Man. SEM images showed that SCu-COF-Man exhibited a typical layered structure, and the surface topology was characterized by interpenetrating macropores and cracks. TEM results revealed a large number of tiny pores in the layered stacking structure. High-resolution TEM (HR-TEM) revealed the simultaneous presence of micropores on the polymer matrix, which were visible through light and dark contrast. At the same time, HR-TEM also revealed widespread lattice fringes with a fringe spacing of 0.23 nm, which may be due to the presence of CuO, consistent with the XRD analysis. This unique structure provides a variety of adsorption sites for bacteria of different shapes and sizes, thereby facilitating the adsorption process. This aggregation can be attributed to the complex hydrogen bonding interactions within the sugar cluster molecules. In addition, consistent with the XPS results, the energy dispersive X-ray spectrum ( Figure 5 ) and the corresponding elemental mapping reveal the uniform distribution of N, Cu and O elements in the C-dominated structural framework.

[0045] (3) Since Cu in SCu-COF-Man is positively charged and can also adsorb negatively charged bacteria, S-COF-Man without copper was prepared to investigate the capture of bacteria by sugar in SCu-COF-Man. S-COF-Man, S-COF-Glc, and S-COF-Gal were prepared by omitting step (5) of ionization modification according to the method of Example 1 and Comparative Examples 1 and 2, and PN-COF was prepared according to the method of step (3) of Example 1. Their binding ability to Escherichia coli and Staphylococcus aureus was investigated. Figure 6 (a) Depicts the four materials at a concentration of 10 8 Capture efficiency of CFU / mL of Escherichia coli or Staphylococcus aureus after 1 hour of interaction. Compared with S-COF-Glc, PN-COF and S-COF-Gal, S-COF-Man showed good capture efficiency for both E. coli and Staphylococcus aureus. Figure 6 (b) ~ Figure 6 (c) The kinetics of bacterial capture were 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 a specified time, with a decrease of about 50% within 40 minutes.

[0046] In order to show the results more intuitively, S-COF-Man was pretreated with BSA and then incubated with Escherichia coli and Staphylococcus aureus (both at a concentration of 10 8CFU / mL). After 1 hour, the supernatant was treated with β-glucuronidase (REG) for 2 hours at room temperature. The solution showed a light pink color due to the release of resorcinol by β-glucuronidase present in the supernatant. Figure 6 (d) ~ Figure 6 As shown in (e), when blank E. coli and S. aureus were treated with REG, the solution was orange-yellow at first, and only turned pink after the addition of cell lysate. This indicates that both strains reacted positively to REG. In the bacterial solution in equilibrium 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) It can be seen that when the supernatant of E. coli and S. aureus cells containing S-COF-Gal was treated with REG, no obvious color change was observed. 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 SCu-COF-Man removes copper, S-COF-Man has better bacterial capture ability than S-COF-Glc, S-COF-Gal and PN-COF.

[0047] Therefore, in addition to using sugar to capture bacteria, SCu-COF-Man also uses surface properties combined with charge effects to make SCu-COF-Man a potential bacterial adsorbent. That is, in addition to bacteria engulfing sugars on the surface of SCu-COF-Man to capture bacteria, the tiny pores and cracks on the surface of SCu-COF-Man provide abundant adsorption sites (positively charged copper), and positively charged copper combined with negatively charged bacteria can also adsorb bacteria. The charge effect promotes the adsorption process between negatively charged bacteria and the positively charged SCu-COF-Man surface through electrostatic interactions, 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 together with the sugars and copper it contains, thereby capturing bacteria faster.

[0048] (4) Using 1,3-diphenylisobenzofuran (DPBF), methylene blue (MB), and dihydrorhodamine 123 (DHR123) as chemical probes, the singlet oxygen ( 1 O2), hydroxyl radicals (•OH) and superoxide anions (O2 •- ), the photoinduced reactive oxygen species (ROS) generation ability of SCu-COF-Man was carefully evaluated. Figure 8 (d) shows the minimal attenuation observed with the pure DPBF probe. Figure 7(a) In sharp contrast, the UV-visible absorption in the presence of SCu-COF-Man shows a clear downward trend, reaching 100% within 1 minute. Figure 8 (a) shows significant 1 O2 generation capacity. Similar to DPBF, Figure 7 (b) UV-vis spectra of the pure MB probe showed only a slight decrease in absorbance after different irradiation times. Figure 8 (b) After the addition of SCu-COF-Man, a significant decrease in absorbance was observed, indicating the generation of •OH. Figure 8 The decay rate in (e) intuitively shows the change in the decay of the curve before and after the introduction of SCu-COF-Man. Figure 8 (c) and Figure 7 (c) It can be seen that the DHR123 solution treated with SCu-COF-Man showed a significant increase compared to the DHR123 solution exposed to laser alone, indicating significant O2 •- Generative capacity. Figure 8 The quantitative decay curves presented in (f) further demonstrate the significantly enhanced O •- In addition, the EPR measurement results provide strong evidence for the formation of the above-mentioned ability. In summary, SCu-COF-Man shows great potential as a photosensitive material with a multifunctional antibacterial platform. 2+ The modification introduced peroxidase-like activity into SCu-COF-Man, which was studied by monitoring the decomposition of H2O2 using 3,3',5,5'-tetramethylbenzidine (TMB) as a visual indicator of color change. Fig. 9 (a) shows that SCu-COF-Man has pH-dependent enzyme activity, and the UV absorption curve shows an increase first and then a decrease, with the peak appearing at pH = 3.5. It is worth noting that SCu-COF-Man also maintains considerable catalytic activity in a bacterial infection environment (pH = 5.5). Similarly, Fig. 9 (b) shows the relationship between the PN-COF enzyme activity and its concentration. The activity increases with increasing concentration (under constant pH = 5.5). Interestingly, the introduction of laser significantly enhances the enzyme activity after irradiation compared to before irradiation, as shown in Figure 2. Fig. 9 (c) This indicates that the photothermal effect further enhances the enzyme activity, and combined with the special bacterial capture ability, a synergistic effect is produced, resulting in severe oxidative damage to the bacteria.

[0049] Glutathione (GSH) produced by cells can bind to and scavenge ROS, thereby reducing the bactericidal effect; therefore, the GSH scavenging ability of SCu-COF-Man was tested. Fig. 9 (d) It can be seen that at the same incubation time (5 min), with increasing concentration, GSH was depleted at a SCu-COF-Man concentration of 100 μg / mL. Fig. 9 (e) SCu-COF-Man at 75 μg / mL, GSH was completely eliminated after 15 minutes of incubation. This indicates that SCu-COF-Man has amazing glutathione peroxidase (GPx)-like activity. Fig. 9 (f) and Fig. 9 This can be observed more intuitively in (g). In order to verify whether the consumption of GSH enhanced the generation of •OH, the changes in •OH concentration at different incubation times after the addition of GSH were detected. Fig. 9 As shown in (h), in the presence of GSH, •OH generation initially increased with increasing incubation time, suggesting that the elimination of GSH can be supplemented with H2O2 to continue the generation of •OH, resulting in a bactericidal effect. Test Example 1: Antibacterial Test The in vitro antibacterial ability of SCu-COF-Man prepared in Example 1 was evaluated by the bacterial plate count method, using PN-COF and SCu-COF-Glc and SCu-COF-Gal prepared in Comparative Examples 1 to 2 as controls, and Escherichia coli and Staphylococcus aureus as model strains.

[0050] First, the above materials were prepared with PBS to a concentration of 190 μg / mL. They were respectively named SCu-COF-Man group, SCu-COF-Glc group, SCu-COF-Gal group and PN-COF group. The total volume of each mixed solution 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 at a power density of 1.5 W cm -2 , the irradiation time 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 was transferred to the solid culture medium from each group, the morphology was observed, and the number of colonies was calculated. The results were compared with the control group (PBS group) to evaluate the antibacterial effect.

[0051] SCu-COF-Man showed the most significant bactericidal effect after incubation at room temperature for 2 hours under PBS pH = 7.35 ( Fig.10 ).

[0052] Secondly, the synergistic antibacterial potential of SCu-COF-Man at different concentrations was investigated ( Fig.11), the preparation solution was PBS 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.

[0053] It can be seen that after 2 hours 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.

[0054] Finally, to emphasize the synergistic antibacterial advantage of SCu-COF-Man, Staphylococcus aureus and Escherichia coli were treated with different treatments, and 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, where the concentrations of SCu-COF-Man and H2O2 were set to 75 μg / mL and 10 μL / mL, respectively. Fig.12 As shown, similar to the control group (I and V) with PBS, the H2O2 groups (II and VI) also showed negligible bactericidal effects regardless of whether they were irradiated with laser light (laser irradiation for 10 minutes), indicating that the H2O2 concentration in this experiment was not sufficient to cause bacterial damage. In contrast, laser irradiation caused significant changes in bacterial survival in the remaining groups (SCu-COF-Man and SCu-COF-Man + H2O2). Specifically, due to the significant change in affinity, the bacterial survival rate was greatly reduced after SCu-COF-Man treatment, which affected the normal metabolism of bacteria and caused damage to the cell structure. In addition, the bacterial mortality rate of SCu-COF-Man treatment increased from 61.02±6.8% and 51.32±6.6% after exposure to laser alone. Notably, the SCu-COF-Man+H2O2+laser group (VIII), which combined the synergistic effects of enzyme activity, photoactivity, and sugar cluster molecular affinity, exhibited the most significant bactericidal performance, with the survival rates of S. aureus and E. coli being 1.20±0.34% and 0.33±0.47%, respectively.

[0055] Test Example 2: Biological Evaluation (1) To visually evaluate the anti-biofilm activity of SCu-COF-Man, a biofilm assay of Staphylococcus aureus was designed, and its ability was quantified using crystal violet staining and enzyme markers. The experimental groups were the same as those 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, and then 10 - 50 μL of crystal violet was added for 15 - 20 minutes. It was washed with PBS until clear, 100 μL of glacial acetic acid was added for 30 minutes, and then aspirated onto a blank plate for photography. 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 to other groups ( Fig.13 ). Generally speaking, these results emphasized the excellent antibacterial ability of SCu-COF-Man, which can simultaneously achieve bactericidal and organism elimination.

[0056] (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 the SYTO-9 and PI dual staining system to label live bacteria and dead bacteria respectively. Grouped into 8 groups according to Experimental Example 1 (the total volume of the mixed solution in each group was 1 mL), 400 μL of the solution was taken out 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, and incubated in the dark at 37 °C for 15 min. 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.

[0057] As Fig.14 (a) shows, consistent with the plate count results, the bacteria in the control group and the H2O2 group in the fluorescence microscope images mainly showed green staining whether or not they received laser irradiation. 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.

[0058] (3) TEM technology was used to evaluate the changes in bacterial status and morphology under different treatments. The samples were divided into 8 groups according to the grouping of Experiment 1. A 2.5 wt% glutaraldehyde solution was added to each group of solutions, fixed at 4°C for 24 h, then washed 3 times with PBS, embedded and blocked with agar. The bacteria were treated with 30 wt%, 50 wt%, 70 wt%, 90 wt%, 95 wt% and 100 wt% ethanol solutions in sequence for 10 min to dehydrate them, then treated with acetone for 3 h, and embedded by gradient penetration. Finally, the bacteria were negatively stained, fixed on a nickel grid and observed by transmission electron microscopy.

[0059] like Fig.14 As shown in (b), in the control group and after H2O2 treatment, the bacterial surface remained intact, Staphylococcus aureus remained spherical, and Escherichia coli remained rod-shaped, regardless of whether it was irradiated by laser. However, in the presence of SCu-COF-Man, the bacterial surface was wrapped by the material, resulting in significant damage to the bacteria, which was attributed to the excellent adhesion of sugar molecules, which shortened 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.

[0060] (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 Experimental Animal Breeding Co., Ltd.). The fresh blood was centrifuged (10,000 rpm, 20 min) and the supernatant was removed to collect the red blood cells. Subsequently, PBS buffer was added for washing. The volume ratio of the added PBS buffer to the red blood cells was about 1:1. After centrifugation, the supernatant was removed and this was repeated 4 times. The red blood cells were then mixed with PBS buffer at a volume ratio of 3:11. The mixed red blood cells were mixed with SCu-COF-Man (concentration of 50-200 μg / mL) at a ratio of 1:9 (v / v) and incubated at 37°C for 4 hours. 100 μL of the supernatant from each tube was added to a 96-well plate and the absorbance was measured using an enzyme 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: Hemolysis volume (%) = (A-An) / (Ap-An) × 100%; A: Add to red blood cells iso -The absorbance obtained by taking the supernatant after CMP-1; An: absorbance obtained by taking the supernatant after adding PBS to red blood cells (negative control); Ap: absorbance of the supernatant after adding distilled water to red blood cells (positive control).

[0061] from Fig.15As can be seen in (a), SCu-COF-Man exhibits a concentration-dependent response when incubated with blood, and the hemolysis percentage increases with increasing concentration. It is worth noting that within the experimental range (50-200 μg / mL), the hemolysis rate remains below 2%, which is well below the recommended 4% threshold.

[0062] (5) NIH / 3T3 cells (from the Cell Bank of the Chinese Academy of Sciences) were plated at 5 × 10 cells per well in a 96-well plate. 3 The cells were inoculated at a density of 100 μL per well, and 100 μL of PBS was used to seal the edges of the plate to prevent excessive evaporation. After incubation for 24 hours, the control group was treated with PBS, and the experimental group was treated with different concentrations of SCu-COF-Man (50, 100, 150, 200, and 300 μg / mL). After standing for 24 hours, the supernatant was discarded, and 10 μL of MTT (tetramethylthiazolyl blue, 5 mg / mL) was added to each well. After incubation in the incubator for 4 hours, the supernatant was aspirated, 100 μL of dimethyl sulfoxide was added, and after standing for about 10 minutes, the absorbance was measured at 540 nm using an enzyme reader. Each group of experiments was repeated 3 times.

[0063] like Fig.15 (b) ~ Fig.15 As shown in (d), even at SCu-COF-Man concentrations as high as 300 μg / mL, cell viability remained at 85%. In contrast, laser irradiation (1.2 W cm -2 The cell viability after 10 min (10 min) decreased slightly but still reached 80%. In the presence of hydrogen peroxide, the cell survival rate was not as good as the former, although it still maintained sufficient cell safety at therapeutic doses. These findings collectively reveal the excellent biocompatibility of SCu-COF-Man.

[0064] Test Example 3: Animal Test The mice were all 5-week-old female KM mice weighing about 25 g (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.) to establish the wound trauma model. The mice were 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. Normally raised mice were used as the blank group (6 mice) for comparison with the other groups mentioned above. Before the formal infection, the weight of each group of mice was weighed. Except for the blank group, the mice in other groups were anesthetized, the hair on the back was shaved, and the wound with a radius of about 5 mm was formed. Staphylococcus aureus (1×10 6CFU / mL), 24 hours after infection, the mice were divided into groups and treated according to the groups.

[0065] Fig.16 (a) shows photos of back wounds on days 1, 3, 6, and 9. On day 1, the backs of mice were punctured and infected with bacterial droplets. Over time, the wounds of mice receiving different treatments showed different degrees of contraction. Compared with the minimal healing progress of the wounds in the control, control + laser, H2O2, and H2O2 + laser groups, the SCu-COF-Man related groups showed a higher degree of wound reduction on day 9, 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) showed the most significant wound healing, achieving 36.22±4.48%, 32.47±4.20%, and 10.60±2.34% wound reduction, respectively. Fig.16 (b) shows the wound area bar graph. Fig.16 (c) The changes in the weight of mice can be seen, indicating that during the treatment period, except for the steady increase in the weight of the blank group, there was no significant fluctuation in the weight of other groups. Fig.17 Cross-sections of back wounds after different treatments on day 9 are shown. In the absence of SCu-COF-Man, the extent of skin healing remained largely unchanged regardless of light exposure. However, in the presence of SCu-COF-Man, the laser-treated group showed a better degree of crust formation than the non-laser group, while the synergistic group even showed signs of new capillaries and skin regeneration. Finally, Fig.18 (a) ~ Fig.18 (f) shows the blood indicators and histological test results of mice treated in different subgroups. The parameters shown in routine blood tests were all within the normal range and there was no statistically significant difference compared with the control group. Fig.18 (g) Histological analysis of the main organs of mice showed 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 effect on organs.

[0066] Test Example 4: Sterilization Speed ​​Test The antibacterial rate was evaluated by the bacterial plate count method, using Escherichia coli and Staphylococcus aureus as model strains.

[0067] The experiment 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 mixed solution 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 at a power density of 1.5 W cm -2 , the irradiation time was 10 min. Finally, the mixed solution of each group was placed in a shaker and incubated at 37°C for 1 h. 100 μL of the mixed solution of each group was transferred to the solid culture medium, the morphology was observed, and the number of colonies was calculated. And compared with the control group (PBS group), the antibacterial effect was evaluated.

[0068] from Fig.19 It can be seen that compared with PBS, about 50% of the bacteria in the Cu-COF group and HLV-Cu group still survived after incubation for 1 hour, while more than 90% of the bacteria in the SCu-COF-Man group were killed, and the sterilization speed was faster than other groups.

[0069] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. 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 that can quickly capture and remove 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 is coordinated with the metal through a four-coordination center, so that the metal is anchored in the covalent organic framework polymer; sugar is grafted on the covalent organic framework polymer; the sugar is a monosaccharide; and the metal is copper.

2. The sugar-grafted metal-covalent organic framework artificial enzyme according to claim 1, characterized in that: The covalent organic framework polymer is obtained by copolymerizing 2,6-bis(2-propynyloxy)naphthalene-1,5-dicarboxaldehyde and tetraaminophenylporphyrin.

3. The sugar-grafted metal-covalent organic framework artificial enzyme according to claim 1, characterized in that: The monosaccharide is mannose.

4. The method for preparing the sugar-grafted metal-covalent organic framework artificial enzyme according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Tetraaminophenylporphyrin and 2,6-bis(2-propynyloxy)naphthalene-1,5-dicarboxaldehyde are added to an organic solvent and heated to react to obtain a dark purple powder PN-COF; (2) adding cuprous iodide, PN-COF, N,N-diisopropylethylamine and azide-modified sugar to a mixture of THF, H2O and tert-butanol to obtain a suspension, stirring the suspension at room temperature overnight under a protective atmosphere, and after the reaction, filtering the solid, washing and drying it to obtain a brown solid COF-sugar; (3) adding COF-sugar and copper acetate into methanol, heating under reflux for reaction, cooling and filtering after the reaction to obtain purple-black powder Cu-COF-sugar; (4) Adding methanol solution to Cu-COF-sugar and dispersing it evenly by ultrasonication; then adding methanol solution containing sodium methoxide, standing overnight at room temperature in a protective atmosphere, filtering, washing, and drying to obtain brown solid SCu-COF-sugar, which is the sugar-grafted metal-covalent organic framework artificial enzyme.

5. The preparation method according to claim 4, characterized in that: In step (1), the molar ratio of tetraaminophenylporphyrin 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 heating reaction time is 5 days.

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

7. The preparation method according to claim 6, characterized in that: The azide-modified sugar is azide-modified mannose.

8. The preparation method according to claim 4, characterized in that: In step (3), the mass ratio of the COF-sugar to copper acetate is 5:1; the temperature of the heating reflux reaction is 65° C., and the heating reflux reaction time is 5 days.

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

10. Use of the sugar-grafted metal-covalent organic framework artificial enzyme according to any one of claims 1 to 3 in the preparation of antibacterial drugs or drugs for removing biofilms, characterized in that: The sugar-grafted metal-covalent organic framework artificial enzyme has a concentration of 75 μg / mL and captures and removes bacteria within 1 to 2 hours.

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