A copper-based metal organic framework material with mucus penetration function and its application

Abstract

The present invention discloses a copper-based metal-organic framework material with mucus penetration function, a preparation method and an application. The present invention utilizes the characteristics of sp<supgt;2< / supgt>-hybridized carbon in the graphene-like structure of NGCD, and modifies fucoidan on the particle surface through hydrophobic interaction. Fucoidan is used as a surface coating to functionalize Cu-MOF@N by non-covalent bond interaction, and Cu-MOF@NF is prepared. Experimental results show that in the treatment of Helicobacter pylori infection, the NGCD released by Cu-MOF@NF during antibacterial can also regulate the expression of gastric inflammatory factors to achieve the anti-inflammatory function, while not affecting the abundance, function and species diversity of the intestinal microbiota. Therefore, it has good application prospects, not only solving the side effect of intestinal flora imbalance caused by antibiotics, but also blocking the periodic growth pattern, providing an effective method for eradicating Helicobacter pylori.

Description

Technical Field

[0001] The invention belongs to the technical field of water treatment, and in particular relates to a copper-based metal organic framework material with mucus penetration function and application thereof. Background Art

[0002] Helicobacter pylori (H.pylori) is a highly prevalent Gram-negative bacterium that infects approximately 50% of the world's population. Its colonization in the gastric mucosa can induce chronic gastritis, and may even develop into chronic atrophic gastritis, intestinal metaplasia and dysplasia, and eventually gastric cancer. Current studies suggest that approximately 90% of non-cardia cancers are related to H. pylori infection.

[0003] Currently, traditional treatment options for Helicobacter pylori include: triple therapy and bismuth-based quadruple therapy. However, the above therapies all have problems such as the drugs cannot effectively penetrate the gastric mucus layer, remove biofilms, and effectively relieve the inflammatory response caused by infection and gastric mucosal damage mediated by oxidative stress. Moreover, both of the above therapies require the use of antibiotics during the treatment process, and the long-term use of antibiotics can easily destroy the homeostasis of intestinal flora and lead to the occurrence of various intestinal diseases. Therefore, there is an urgent need to develop a new alternative to antibiotics to achieve the following four requirements: I. Effectively penetrate the gastric mucus layer; II. Block The cyclical growth pattern of the intestine prevents persistent infection; III. It removes reactive oxygen species and regulates inflammatory responses; IV. The entire antibacterial process does not affect the intestinal microbial flora. Summary of the invention

[0004] In view of the problems existing in the background technology, the purpose of the present invention is to provide a copper-based metal organic framework material with mucus penetration function and application thereof.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a copper-based metal organic framework material with mucus penetration function, denoted as Cu-MOF@NF, wherein the copper-based metal organic framework material is composed of copper-based metal organic framework-loaded nitrogen-doped carbon dots (Cu-MOF@N) and fucoidan sulfate, and the preparation method thereof comprises the following steps:

[0007] S1. Synthesis of copper-based metal organic framework material loaded with nitrogen-doped carbon dots, denoted as Cu-MOF@N: copper-based metal organic framework material (denoted as Cu-MOF) and nitrogen-doped carbon dots (denoted as NGCD) were added to ethanol, stirred at room temperature, and then ultrasonicated in the dark, stirred overnight, to obtain copper-based metal organic framework material loaded with nitrogen-doped carbon dots (denoted as Cu-MOF@N);

[0008] S2. Synthesis of Cu-MOF@NF: Disperse Cu-MOF@N in ethanol to obtain an ethanol suspension of Cu-MOF@N, add fucoidan to the ethanol, disperse it by ultrasonication in the dark, and then add it to the ethanol suspension of Cu-MOF@N, stir at room temperature in the dark, wash with ethanol, and freeze-dry to obtain Cu-MOF@NF.

[0009] Preferably, in step S1, the mass ratio of Cu-MOF to NGCD is 1:2-4; and the ultrasonic time in the dark is 20-30 min.

[0010] Preferably, in step S2, the mass ratio of Cu-MOF@N to Fucoidan is 1:5; the ultrasonic dispersion time in the dark is 25-40 min, and the light-proof stirring time is 18-24 h.

[0011] Preferably, the nitrogen-doped carbon dots (referred to as NGCD) are synthesized by a one-step hydrothermal method, specifically comprising the following steps: dissolving glucose and urea in deionized water, and then placing the mixture in a polytetrafluoroethylene-lined stainless steel reactor to perform a hydrothermal reaction, then centrifuging the reaction product to remove unreacted substances, filtering the supernatant through a nylon filter to remove impurities, and freeze-drying the filtrate to obtain NGCD;

[0012] Preferably, the weight ratio of glucose to urea is 2:1.

[0013] Preferably, the temperature of the hydrothermal reaction is 180-220° C., and the hydrothermal reaction time is 4-8 h.

[0014] The second aspect of the present invention provides a copper-based metal organic framework material with mucus penetration function obtained by the above preparation method.

[0015] The third aspect of the present invention provides the use of the above copper-based metal organic framework material with mucus penetration function in the preparation of a drug for treating Helicobacter pylori infection.

[0016] The present invention has the following beneficial effects:

[0017] The present invention first combines Cu-MOF and nitrogen-doped carbon dots (referred to as NGCD) to prepare a copper-based metal organic framework material loaded with nitrogen-doped carbon dots (referred to as Cu-MOF@N), and then composites it with fucoidan sulfate to prepare Cu-MOF@NF. 2Taking advantage of the properties of hybrid carbon, fucoidan sulfate (FU) was modified on the particle surface through hydrophobic interaction (van der Waals force). Fucoidan sulfate was used as a surface coating to functionalize Cu-MOF@N through non-covalent bonding, which could improve the water solubility and stability and promote the mucus penetration function of Cu-MOF@NF.

[0018] The experimental results show that the Cu-MOF@NF nanoparticles have good mucus penetration, antibacterial and anti-biofilm properties in vivo and in vitro. In the treatment of Helicobacter pylori infection, the Cu-MOF@NF nanoparticles after oral administration can penetrate the mucus layer and inhibit the adhesion of Helicobacter pylori to gastric epithelial cells; the natural degradation of the Cu-MOF@NF promotes the degradation of the copper-based metal organic framework, thereby releasing Cu 2+ and NGCD. Cu 2+ It can penetrate into the biofilm and degrade the polysaccharides in the biofilm matrix; at the same time, it can effectively kill Helicobacter pylori in the biofilm and planktonic Helicobacter pylori by reducing the ATP level in the bacteria and increasing the permeability of the bacterial membrane, so as to block The cyclical growth pattern of the intestinal microbiota is changed to prevent repeated and persistent infections. Different from the traditional triple therapy, the Cu-MOF@NF prepared by the present invention can release NGCD while being antibacterial and can also regulate the expression of gastric inflammatory factors to achieve anti-inflammatory function. At the same time, Cu-MOF@NF does not affect the abundance, function and species diversity of intestinal microbial flora. Therefore, the Cu-MOF@NF proposed by the present invention has good application prospects. It not only solves the side effects of intestinal flora imbalance caused by antibiotics, but also blocks The cyclical growth pattern provides an effective method for eradication of Helicobacter pylori. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 Transmission electron microscopy images of Cu-MOF@N at low magnification (a) and high magnification (b);

[0021] Figure 2 XRD pattern of NGCD (a) and size distribution of NGCD in Cu-MOF@N (b);

[0022] Figure 3 Element mapping diagram corresponding to Cu-MOF@N;

[0023] Figure 4XRD patterns of Cu-MOF and Cu-MOF@N;

[0024] Figure 5 XPS spectra of Cu-MOF@N (a) and XPS spectra of Cu2p in Cu-MOF@N (b);

[0025] Figure 6 Infrared spectra of Cu-MOF@NF, Cu-MOF@N, Cu-MOF and FU;

[0026] Figure 7 The long-term stability test results of Cu-MOF, Cu-MOF@N, and Cu-MOF@NF in simulated gastric fluid;

[0027] Figure 8 The results of the in vitro Cu-MOF@NF mucus penetration and H.pylori adhesion inhibition function are shown in the figure: a) Transwell schematic diagram of Cu-MOF@NF penetrating the gastric mucus layer. The fluorescence quantification results of the nanoparticles of each group in the lower chamber of the Transwell in the simulated gastric mucus; c) Copper ion quantification diagram after the material penetrates the agar block. d) Confocal image of mucus penetration of Cu-MOF@NF in mouse stomach; e) Fluorescence confocal image of H.pylori binding to HFE145 cells after each group of nanoparticles were first incubated with HFE145 cells and then treated with H.pylori; f) Quantitative analysis of the average fluorescence intensity of Hp in e); Data are mean ± standard deviation (n≥3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0028] Fig. 9 : Fig. 9 (a) Fluorescence images of each group of nanoparticles first incubated with Ges-1 cells and then treated with Hp, showing the binding of Hp to Ges-1 cells; Fig. 9 (b) is the quantitative analysis of the mean fluorescence intensity of Hp in (a); the data are mean ± SD (n ≥ 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001);

[0029] Fig.10 : Fig.10 (a) The H. pylori colony plate images after different concentrations of Cu-MOF@NF were treated in ATCC43504 and PMSS1; Fig.10 (b) for Fig.10 (a) Quantification of antibacterial activity; data are mean ± SD (n ≥ 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001);

[0030] Fig.11 The results of the in vitro study on the antibacterial activity and antibacterial mechanism of nanoparticles are as follows: Fig.11 (a) is a picture of H. pylori colony plates after being treated with different samples under different pH conditions; Fig.11 (b) for Fig.11 (a) Quantification of antibacterial activity; Fig.11 (c) Live (green): dead (red) fluorescence images of H. pylori exposed to each group of nanoparticles; Fig.11 (d) SEM images of the physiological morphological changes of H. pylori after different treatments; Fig.11 (e) Colony plate image of clinical drug-resistant strains H. pylori 532 and 536 treated with Cu-MOF@NF; Fig.11 (f) Fig.11 (e) Quantification of antibacterial activity. g) ATP metabolism level of H. pylori after treatment with nanoparticles in each group; Fig.11 (h) Changes in H. pylori bacterial membrane permeability measured by β-galactosidase; data are mean ± SD (n ≥ 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001);

[0031] Fig.12 Biofilm Removal for Nanoparticles in Vitro: Fig.12 (a) CLSM images of H. pylori ATCC43504 biofilm treated with nanoparticles of various groups; Fig.12 (b) is the fluorescence analysis of SYTO9 and PI in Figure a); Fig.12 (c) Thickness of mature H. pylori biofilm in 3D confocal image stained with SYTO9; Fig.12 (d) The polysaccharide concentration in EPS of H. pylori mature biofilm was determined by phenol-sulfuric acid method after different treatments; Fig.12 (e) The inhibition rate of crystal violet-stained mature biofilm EPS under different antibacterial strategies Fig.12 (f) Pictures of crystal violet-stained mature biofilm EPS under different antibacterial strategies; Fig.12 (g) shows the killing effect of nanoparticles on H. pylori dispersed in the biofilm; Fig.12 (h) is a scanning electron microscopy image of the H. pylori biofilm structure after nanoparticle treatment; the data are mean ± standard deviation (n≥3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0032] Fig.13Results of active oxygen free radical scavenging activity and anti-inflammatory function of Cu-MOF@NF: Fig.13 (a), (b), and (c) are the removal of O by Cu-MOF@NF. 2 · ﹣ , ·OH, and ·NO activity results; 13(d) is the content of ROS in HFE145 cells after treatment with Cu-MOF@NF (180 μg / mL); 13(e) is the DCFH-DA quantitative statistical chart in Figure 13(d); Fig.13 (f), 13(g) 13(i) are RT-PCR detections of mRNA levels of inflammatory cytokines in HFE145 cells induced by LPS, which are IL-1β, IL-6, IL-8 and TNF-α, respectively; data are mean ± SD (n≥3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0033] Fig.14 : Fig.14 (a) The content of ROS in GES-1 cells after treatment with Cu-MOF@NF (180 μg / mL); Fig.14 (b) for Fig.14 (a) Quantitative statistical graph of DCFH-DA; data are mean ± SD (n ≥ 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001);

[0034] Fig.15 :Cytotoxicity evaluation of Cu-MOF@NF on HFE145 and Ges-1 cells for 48 h. Data are mean ± SD (n ≥ 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001);

[0035] Fig.16 For the treatment of H.pylori infection in vivo: Fig.16 (a) Schematic diagram of the modeling and treatment of H. pylori-infected mice; Fig.16 (b) Silver staining of H. pylori colonization in mouse gastric mucosa; Fig.16 (c) quantification of antibacterial activity against H. pylori in vivo; Fig.16 (d) H&E staining of the stomach of mice in different treatment groups; Fig.16 (e) RT-PCR analysis shows the mRNA levels of inflammatory cytokines (IL-1β) in gastric tissues of mice in the indicated groups; Fig.16 (f) RT-PCR analysis shows the mRNA levels of inflammatory cytokines (IL-6) in the gastric tissues of the indicated groups of mice; Fig.16(g) RT-PCR analysis shows the mRNA levels of inflammatory cytokines (TNF-α) in gastric tissues of mice in the indicated groups; data are mean ± SD (n ≥ 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001);

[0036] Fig.17 Plasma creatinine (a), urea (b), aspartate aminotransferase (c), alanine aminotransferase (d) and alkaline phosphatase (e) levels of mice after treatment and changes in body weight of mice during treatment (n=6) (f);

[0037] Fig.18 H&E staining results of the main organs of mice;

[0038] Fig.19 Protection of intestinal flora by Cu-MOF@NF: Fig.19 (a) is the intestinal flora richness CHO1. If the number of difference groups is greater than 5, compact letter displays (CLDs) are used. Two groups that share one or more letters are not "significantly" different. Fig.19 (b) NMDS plot showing the β-diversity of the gut microbiome; Fig.19 (c) is the histogram of the relative abundance of intestinal microbiota at the family level in the OCA treatment group; Fig.19 (d) Heat map of relative abundance of the top 15 OTUs at the genus level; Fig.19 (e) The evolutionary branch diagram with circular radiation from inside to outside represents the classification level from phylum to species. The red nodes in the branches represent the microbial taxa that play a key role in the Cu-MOF@NF group, and the green nodes represent the microbial taxa that play a key role in the OCA treatment group; Fig.19 (f) The green and red areas in the LDA value distribution histogram represent different groups. The red nodes in the branches show the significant microbial taxa in the Cu-MOF@NF group, and the green nodes show the significant microbial taxa in the OCA treatment group. All species with LDA scores greater than 3.6 are only presented, and the species names are represented by letters in the figure;

[0039] Fig. 20 Schematic diagram of the synthesis route of copper-based metal organic framework material, namely Cu-MOF@NF. DETAILED DESCRIPTION

[0040] In the following description, specific details such as specific system structures, technologies, etc. are provided for the purpose of illustration rather than limitation, so as to provide a thorough understanding of the embodiments of the present invention. However, it should be clear to those skilled in the art that the present invention may be implemented in other embodiments without these specific details. In other cases, detailed descriptions of well-known systems, devices, circuits, and methods are omitted to prevent unnecessary details from obstructing the description of the present invention.

[0041] Example 1

[0042] The embodiment of the present invention provides a copper-based metal organic framework material with mucus penetration function, denoted as Cu-MOF@NF, which is self-assembled by a copper-based metal organic framework loaded with nitrogen-doped carbon dots (denoted as Cu-MOF@N) and fucoidan sulfate. The synthesis route is shown in Fig. 20 , and its preparation method comprises the following steps:

[0043] A1. Synthesis of Cu-MOF: 0.1 mmol of cupric chloride dihydrate and 0.1 g of polyvinyl pyrrolidone were dissolved in 40 mL of water, and then 2.5 mL of sodium hydroxide (0.2 M) was slowly added dropwise; the solution was then magnetically stirred for 5 min, and then 2.5 mL of ascorbic acid (0.1 M) was slowly added dropwise, and stirred for another 5 minutes after addition. After washing with ethanol three times, the yellow liquid was redispersed in 10 mL of ethanol for later use; 0.5 mmol of terephthalic acid (H 2 BDC) was completely dissolved in 5 mL of ethanol and 5 mL of DMF, 10 mL of the yellow liquid (ethanol) prepared above was added to the solution, and the reaction was carried out for 5 h under full contact with oxygen at room temperature, and the yellow liquid gradually turned into a blue liquid; finally, the blue product was collected by centrifugation, and then washed 3 times with methanol, and then washed 3 times with ethanol, and freeze-dried to obtain Cu-MOF;

[0044] A2. One-step hydrothermal method to synthesize nitrogen-doped carbon dots, denoted as NGCD: 1 g of glucose and 0.5 g of urea were added to 60 mL of deionized water, stirred to dissolve, and then placed in a polytetrafluoroethylene-lined stainless steel reactor (100 mL) to react at 200 °C for 6 h. After cooling, the reaction product was centrifuged at 10,000 rpm for 30 min to remove unreacted substances, and then the supernatant was filtered through a nylon filter (pore size: 0.22 μM) to remove impurities, and then the filtrate was freeze-dried to obtain NGCD black powder, which was stored in a refrigerator;

[0045] A3. Synthesis of copper-based metal organic framework materials loaded with nitrogen-doped carbon dots, denoted as Cu-MOF@N: Cu-MOF (5 Mg) was dissolved in an ethanol solution (10 mL), and then 10 mL of an ethanol solution containing NGCD (10 Mg) was added, magnetically stirred at room temperature for 1 h, ultrasonicated in the dark for 20 min using an ultrasonic cleaner (40 kHz), and then stirred overnight, then collected by centrifugation, eluted with ethanol 3 times, and finally freeze-dried to obtain Cu-MOF@N;

[0046] A4. Synthesis of Cu-MOF@NF: The synthesized Cu-MOF@N (3 mg) was dispersed in 10 ml of ethanol. Fucoidan (fucoidan sulfate) was then dissolved in 1 ml of ethanol and ultrasonicated for 30 min in the dark using an ultrasonic cleaner (40 kHz). The mixture was added to the ethanol suspension of Cu-MOF@N and magnetically stirred for 24 h at room temperature in the dark. The mixture was then washed with ethanol for three times and freeze-dried to obtain Cu-MOF@NF.

[0047] The Cu-MOF@N, NGCD and Cu-MOF@NF prepared in Example 1 were characterized. Figure 1-6 .

[0048] like Figure 1 As shown, the SEM image of Cu-MOF (a) and the XRD image of NGCD (b) are shown. Figure 1 (a) The results show that the size of Cu-MOF is 300-500nm thin nanosheets. Figure 1 (b) The results show that the broad diffraction peak near 2θ=23.12° coincides with the characteristic peak of NGCD, indicating that NGCD has been successfully synthesized.

[0049] like Figure 2 The following are the low-magnification (a) and high-magnification (b) transmission electron microscopy images of Cu-MOF@N and the size and distribution of NGCD in Cu-MOF@N (c). Figure 2 The results show that the present invention utilizes the unsaturated surface coordination Cu(II) ions in Cu-MOF to form coordination bonds with the carbonyl group of the enone nitrogen group and the oxygen or nitrogen of the amino group in the nitrogen group in NGCD, thereby chemically adsorbing NGCD, and after NGCD is adsorbed on the Cu-MOF nanosheets, Cu-MOF@N is formed. In Cu-MOF@N, the Cu-MOF nanosheets can still maintain their morphology, and the average size of NGCD is 2.45nm, which is evenly distributed on the outer surface of the Cu-MOF nanosheets.

[0050] like Figure 3 As shown, this is the element mapping diagram corresponding to Cu-MOF@N. Figure 3The results can intuitively present the distribution of Cu, C, N and O elements in Cu-MOF@N.

[0051] like Figure 4 As shown, the XRD patterns of Cu-MOF and Cu-MOF@N are Figure 4 The results show that the crystal peaks of Cu-MOF@N are consistent with the (110), (20-1) and (40-2) main crystal planes of Cu-MOF alone and the characteristic peaks of the (002) crystal plane of NGCD.

[0052] like Figure 5 As shown in the figure, the XPS graph of C1s, N1s, O1s in Cu-MOF@N (a) and the XPS graph of Cu2p in Cu-MOF@N (b). Figure 5 (a) The results show that the C1s spectrum shows two characteristic peaks, corresponding to the sp at 288.5 and 284.8 eV, respectively. 2 The characteristic peaks of hybrid carbon (NC=N) and graphene carbon (C=C) at 398.05 eV match the N1s binding energy of nitrogen bonds (CN and NH) in the amide or amino groups of NGCD, and also indicate that NGCD has been successfully adsorbed on the Cu-MOF nanosheets. Figure 5 (b) The results show that by analyzing the valence state of the Cu element in Cu-MOF@N, it can be seen that the main species distributed in MOF are divalent copper (II) species.

[0053] In the present invention, the graphene-like structure sp of NGCD is used 2 The properties of hybrid carbon are used to modify the surface of the particles with fucoidan sulfate (hereinafter, fucoidan sulfate is abbreviated as FU) through hydrophobic interaction (van der Waals force). Fucoidan sulfate is used as a surface coating to functionalize Cu-MOF@N through non-covalent bonding to improve water solubility and stability, and promote the mucus penetration function of Cu-MOF@NF.

[0054] like Figure 6 As shown, the Fourier transform infrared spectra of FU, Cu-MOF, Cu-MOF@N and Cu-MOF@NF. Figure 6 The results show that FU and Cu-MOF@NF have a strong affinity at 968 cm -1 Both sides show the sugar ring stretching vibration peak, which corresponds to the vibration of the COC bond in the α-glucan chain of alginate polysaccharide, indicating that FU is successfully encapsulated. In summary, the present invention has successfully synthesized Cu-MOF@NF.

[0055] 2. Performance evaluation

[0056] (1) Study on the long-term stability of Cu-MOF@NF in simulated gastric fluid

[0057] Considering the special environment in the stomach, the present invention placed three nanomaterials, Cu-MOF, Cu-MOF@N and Cu-MOF@NF, in simulated gastric fluid (SGF) to investigate their stability. Figure 7 shown.

[0058] Depend on Figure 7 The results show that Cu-MOF and Cu-MOF@N are unstable in the simulated stomach and have deposition, while Cu-MOF@NF is almost unchanged in the simulated gastric fluid and the solution is uniform. It is speculated that this is because Cu-MOF and Cu-MOF@N lack a protective layer, and the organic framework of pure Cu-MOF degrades under acidic conditions, resulting in a certain degree of agglomeration. In the present invention, Cu-MOF@NF utilizes the graphene-like structure sp of NGCD. 2 The properties of hybrid carbon can modify FU on the particle surface through hydrophobic interaction (van der Waals force), where FU can act as a surface coating to functionalize Cu-MOF@N through non-covalent bonding, thereby improving the water solubility and long-term stability of Cu-MOF@NF.

[0059] Nanomaterials can only work if they penetrate the gastric mucus layer and reach the site where Helicobacter pylori is implanted (see Figure 8 ). In general, negatively or neutrally charged particles have little interaction with mucus, and therefore can easily penetrate the mucus layer. The present invention designs FU as a surface coating, which not only improves the long-term stability of Cu-MOF@NF, but also greatly increases the permeability of Cu-MOF@NF and inhibits Hp adhesion. This is mainly because FU can wrap Cu-MOF@N through hydrophobic interaction (van der Waals force), and its Zeta potential value is -1.8±0.52mV, which is a weak negative charge, which can reduce the interaction with the mucus layer, making it easier to penetrate the mucus layer.

[0060] (2) Evaluation of mucus permeability and adhesion inhibition function of Cu-MOF@NF

[0061] In order to study the permeability of Cu-MOF@NF to mucus, the present invention first verified the ability of FITC fluorescently labeled NGCD, FU, Cu-MOF, Cu-MOF@N, and Cu-MOF@NF to penetrate simulated gastric mucus by using a Transwell device, and quantified it by measuring the intensity of fluorescence in the lower chamber. The results are shown in Figure 2. Figure 8 shown.

[0062] like Figure 8 As shown in (b), stronger fluorescence was detected in the lower chamber in the FU and Cu-MOF@NF groups compared with the other groups, and their quantitative results showed obvious statistical differences.

[0063] Then, the amount of copper ions in the bottom layer of agarose gel was analyzed by ICP-AES to quantify the permeability of mucus. It can be observed that the Cu-MOF@NF group has the most copper ions (see Figure 8 (b)), which indicates that FU-coated cells have better mucus permeability than bare cells.

[0064] In order to observe the permeation effect of Cu-MOF@NF in the stomach of mice, FITC-labeled Cu-MOF@NF was gavaged 2 hours later, and the stomach of mice was taken for immunofluorescence. The gastric mucus layer was co-localized with MUC5AC mucus glycoprotein-specific antibody, and then observed with confocal microscopy. It can be seen that there are a lot of FITC-labeled Cu-MOF@NF nanomaterials in the lower layer of the gastric mucus layer MUC5AC antibody expression in the Cu-MOF@NF group compared with the simple FITC gavage group (see Figure 8 (c)), which indicates that the nanomaterial Cu-MOF@NF has a good ability to penetrate the mouse gastric mucus, which provides good feasibility for in vivo treatment in mice.

[0065] In order to verify the inhibitory effect of nanomaterial Cu-MOF@NF on Helicobacter pylori adhesion, the present invention used Helicobacter pylori antibody for localization through immunofluorescence experiment, and studied the adhesion of Helicobacter pylori on gastric epithelial cells HEF-145 and GES-1 after being treated with Cu-MOF@NF nanomaterial. The results are shown in Figure 8 and Fig. 9 .

[0066] Depend on Figure 8 (e) and Fig. 9 (a) It can be seen that the cells in the blank group grew well and no bacteria adhered, while the Helicobacter pylori (red) in the control group gathered around the cells (blue), indicating that the bacteria were widely adhered to the cell surface. It is worth noting that there was almost no Hp adhesion in the FU group and the Cu-MOF@NF group. Figure 8 (f) and Fig. 9 (b) Further quantification of fluorescence intensity shows that the adhesion of H. pylori is significantly reduced when treated with FU and Cu-MOF@NF, that is, as a sulfated polysaccharide containing fucosyl, FU can reduce the interaction between H. pylori and epithelial cells, thereby inhibiting the adhesion of H. pylori to cells. The possible mechanism is the competitive binding of FU with H. pylori BABA.

[0067] (3) Antibacterial activity of Cu-MOF@NF and its mechanism of action

[0068] The present invention first evaluated the in vitro antibacterial properties of Cu-MOF@NF. Fig.10 As shown. Fig.10 (a) and Fig.10(b) The results show that the antibacterial effect of Cu-MOF@NF on Helicobacter pylori strain ATCC43504 and virulence protein CagA-positive strain PMSS1 is concentration-dependent, and the optimal antibacterial concentration is 180 μg / mL.

[0069] Generally, the pH value of gastric acid is between 1 and 2. Therefore, the present invention studies the antibacterial effects of each group of materials (NGCD, FU, Cu-MOF, Cu-MOF@N, Cu-MOF@NF) under different pH conditions. Fig.11 As shown. Fig.11 (a) and Fig.11 (b) The results show that the Cu-MOF group has antibacterial effects at pH 2 and pH 7, while the FU and NGCD groups have almost no antibacterial effects, indicating that the antibacterial effect of the material is mainly attributed to Cu-MOF, but the antibacterial effect of the bare Cu-MOF group is slightly higher than that of the Cu-MOF@N and Cu-MOF@NF groups. This may be attributed to the release of more copper ions in the bare Cu-MOF group at the same concentration.

[0070] Depend on Fig.11 (c) and Fig.11 (d) The results show that the antibacterial activity of the material was further evaluated by live / dead bacterial staining, and fluorescence microscopy also showed a large amount of red fluorescence in the bacteria treated with the Cu-MOF group, which also showed that the Cu-MOF group had good antibacterial activity against Helicobacter pylori. Fig.11 (e) shows that compared with the control group, the biofilm of Helicobacter pylori was significantly sunken and ruptured after treatment with Cu-MOF.

[0071] Based on the good antibacterial properties of Cu-MOF@NF, the present invention also evaluated the antibacterial effect of Cu-MOF@NF on multidrug-resistant Helicobacter pylori strains 532 and 536. Fig.11 shown.

[0072] Depend on Fig.11 (f) and Fig.11 (g) The results show that Cu-MOF@NF also has antibacterial effects on multidrug-resistant Helicobacter pylori strains 532 and 536, judging from the agar plate colony map and colony counting results.

[0073] Then, the antibacterial mechanism of Cu-MOF@NF was studied, and the results were as follows Fig.11 Studies have shown that changes in ATP levels will affect cell function. Usually, when cells are in apoptosis, necrosis, or some toxic state, ATP levels will decrease. Fig.11 (g) and Fig.11(h) The results show that compared with other groups, the Cu-MOF group can significantly reduce the ATP level. The corresponding bacterial cell membrane permeability experimental results are consistent with the above results. Compared with other groups, the Cu-MOF group can significantly increase the permeability of bacterial membranes. In summary, Cu-MOF@NF can achieve anti-Helicobacter pylori effects by reducing ATP levels and increasing bacterial membrane permeability.

[0074] (4) Anti-biofilm effect of Cu-MOF@NF on Helicobacter pylori

[0075] Biofilms can serve as shelters for internal bacteria, preventing effective penetration of antimicrobial agents. Therefore, if the nanoparticles prepared by the present invention can effectively penetrate into the biofilm, their killing efficiency within the biofilm will be greatly improved. Based on the good antibacterial effect of Cu-MOF@NF on planktonic Helicobacter pylori, the present invention evaluated the destructive effect of Cu-MOF@NF on Helicobacter pylori biofilm. Specifically, the present invention used a laser confocal scanning microscope to evaluate the effect of the material on bacterial biofilms, and used Syto 9 and PI to observe and quantify the bacteria in the biofilm. The results are shown in Figure 2. Fig.12 .

[0076] Depend on Fig.12 (a) The results show that the biofilms in the control group, FU and NGCD groups only show live Helicobacter pylori, showing uniform green fluorescence. However, after the biofilms were treated with the Cu-MOF group, the number of dead bacteria increased significantly. Fig.12 In (b) and 12(c), the quantitative results of the percentage of viable bacteria and biofilm thickness showed that the Cu-MOF-containing group reduced the bacteria in the biofilm by about 90% and significantly reduced the biofilm thickness.

[0077] Biofilm is a three-dimensional multicellular community of bacteria embedded in their own secreted extracellular polymers (EPS), which are mainly composed of polysaccharides and proteins. Therefore, the present invention also studies the degradation of polysaccharides in the biofilm after the biofilm is incubated with the material. The remaining polysaccharides in the biofilm were quantified using the phenol-sulfuric acid method. The results are as follows: Fig.12 (d) Fig.12 (d) The results showed that the amount of polysaccharides in the Cu-MOF group after treatment was significantly less than that in the control group, FU and NGCD groups, so the significant reduction in biofilm thickness may be attributed to the degradation of polysaccharides in the biofilm.

[0078] In addition, Helicobacter pylori That is, after Cu-MOF@NF destroys the biofilm to release a large number of plankton, if the dispersed H. pylori are not killed in time, they will spread to other places, causing and aggravating a wider infection. Therefore, it is crucial to evaluate the survival rate of H. pylori dispersed in the biofilm. Fig.12 (e) The results showed that after the nanoparticles were incubated with the Helicobacter pylori biofilm for 24 h, the supernatant was collected. From the colony counting results on the agar plate, it can be seen that almost all the dispersed H. pylori were killed after the biofilm was treated with the Cu-MOF group, which significantly reduced the risk of persistent infection.

[0079] When a small amount of residual H. pylori is present, there is still a risk of biofilm regeneration. Therefore, the ability to inhibit the reestablishment of H. pylori biofilm was also evaluated, and the biofilm inhibition rate was determined by crystal violet staining. Fig.12 (f) The results showed that the Cu-MOF group had a significant inhibitory effect on the formation of biofilm.

[0080] Then the OD value is quantitatively determined and quantitative analysis is performed. Fig.12 (g) It can be seen that the inhibition rate of biofilm in the Cu-MOF@NF group is significantly higher than that in the bare Cu-MOF and Cu-MOF@N groups, with an inhibition rate of about 77%. This may be attributed to the bactericidal effect of copper ions, which significantly reduced the amount of bacteria. At the same time, FU in the Cu-MOF@NF group can competitively inhibit the adhesion of H. pylori and reduce the interaction between bacteria, which has the potential to prevent the regeneration of biofilm.

[0081] Subsequently, the structure of the Helicobacter pylori biofilm was directly observed by scanning electron microscopy. Fig.12 (h) It can be seen that the biofilm structure of the control group is dense and the bacterial biofilm structure is intact. The biofilm of the Cu-MOF group is significantly reduced and the Helicobacter pylori is obviously spherical, and the biofilm is concave. The above findings indicate that Cu-MOF@NF may be a potential candidate for antibacterial biofilm.

[0082] (5) Cu-MOF@NF reduces Helicobacter pylori-induced oxidative stress and has anti-inflammatory function

[0083] The overexpression of inflammatory factors and the oxidative stress mediated by excessive accumulation of reactive oxygen species caused by H. pylori infection have been shown to be closely related to the occurrence of gastric diseases such as chronic gastritis, peptic ulcers and even gastric cancer. The increase of reactive oxygen species (ROS) and reactive nitrogen species (RNS) will cause an imbalance between oxidation and antioxidant activity in the body. Here, the present invention studies the removal of RONS by Cu-MOF@NF by monitoring single radical reactions. The results are shown in Fig.13 and Fig.14 .

[0084] Fig.13 Electron spin resonance (ESR) was used to show that Cu-MOF@NF could remove O 2 The scavenging ability of Cu-MOF@NF for RONS is attributed to the fact that the surface of nitrogen-doped carbon dots loaded in Cu-MOF@NF has many functional groups, such as hydroxyl, carbonyl, hydroxylamine, etc., which can directly react with free radicals and transform into stable compounds, thereby achieving the effect of scavenging reactive oxygen and reactive nitrogen.

[0085] Then, see Fig.13 (d), 13(e) and Fig.14 Fluorescence images of ROS UP-treated cells with and without Cu-MOF@NF were displayed using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe on gastric epithelial cells HEF-145 and GES-1. ROS UP-treated cells showed stronger fluorescence than healthy cells, which meant excessive ROS production. After treatment with Cu-MOF@NF, the fluorescence signal was significantly weakened due to the effective removal of ROS, approaching the level of blank cells, confirming the role of Cu-MOF@NF in alleviating oxidative stress.

[0086] Studies have shown that nitrogen-doped carbon dots can reduce inflammatory responses by inhibiting the activation of the NF-κB signaling pathway. The present invention also uses RT-qPCR to detect the mRNA expression levels of inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α in HFE145 cells induced by LPS. Fig.13 (f) and Fig.13 (g) It can be seen that the mRNA levels of inflammatory cytokines IL-1β, IL-6, IL-8 and TNF-α after Cu-MOF@NF treatment were significantly lower than those in the LPS group and were close to the blank cell levels. Therefore, Cu-MOF@NF has a significant effect in clearing RONS and inhibiting inflammatory factors to relieve inflammation.

[0087] Since good biocompatibility is the prerequisite for ensuring the application of biomaterials in vivo, the present invention also preliminarily studied the cytotoxicity of Cu-MOF@NF using gastric mucosal epithelial cells (HFE145 and GES-1). Fig.15 It was shown that after culturing gastric mucosal epithelial cells (HFE145 and GES-1) for 2 days, the cell survival rate of Cu-MOF@NF was still above 80%, indicating that it has good biosafety.

[0088] (f) Cu-MOF@NF treats Helicobacter pylori infection in mice

[0089] The present invention evaluates the efficacy of Cu-MOF@NF in treating Helicobacter pylori infection in vivo. Specifically, C57BL / 6 mice were randomly divided into a healthy mouse group, a PBS group, a Cu-MOF group, a Cu-MOF@N group, a Cu-MOF@NF group, and a triple therapy group. The results are as follows Fig.16 shown.

[0090] Fig.16 (a) shows that the healthy mice were gavaged with Brucella broth, and the other five groups were gavaged with 1×10 8 cfu mL -1 The solution was administered intragastrically once every other day for 8 consecutive times. Fig.16 (b) and Fig.17 It was shown that 4 weeks after infection, silver staining, plate smear and Gram staining results showed that Helicobacter pylori successfully colonized the mouse stomach.

[0091] Next, the corresponding oral administration was performed, and the mice were killed on the second day after drug withdrawal for subsequent verification. Fig.16 (c) It can be seen that Cu-MOF, Cu-MOF@N and Cu-MOF@NF all reduced the colonization of Helicobacter pylori to a certain extent, among which Cu-MOF@NF had the best antibacterial effect and was second only to triple therapy.

[0092] from Fig.16 (b) It can be seen that H&E staining of gastric tissue showed a large number of inflammatory cell infiltrations and glandular thickening in the submucosal layer of the PBS group, Cu-MOF group, Cu-MOF@N group, and triple therapy group, while no obvious inflammatory lesions were observed in the Cu-MOF@N group.

[0093] In addition, the present invention uses RT-qPCR to detect the expression of inflammatory cytokines in mouse gastric tissue. Fig.16 (e), 16(h), 16(g) show that Cu-MOF@NF treatment effectively reduced the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, which is attributed to the antioxidant and anti-inflammatory ability of the Cu-MOF@NF group to penetrate into the submucosal layer and release free NGCD. However, due to the poor mucus permeability of the Cu-MOF@N group, the released NGCD could not penetrate into the submucosal layer, so its antioxidant and anti-inflammatory effects were not observed. The above results show that Cu-MOF@NF can regulate the inflammatory response of the gastric mucosa.

[0094] In order to study the biosafety of Cu-MOF@NF, the body weight and blood biochemical indexes of mice were observed. Blood biochemical indexes were tested 7 days after treatment, including creatinine (Crea) and urea (Urea) reflecting kidney function, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) reflecting liver function. Fig.17 (a)-(e) The results show that blood biochemical indicators confirm that Cu-MOF@NF has no adverse effects on the liver and kidney functions of mice. Fig.17 (f) The results showed that there was no difference in body weight among the mice groups before and after treatment.

[0095] In addition, the in vivo tissue toxicity of Cu-MOF@NF was evaluated by histological analysis of major organs (heart, liver, spleen, lung, and kidney). Fig.18 The H&E staining results showed that compared with normal mice, the main organs of the Cu-MOF@NF group mice showed no obvious histological abnormalities or inflammatory lesions. This indicates that Cu-MOF@NF has no obvious toxic side effects on mice after a short-term 7-day treatment. In summary, Cu-MOF@NF has good biocompatibility and can be used as a safe drug for the treatment of Helicobacter pylori infection.

[0096] (g) Protection of intestinal flora and biosafety research

[0097] Traditional antibiotic treatment often affects the dynamic balance of intestinal flora and leads to various intestinal diseases. Therefore, the feces of mice in each group were collected after treatment, and 16S rRNA sequencing was used to evaluate the abundance, diversity and colony structure of intestinal microorganisms. Fig.19 shown.

[0098] Depend on Fig.19 (a) It can be seen that the α diversity (Chao1) of the Cu-MOF@NF group was not significantly different from that of the healthy group of mice, but there was a significant difference in the OCA treatment group.

[0099] Principal coordinates analysis was performed to analyze the beta diversity of the intestinal microbiota. Fig.19 The non-metric multidimensional scaling (NMDS) plot of (b) shows that the OCA-treated group and healthy mice are clearly separated. However, the intestinal microbiota of Cu-MOF@NF-treated mice is more similar to that of healthy mice than that of healthy mice. The above results indicate that Cu-MOF@NF has little effect on the overall microbial abundance of the intestinal flora.

[0100] The present invention also conducts an in-depth analysis of the intestinal microbial flora. Fig.19(c) The results show that the relative abundance of pathogenic bacteria at the family level in the OCA treatment group increased significantly, such as Enterobacteriaceae (harmful to intestinal homeostasis). At the same time, the relative abundance of protective bacteria, such as Muribaculaceae and Lachnospiraceae (which relieve intestinal inflammation, protect intestinal energy metabolism, maintain intestinal mucosal integrity and stabilize immune status by producing short-chain fatty acids) decreased significantly, but there was no significant difference in relative abundance between the Cu-MOF@NF group and the healthy group of mice. Fig.19 (d) The results showed that compared with the OCA-treated mice, the Cu-MOF@NF group treatment significantly reduced the relative abundance of Escherichia_Shigella, Enterobacter, and Klebsiella (which are known for producing extracellular toxins and triggering intestinal inflammation).

[0101] Linear discriminant analysis (LDA) effect size (LFSE) analysis was used to determine the specialized microbial communities between the Cu-MOF@NF group and the OCA-treated group. Fig.19 (e)-(f) The results show that there are significant differences in the intestinal microbial flora between the two groups. The dominant microbial flora in the OCA-treated mice include harmful Bacteroides, Enterobacteriaceae, and Proteus, while in the Cu-MOF@NF group, a variety of probiotics, such as Muribaculaceae and Lachnospiraceae, play a major role. In other words, after Cu-MOF@NF treatment, harmful bacteria are inhibited, while beneficial bacteria obtain favorable conditions for survival and proliferation, thus playing a vital role in protecting the balance of intestinal microecology.

[0102] In summary, the Cu-MOF@NF (nanoparticles) prepared by the present invention have good mucus penetration, antibacterial and anti-biofilm properties in vivo and in vitro. In the treatment of Helicobacter pylori infection, Cu-MOF@NF nanoparticles after oral administration can penetrate the mucus layer and inhibit the adhesion of Helicobacter pylori to gastric epithelial cells. The natural degradation inside the Cu-MOF@NF promotes the degradation of the copper-based metal organic framework, thereby releasing Cu2+ and NGCD. Cu 2+ It can penetrate into the biofilm and degrade the polysaccharides in the biofilm matrix. At the same time, it can effectively kill Helicobacter pylori in the biofilm and floating Helicobacter pylori by reducing the ATP level in the bacteria and increasing the permeability of the bacterial membrane. The cyclical growth pattern of the gastric mucosa can prevent repeated persistent infections. Importantly, unlike traditional triple therapy, the NGCD released by Cu-MOF@NF can regulate the expression of gastric inflammatory factors while being antibacterial, achieving anti-inflammatory function. At the same time, Cu-MOF@NF does not affect the abundance, function and species diversity of intestinal microbial flora. Therefore, the Cu-MOF@NF nanoparticles we proposed have good application prospects. They not only solve the side effects of intestinal flora imbalance caused by antibiotics, but also block The cyclical growth pattern provides an effective method for eradication of Helicobacter pylori.

[0103] The present invention is not limited to the above-mentioned specific implementation modes. Various changes made by ordinary technicians in this field based on the above-mentioned concepts without creative work are all within the protection scope of the present invention.

Claims

1. A method for preparing a copper-based metal organic framework material with mucus penetration function, It is characterized in that The following steps are involved: S1. Synthesis of Cu-MOF@N: Cu-MOF and nitrogen-doped carbon dots were added to ethanol, stirred at room temperature, and then sonicated in the dark and stirred overnight to obtain Cu-MOF@N. S2. Synthesis of Cu-MOF@NF: Disperse Cu-MOF@N in ethanol to obtain an ethanol suspension of Cu-MOF@N, add fucoidan sulfate to ethanol, disperse it by ultrasonication in the dark, then add it to the ethanol suspension of Cu-MOF@N, stir in the dark at room temperature, and self-assemble to obtain Cu-MOF@NF.

2. A method for preparing a copper-based metal organic framework material with mucus penetration function according to claim 1, It is characterized in that In step S1, the mass ratio of the Cu-MOF to the nitrogen-doped carbon dots is 1:2-4; and the time of ultrasonication in the dark is 20-30 min.

3. The method for preparing a copper-based metal organic framework material with mucus penetration function according to claim 1, It is characterized in that In step S2, the mass ratio of Cu-MOF@N to fucoidan sulfate is 1:5; the ultrasonic dispersion time in the dark is 25-40 minutes, and the light-proof stirring time is 18-24 hours.

4. The method for preparing a copper-based metal organic framework material with mucus penetration function according to claim 1, It is characterized in that The nitrogen-doped carbon dots are synthesized by a one-step hydrothermal method, and the specific steps are: glucose and urea are dissolved in deionized water, and then placed in a stainless steel reactor lined with polytetrafluoroethylene for hydrothermal reaction, and then the reaction product is centrifuged to remove unreacted substances, and the supernatant is filtered through a nylon filter to remove impurities, and the filtrate is freeze-dried to obtain nitrogen-doped carbon dots.

5. The method for preparing a copper-based metal organic framework material having mucus penetration function according to claim 4, It is characterized in that The temperature of the hydrothermal reaction is 180-220°C, and the hydrothermal reaction time is 4-8h; The weight ratio of glucose to urea is 2:

1.

6. A copper-based metal-organic framework material with mucus penetration function, It is characterized in that The compound is prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the copper-based metal organic framework material as claimed in claim 6 in the preparation of a drug for treating Helicobacter pylori infection.

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