An antibacterial MOF drug, its preparation method and application

Abstract

The present invention provides an antibacterial MOF drug, its preparation method and application. It dissolves fumarate and gallium salt in water respectively, then slowly drops the gallium salt solution into the fumarate solution, reacts at room temperature for 0.5 - 1.5 h, then centrifuges and separates. The collected product is washed and dried to obtain the antibacterial MOF drug. The drug of the present invention uses gallium ions as the central atom and fumaric acid as the ligand, can specifically respond to bacteria, is non-toxic and harmless to cells and mice, and has very good bactericidal effects in vitro and in vivo.

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and in particular to an antibacterial MOF drug and a preparation method and application thereof. Background Art

[0002] Pseudomonas aeruginosa (P. aeruginosa) is a common Gram-negative conditional pathogen. It is highly pathogenic to people with low immunity (such as cancer patients, AIDS patients, burn patients, etc.), patients with cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), etc. It can cause severe acute or chronic infections and is one of the three major pathogens of acquired hospital infections. At present, the main clinical treatment for P. aeruginosa infection is the use of antibacterial drugs for treatment. However, due to the widespread use and even abuse of antibiotics in clinical practice, P. aeruginosa has a variety of drug resistance mechanisms, including the production of antimicrobial enzymes (such as β-lactamase, aminoglycoside deactivating enzyme, etc.), mutations in antibiotic target sites, reduced outer membrane permeability, active efflux pump system, and the formation of biofilms. These rich resistance mechanisms enable Pseudomonas aeruginosa to tolerate a variety of antibiotics, which brings great difficulties to clinical anti-infection treatment. Therefore, finding possible drug targets and developing new antibacterial drugs has become a major problem that needs to be solved urgently.

[0003] Gallium is a diagnostic and chemotherapeutic drug with a long history. Its compounds were first used in clinical practice in the 1860s. Research has found that radioactive gallium citrate ( 67 Gallium (Ga) will concentrate in the area where the tumor grows rapidly and can be used as a tumor locator and tumor-affinity scanning agent. In recent years, research on the antibacterial properties of gallium preparations has continued to expand, and gallium nitrate, gallium protoporphyrin, gallium maltol, and gallium deferoxamine have all been found to have good antibacterial activity.

[0004] Unlike most antibiotics that have a single target in bacteria, gallium is a multi-target drug that interferes with iron metabolism and impairs a variety of iron-dependent functions. Iron is essential for bacteria, and resistance mechanisms such as target mutations, drug modifications, or alternative metabolic pathways are unlikely to enable bacteria to overcome the inhibitory effects of gallium. On the contrary, reducing permeability and active efflux through mutations in membrane transport proteins may theoretically become gallium resistance mechanisms. However, the similarity between gallium and iron means that resistant bacteria need to pay a huge adaptive cost. Therefore, reducing gallium intake will also reduce iron intake. Therefore, it is difficult for bacteria to develop resistance to gallium. Gallium is a very promising antibacterial drug.

[0005] Metal-Organic Framework (MOF) is a material with a three-dimensional porous structure composed of metal ions as nodes and organic ligands as supports. In recent years, MOF materials have received attention in the field of antibacterial applications and have become a new favorite in the research of antibacterial agents. This is mainly due to their good biodegradability, outstanding specificity, as well as high loading and sustained release capabilities. In vivo, MOF materials can be released under specific conditions, achieving targeted delivery effects. Selecting appropriate ligands to synthesize MOF materials with gallium can not only utilize the non-toxic and antibacterial properties of gallium but also achieve the function of targeted release. Therefore, how to provide an antibacterial drug centered on gallium with good effects has become the research direction for us. Summary of the Invention

[0006] The purpose of the present invention is to provide an antibacterial MOF drug, its preparation method, and applications, in order to solve the problems in the prior art such as the generation of various drug resistance mechanisms in bacteria due to the inappropriate use of antibiotics, and the unsatisfactory antibacterial effects of existing antibacterial drugs.

[0007] The technical solution of the present invention is: an antibacterial MOF drug, which is prepared by the following method:

[0008] Dissolve fumarate and gallium salt in water respectively, then slowly drip the gallium salt solution into the fumarate solution, react at room temperature for 0.5 - 1.5 h, then perform centrifugal separation. The collected product is washed and dried to obtain the antibacterial MOF drug.

[0009] The molar ratio of the fumarate to the gallium salt used is 1:1 - 1.2.

[0010] The fumarate is sodium fumarate, and the gallium salt is gallium nitrate.

[0011] The centrifugation conditions are: centrifuging at a speed of 7000 - 9000 r / min for 5 - 15 min.

[0012] The drying conditions are: drying at 55 - 65 °C for 10 - 14 h.

[0013] A preparation method of an antibacterial MOF drug, which is to dissolve fumarate and gallium salt in water respectively, then slowly drip the gallium salt solution into the fumarate solution, react at room temperature for 0.5 - 1.5 h, then perform centrifugal separation. The collected product is washed and dried to obtain the antibacterial MOF drug.

[0014] The molar ratio of the fumarate to the gallium salt used is 1:1 - 1.2.

[0015] The fumarate is sodium fumarate, and the gallium salt is gallium nitrate.

[0016] The centrifugation conditions are: centrifuging at a speed of 7000 - 9000 r / min for 5 - 15 min; the drying conditions are: drying at 55 - 65 °C for 10 - 14 h.

[0017] The application of the above antibacterial MOF drug in products against Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli.

[0018] The present invention provides an antibacterial drug and a preparation method thereof. The drug uses gallium ions as the central atom and fumaric acid as the ligand, can specifically respond to bacteria, is non-toxic and harmless to cells and mice, and has very good bactericidal effects in vitro and in vivo and is not prone to drug resistance. Description of the Drawings

[0019] Figure 1 Characterization of the antibacterial drug. A is a scanning electron microscope, B is a transmission electron microscope, and C - F are elemental analyses.

[0020] Figure 2 X-ray diffraction analysis of the antibacterial drug.

[0021] Figure 3 Targeted release of the antibacterial drug to bacteria.

[0022] Figure 4 Bactericidal effect of the antibacterial drug on bacteria. A is the result of the in vitro bactericidal experiment of planktonic bacteria, B is the result of the in vitro bactericidal experiment of biofilm, and C is the result of the in vivo bactericidal experiment in mice.

[0023] Figure 5 Toxicity detection of the antibacterial drug. A is the cytotoxicity detection of A549 cells, B is the cytotoxicity detection of BEAS - 2B cells, and C is the in vivo toxicity detection in mice. Detailed Embodiments

[0024] The following further elaborates on the present invention in combination with examples. The processes and methods not described in detail in the following examples are conventional methods well known in the art. The raw materials or reagents used in the examples are commercially available products except as otherwise specified and can be obtained through commercial channels. The wild-type Pseudomonas aeruginosa PA14 used in the present invention is a well-known strain. The cell lines used are A549 and BEAS - 2B, both of which are well-known cell lines. The experimental animals are Balb / c female mice, 6 - 8 weeks old, with a body weight of 20 g.

[0025] Example 1

[0026] Selection of ligands:

[0027] Prepare a basal medium with glucose and sodium fumarate as the sole carbon sources respectively, sterilize it and reserve for use. Dilute gallium nitrate in a two-fold manner with the two media to prepare 10 dilution concentrations of 0.78 - 400 mg / L. Add the diluted liquid medicine into a sterile 96-well plate, 100 μl per well, then add 100 μl of bacterial liquid into each well. At the same time, set up negative controls and blank controls. Incubate the 96-well plate at 37 °C for 20 h, observe and record the results, and calculate the minimum inhibitory concentration (MIC) values under different carbon sources. The results are shown in Table 1.

[0028] Table 1: Effects of Different Carbon Sources on the Sensitivity of Pseudomonas aeruginosa to Ga(NO3)3

[0029]

[0030] As shown in Table 1, compared with glucose, when sodium fumarate is used as the single carbon source, the MIC of Pseudomonas aeruginosa to Ga(NO3)3 decreases by 4 times, indicating that sodium fumarate can significantly enhance the sensitivity of Pseudomonas aeruginosa to gallium.

[0031] Example 2

[0032] Preparation method of antibacterial drug:

[0033] Dissolve 0.005 mol of sodium fumarate and 0.005 mol of gallium nitrate hydrate in 15 mL of water respectively. Slowly drip the gallium nitrate solution into the sodium fumarate solution, react at room temperature for 1 h, centrifuge at a speed of 8000 r / min for 10 min, collect the product, wash and centrifuge it three times repeatedly, and dry it at 60 °C for 12 h to obtain a white powder product. Characterize the obtained product, and the results are as Figure 1-2 shown.

[0034] Example 3

[0035] Preparation method of antibacterial drug:

[0036] Dissolve 0.005 mol of sodium fumarate and 0.005 mol of gallium nitrate hydrate in 15 mL of water respectively. Slowly drip the gallium nitrate solution into the sodium fumarate solution, react at room temperature for 1.5 h, centrifuge at a speed of 8000 r / min for 10 min, collect the product, wash and centrifuge it three times repeatedly, and dry it at 60 °C for 12 h to obtain a white powder product. Characterize the obtained product, and it has properties similar to those of the product in Example 2.

[0037] Example 4

[0038] Preparation method of antibacterial drug:

[0039] Dissolve 0.005 mol of sodium fumarate and 0.006 mol of gallium nitrate hydrate in 15 mL of water respectively. Slowly drop the gallium nitrate solution into the sodium fumarate solution, react at room temperature for 0.5 h, centrifuge at a speed of 8000 r / min for 10 min, wash the collected product with water and centrifuge it three times repeatedly, and dry it at 60 °C for 12 h to obtain a white powder product. The obtained product was characterized and it had properties similar to those of the product in Example 2.

[0040] Example 5

[0041] Response of antibacterial agents to bacteria:

[0042] Take 10 mg of the antibacterial agent prepared in Example 2 and add it to a dialysis bag. Place the dialysis bag in 50 ml of PBS and PBS containing Pseudomonas aeruginosa PA14 respectively, and culture it in a shaker at 37 °C for 3 days. Take 0.5 ml of the sample at each time point and determine the gallium ion concentration by graphite furnace atomic absorption method. The results are as Figure 3 shown. It can be Figure 3 seen that in the presence of bacteria, the degradation rate of the MOF material is significantly accelerated, indicating that the MOF material can respond to bacteria.

[0043] Example 6

[0044] Antibacterial performance detection:

[0045] 1. Planktonic bacteria bactericidal experiment

[0046] The overnight cultured PA14 was diluted at a ratio of 1:1000, and 200 mg / L of the synthesized MOF drug was added, and cultured at 37 °C. At the same time, the bacterial liquid was taken and diluted for counting at each time point.

[0047] 2. Biofilm bactericidal experiment

[0048] The overnight cultured PA14 was diluted at a ratio of 1:1000, then 150 μl was taken and added to a 96-well plate, and cultured statically at 37 °C for 24 h. Remove the culture medium, add fresh culture medium containing the antibacterial agent, continue to culture statically, remove the upper layer of planktonic bacteria after 24 h, wash 3 times with physiological saline, add 150 μl of physiological saline, ultrasonically treat to dissociate the biofilm, and perform dilution drop plate.

[0049] 3. In vivo bactericidal experiment in mice

[0050] Depilate the back of the mouse, infect the mouse by subcutaneous injection of bacteria, and inject the synthesized antibacterial agent into the wound site formed on the back of the mouse 5 days later, and observe the recovery of the back infection of the mouse.

[0051] The experimental results are as Figure 4 shown. It can be Figure 4It can be seen that in vitro, the MOF material can effectively kill Pseudomonas aeruginosa bacteria and remove biofilms. At the same time, the MOF material can effectively treat Pseudomonas aeruginosa infections caused by Pseudomonas aeruginosa.

[0052] Example 7

[0053] Toxicity detection of antibacterial drugs:

[0054] 1. In vitro cytotoxicity detection

[0055] Incubate 100 mg / L and 200 mg / L of antibacterial drugs with A549 cells and BEAS-2B cells for 12 h, 24 h, and 48 h respectively, perform cell counting, and determine the toxicity of the antibacterial drugs to the cells.

[0056] 2. Detection of toxicity in mice

[0057] After depilating the back of the mice, inject the antibacterial drugs into the mice by subcutaneous injection. After 1 week, observe the condition of the mice's backs, and at the same time take skin sections to detect the toxicity of the antibacterial drugs to the mice.

[0058] The experimental results are as Figure 5 shown. It can be seen from Figure 5 that the MOF material has no toxicity to A549 and BEAS-2B cells, and subcutaneous injection of the MOF material has no toxicity to mice.

Claims

1. A preparation method of an antibacterial MOF drug, characterized in that, Dissolve the fumarate and gallium salt in water respectively, then slowly add the gallium salt solution dropwise to the fumarate solution, react at room temperature for 0.5 - 1.5 h, then centrifuge, and the collected product is washed and dried to obtain the antibacterial MOF drug.

2. The preparation method according to claim 1, wherein The molar ratio of the fumarate to the gallium salt used is 1:1 - 1.

2.

3. The preparation method according to claim 1, characterized in that, The fumarate is sodium fumarate, and the gallium salt is gallium nitrate.

4. The preparation method according to claim 1, characterized in that, The centrifugation conditions are: centrifuge at a speed of 7000 - 9000 r / min for 5 - 15 min; the drying conditions are: dry at 55 - 65 °C for 10 - 14 h.

5. Use of the antibacterial MOF drug prepared by the preparation method according to any one of claims 1 - 4 in the preparation of products against Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli.

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