Injectable copper ion mediated antibacterial hydrogel as well as preparation method and application thereof

Abstract

The invention discloses injectable copper ion mediated antibacterial hydrogel and a preparation method and application thereof, and relates to the technical field of medicinal chemistry, the injectable copper ion mediated antibacterial hydrogel is formed by self-assembly of 4 '-[(4-(4-morpholinyl butoxy) phenyl)]-2, 2': 6 '2 ''-terpyridyl ligand L and copper ions, and the antibacterial hydrogel has a porous structure; the preparation method comprises the following steps: mixing a 4 '-[(4-(4-morpholinyl butoxy) phenyl)]-2, 2': 6 '2 ''-terpyridyl ligand L with a copper bromide aqueous solution to prepare hydrogel; the invention also provides an application of the antibacterial hydrogel in preparation of antibacterial drugs. The process for preparing the hydrogel is simple, the prepared hydrogel is excellent in antibacterial performance and has good biocompatibility, in-vivo greater wax moth larva infection treatment tests also prove that the hydrogel has a good in-vivo antibacterial effect, and the survival rate of greater wax moth larvae can be increased.

Description

Technical Field

[0001] The present invention relates to an injectable copper ion-mediated antibacterial hydrogel, its preparation method and antibacterial application, which has good inhibitory effects on Staphylococcus aureus and Escherichia coli, and has good biosafety; it has self-assembly performance and can be prepared into a carrier-free hydrogel drug, belonging to the field of pharmaceutical chemistry. Background Art

[0002] Due to its excellent properties such as soft texture, good biocompatibility, stimulus response, and drug slow release, the development prospect of hydrogels in the antibacterial field has attracted the attention of researchers in recent years. When applied to anti-infection treatment, hydrogels can be locally used at the infection site and slowly, continuously or even controllably release antibacterial agents, avoiding the toxicity caused by multiple administrations and systemic administrations and improving the bioavailability of drugs.

[0003] A hydrogel is a material composed of a three-dimensional hydrophilic water-soluble network with high water content. Water-soluble or hydrophilic substances can form hydrogels through certain chemical cross-linking or physical cross-linking. Small molecule metal hydrogels are three-dimensional network structure materials formed by the coordination of metal ions with small molecule ligands.

[0004] In the research of metal pharmaceutical chemistry, it is found that metal copper (II) complexes can be modified with known anti-inflammatory drugs to improve their antibacterial effects against Gram-negative bacteria and Gram-positive bacteria. The reason is that copper ions have natural antibacterial properties, can destroy the bacterial cell membrane by producing reactive oxygen species (ROS), enter the bacteria and inhibit protein synthesis, thereby achieving the bactericidal effect. Further research finds that the introduction of metal ions can also significantly improve the antibacterial activity of hydrogels. These properties make copper ion hydrogels have broad application prospects in the biomedical field. Summary of the Invention

[0005] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art, and provide an injectable copper ion-mediated antibacterial hydrogel, its preparation method and antibacterial application, specifically design an injectable copper ion-mediated antibacterial hydrogel, its preparation method and antibacterial application.

[0006] The technical solution of the present invention is as follows: In the first aspect, the present invention provides an injectable copper ion-mediated antibacterial hydrogel, which is self-assembled by 4'-[(4-(4-morpholinobutoxy)phenyl)]-2,2':6'2"-terpyridine ligand L and copper ions, and the antibacterial hydrogel has a porous structure.

[0007] Among them, "self-assembly" refers to the process in which the 4'-[(4-(4-morpholinobutoxy)phenyl)]-2,2':6'2"-terpyridine ligand L and copper ions spontaneously form an ordered structure through non-covalent interactions.

[0008] Optionally, the structural formula of the 4'-[(4-(4-morpholinobutoxy)phenyl)]-2,2':6'2"-terpyridine ligand L is as follows: 。

[0009] Optionally, the molar ratio of the 4'-[(4-(4-morpholinobutoxy)phenyl)]-2,2':6'2"-terpyridine ligand L to copper ions is 0.005~0.02:0.005~0.02. Preferably, the molar ratio of the 4'-[(4-(4-morpholinobutoxy)phenyl)]-2,2':6'2"-terpyridine ligand L to copper ions is 0.012~0.018:0.012~0.018. More preferably, the molar ratio of the 4'-[(4-(4-morpholinobutoxy)phenyl)]-2,2':6'2"-terpyridine ligand L to copper ions is 0.015:0.015.

[0010] Optionally, the preparation method of the 4'-[(4-(4-morpholinobutoxy)phenyl)]-2,2':6'2"-terpyridine ligand L is as follows: 。

[0011] In a second aspect, the present invention provides a method for preparing the antibacterial hydrogel, comprising the following steps: Mix the 4'-[(4-(4-morpholinobutoxy)phenyl)]-2,2':6'2"-terpyridine ligand L with an aqueous copper bromide solution to obtain the antibacterial hydrogel.

[0012] Optionally, the 4'-[(4-(4-morpholinobutoxy)phenyl)]-2,2':6'2"-terpyridine ligand L and the aqueous copper bromide solution are mixed under ultrasonic conditions to obtain the hydrogel; wherein, the power of ultrasonic is 300W, the ultrasonic frequency is 40kHz, and the ultrasonic time is 3 minutes.

[0013] Optionally, the concentration of the aqueous copper bromide solution is 9.98 μmol / mL~40.0 μmol / mL.

[0014] In a second aspect, the present invention provides an application of the antibacterial hydrogel in the preparation of antibacterial drugs or bacteriostatic drugs.

[0015] Optionally, the antibacterial or bacteriostatic drug is a drug that inhibits the growth of Staphylococcus aureus and Escherichia coli.

[0016] The present invention has at least one of the following beneficial effects: The present invention uses ligand L, 4ʹ-[(4-(4-morpholinobutoxy)phenyl)]-2,2ʹ:6ʹ2ʹʹ-terpyridine, and an aqueous solution of copper bromide as raw materials to self-assemble a hydrogel with a porous structure under ultrasonic conditions. The obtained hydrogel has excellent antibacterial properties against Staphylococcus aureus and Escherichia coli. The hydrogel prepared by the present invention has a simple process, good biocompatibility, and good in vivo antibacterial effects. The in vivo antibacterial effect of the hydrogel was also demonstrated in the in vivo treatment experiment of Galleria mellonella larvae infection, which can improve the survival rate of Galleria mellonella larvae and has good biosafety. Description of the Drawings

[0017] Figure 1 The figure shows the physical picture of the prepared hydrogel; among them, A is the inverted physical picture of the hydrogel; B is the physical picture of the hydrogel inhaled by the syringe. Figure 2 The figure shows the scanning electron microscope image of the hydrogel magnified 1000 times. Figure 3 The figure shows the bacteriostatic circle experiment effect pictures of the hydrogel against Staphylococcus aureus and Escherichia coli; among them, A is the bacteriostatic circle of Staphylococcus aureus; B is the bacteriostatic circle of Escherichia coli. Figure 4 The figure shows the hemolysis test result pictures of the hydrogel; among them, A is the physical picture of the hemolysis test result; B is the column chart of the hemolysis test result. Figure 5 The figure shows the toxicity result pictures of the hydrogel and ligand L on Galleria mellonella larvae; among them, A is the toxicity result picture of the antibacterial hydrogel, cefalexin, copper bromide, and water prepared in Example 2 on Galleria mellonella larvae; B is the toxicity result picture of 4'-[(4-(4-morpholinobutoxy)phenyl)]-2,2':6'2"-terpyridine ligand L and DMSO on Galleria mellonella larvae. Figure 6 The figure shows the treatment result pictures of the hydrogel and ligand L on Galleria mellonella larvae; among them, A is the treatment result picture of the antibacterial hydrogel, cefalexin, copper bromide, and water prepared in Example 2 on Galleria mellonella larvae; B is the treatment result picture of 4'-[(4-(4-morpholinobutoxy)phenyl)]-2,2':6'2"-terpyridine ligand L and DMSO on Galleria mellonella larvae. Detailed Embodiments

[0018] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0019] Example 1 In the preparation method of an injectable copper ion-mediated antibacterial hydrogel as described above, the synthesis of the 4'-[(4-(4-morpholinobutoxy)phenyl)]-2,2':6'2"-terpyridine ligand L involves two key intermediates. First, through intermediate J, and then through intermediate K, and finally the target ligand L is formed. The preparation method includes the following steps: The reaction is shown in the following reaction route: ; Wherein, r.t. represents room temperature conditions.

[0020] Step 1: Preparation of intermediate J. The structural formula of intermediate J is as follows: The specific steps are as follows: Dissolve the raw material p-hydroxybenzaldehyde (3.60 g, 30 mmol) in 45 mL of an ethanol aqueous solution (volume ratio 2:1). Under stirring conditions, add 2-acetylpyridine (7.21 g, 60 mmol), sodium hydroxide (2.88 g, 72 mmol) and 90 mL of ammonia water, and react overnight. Stop the reaction. The solution shows dark yellow, accompanied by yellow crystals and flocculants. Rotate and evaporate the reaction solution on a rotary evaporator to remove ammonia water and ethanol. Adjust the pH to 5-6 with dilute hydrochloric acid. The solution turns green and precipitates are formed. Filter by suction, retain the filter cake. Transfer the filter cake to a beaker, add 50 mL of methanol, stir for 15 minutes, centrifuge the precipitate, wash it with methanol 3 times and then centrifuge again, and dry it to obtain intermediate J as a white solid. Yield: 4.02 g, 41.84%.

[0021] Step 2: Preparation of intermediate K. The structural formula of intermediate K is as follows: The specific steps are as follows: First, weigh the raw material (J) (1.00 g, 3.08 mmol) and add it to 150 mL of acetonitrile solution. While stirring, add potassium carbonate (0.85 g, 6.15 mmol) and 1,4-dibromobutane (1.12 mL, 9.23 mmol). Heat to 84 °C and react for 24 hours. Stop the reaction, and there are flocculates in the solution. Remove the excess acetonitrile from the reaction solution under a rotary evaporator. Dissolve the product in 50 mL of dichloromethane and wash it with an aqueous solution three times. Separate and retain the organic phase, add anhydrous sodium sulfate for drying, filter by suction, and concentrate the organic phase. Purify the crude product by column chromatography on neutral alumina using a petroleum ether / dichloromethane mixture (4:1, v / v). Remove the solvent under reduced pressure to obtain intermediate K, which is a white solid, yield: 1.62 g, 58.7%.

[0022] Step 3: Preparation of ligand L, and the structural formula of ligand L is as follows: Dissolve intermediate K (0.60 g, 1.29 mmol) in 150 mL of acetonitrile solution, then add potassium carbonate (0.89 g, 6.45 mmol) and 0.5 mL of morpholine (5.17 mmol), and react at 84 °C for 24 hours. After stopping the reaction, remove the excess acetonitrile from the reaction solution under a rotary evaporator. Dissolve the product in 50 mL of dichloromethane and wash it with an aqueous solution three times. Separate and retain the organic phase, add anhydrous sodium sulfate for drying, filter by suction, and concentrate the organic phase. Recrystallize the crude product with ethanol to obtain ligand L, which is a light white powder, yield: 0.4 g, 66.3%.

[0023] Characterization shows that: high-resolution mass spectrometry (ESI) m / z: C 29 H 31 N 4 O 2 The theoretical value is 467.2447, [M + H] + The calculated value is 467.2441. 1H NMR spectrum: 1 1H NMR (400 MHz, CDCl 3) δ 8.77 – 8.70 (m, 4H), 8.67 (d, J =8.0 Hz, 2H), 7.88 (t, J = 7.4 Hz, 4H), 7.39 – 7.31 (m, 2H), 7.01 (d, J = 8.7Hz, 2H), 4.06 (t, J = 6.3 Hz, 2H), 3.78 – 3.70 (m, 4H), 2.55 – 2.38 (m, 6H),1.93 – 1.82 (m, 2H), 1.72 (dd, J = 14.5, 7.4 Hz, 2H).

[0024] Example 2 A preparation method of an injectable copper ion-mediated antibacterial hydrogel, comprising the following steps: Mix a 4'-[(4-(4-morpholinobutoxy)phenyl)]-2,2':6',2"-terpyridine ligand L and an aqueous copper bromide solution (concentration of 13.89 μmol / mL) in a molar ratio of 0.015:0.015, and under ultrasonic conditions, the power of ultrasonic is 300 W, the ultrasonic frequency is 40 kHz, and the ultrasonic time is 3 minutes to obtain a hydrogel.

[0025] To demonstrate the effect of the present invention, the physical pictures of the formed hydrogel were taken respectively. Figure 1 It can be seen that the prepared hydrogel is green, clear and transparent, and the hydrogel has injectability.

[0026] Example 3 In this example, the antibacterial hydrogel prepared in Example 2 was observed for its morphology, and the method is as follows: The antibacterial hydrogel prepared in Example 2 was frozen in a -80 °C refrigerator for three days, and then placed in a freeze dryer for 24 hours, and the morphology of the prepared hydrogel was observed under a scanning electron microscope.

[0027] Figure 2 The picture of the freeze-dried hydrogel was taken at a magnification of 1000 times under an electron scanning electron microscope. The clearly visible holes and channels in the image indicate that the hydrogel has high porosity.

[0028] Example 4 In this example, the antibacterial hydrogel prepared in Example 2 was subjected to an in vitro antibacterial activity test, and the method is as follows: Inhibitory zone: The Newman strain of Staphylococcus aureus and Escherichia coli were cultured in TSB medium until the logarithmic phase, and the number of bacterial cells was diluted 1000 times with fresh TSB medium. Subsequently, 100 μL of the bacterial solution was pipetted onto an agar plate and spread, and 100 μL of the antibacterial hydrogel prepared in Example 2 was injected into the punched holes and incubated for 20 hours.

[0029] The results are as Figure 3 shown.

[0030] Table 1 shows the size of the inhibitory zones of the antibacterial hydrogel prepared in Example 2 against different bacteria From Figure 3 Table 1, it can be seen that the antibacterial hydrogel prepared in Example 2 has good antibacterial effects against Staphylococcus aureus and Escherichia coli.

[0031] Example 5 In this example, a safety test was carried out on the antibacterial hydrogel prepared in Example 2, and the method is as follows: (1) Hemolytic toxicity test: Fresh and sterile rabbit red blood cells were centrifuged at 2000 rmp for 2 min, washed three times with PBS for further use. Then, a solution containing 5% rabbit red blood cells and 100 μL of the hydrogel prepared in Example 2 was incubated at 37 °C for 30 min. After incubation, it was centrifuged at 2000 rmp for 2 min and transferred to a 96-well plate. The OD 540 value was measured under a microplate reader. 0.1% Triton X-100 was used as a positive control, and PBS was used as a negative control. Each group was repeated 3 times.

[0032] (2) Toxicity test on Galleria mellonella larvae: After the Galleria mellonella larvae were placed in a 37 °C incubator and observed for 12 hours, Galleria mellonella larvae with comparable activity were selected for subsequent experiments. Different compounds (the antibacterial hydrogel prepared in Example 2, cefalexin, copper bromide, 4'-[(4-(4-morpholinobutoxy)phenyl)]-2,2':6'2"-terpyridine ligand L, DMSO) were formulated into a concentration of 10 mg / mL, and water and DMSO were used as blank controls. 3 μL of the compound, water, and DMSO were pipetted into the Galleria mellonella larvae, and then they were placed in a 37 °C incubator, and their survival was monitored within 7 days.

[0033] Figure 4 The figure shows the results of the hemolysis test of the hydrogel; among them, A is a physical picture of the hemolysis test results; B is a bar chart of the hemolysis test results; From Figure 4 this, it can be seen that the hemolysis rate of the hydrogel is 1.73%, and there is no hemolytic toxicity, indicating that the hydrogel has a certain degree of safety.

[0034] Figure 5 The figure shows the toxicity results of the hydrogel and ligand L on Galleria mellonella larvae; among them, A is the toxicity results figure of the antibacterial hydrogel, cefalexin, copper bromide and water prepared in Example 2 on Galleria mellonella larvae; B is the toxicity results figure of 4'-[(4-(4-morpholinobutoxy)phenyl)]-2,2':6'2"-terpyridine ligand L and DMSO on Galleria mellonella larvae; from Figure 5 It can be seen that the survival rate of all the worms injected with 3 μL of 10 mg / mL compound is 100% compared with those injected with water or DMSO as the blank control. This indicates that the hydrogel has good in vivo safety.

[0035] Example 6 In this example, the antibacterial hydrogel prepared in Example 2 was subjected to a Galleria mellonella infection treatment test, and the method is as follows: Galleria mellonella larvae infection treatment test: Select worms with comparable activity in the same steps as above. Then inject 3 μL of the logarithmic-phase Staphylococcus aureus suspension (OD 600 = 0.5) into the left hind leg of the Galleria mellonella larvae. One hour after infection, inject 3 μL of 10 mg / mL different compounds into the right hind leg of the Galleria mellonella larvae. Place them in the dark in a 37 °C constant temperature incubator and observe the survival of the worms within 7 days.

[0036] Figure 6 The figure shows the treatment results of the hydrogel and ligand L on Galleria mellonella larvae; among them, A is the treatment results figure of the antibacterial hydrogel, cefalexin, copper bromide and water prepared in Example 2 on Galleria mellonella larvae; B is the treatment results figure of 4'-[(4-(4-morpholinobutoxy)phenyl)]-2,2':6'2"-terpyridine ligand L and DMSO on Galleria mellonella larvae. From Figure 6 It can be seen that for the worms treated with ligand L of the synthesized hydrogel, the survival rate after seven days is only 30%, and the survival rate of the worms treated with copper bromide is also only 20%. However, under the treatment of 3 μL of the hydrogel, the survival rate of the worms within seven days is 90%, showing a treatment effect similar to that of cefalexin, which proves its good in vivo antibacterial potential.

[0037] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and all should be covered within the protection scope of the present invention.

Claims

1. An injectable copper ion-mediated antibacterial hydrogel, characterized in that: The antibacterial hydrogel is formed by self-assembly of 4'-[(4-(4-morpholinylbutyloxy)phenyl)]-2,2':6'2"-terpyridine ligand L and copper ions, and has a porous structure.

2. The antibacterial hydrogel according to claim 1, characterized in that: The structural formula of the 4'-[(4-(4-morpholinylbutyloxy)phenyl)]-2,2':6'2"-terpyridine ligand L is as follows: 。 3. The antibacterial hydrogel according to claim 1, characterized in that: The molar ratio of the 4'-[(4-(4-morpholinylbutyloxy)phenyl)]-2,2':6'2"-terpyridine ligand L to the copper ion is 0.005-0.02:0.005-0.

02.

4. The antibacterial hydrogel according to claim 1, characterized in that The molar ratio of the 4'-[(4-(4-morpholinylbutyloxy)phenyl)]-2,2':6'2"-terpyridine ligand L to the copper ion is 0.012-0.018:0.012-0.

018.

5. The antibacterial hydrogel according to claim 1, characterized in that: The preparation method of the 4'-[(4-(4-morpholinylbutyloxy)phenyl)]-2,2':6'2"-terpyridine ligand L is as follows: 。 6. The method for preparing the antibacterial hydrogel according to any one of claims 1 to 5, characterized in that: The following steps are involved: The antibacterial hydrogel is prepared by mixing 4'-[(4-(4-morpholinylbutyloxy)phenyl)]-2,2':6'2"-terpyridine ligand L with a copper bromide aqueous solution.

7. The preparation method according to claim 6, characterized in that: The 4'-[(4-(4-morpholinylbutyloxy)phenyl)]-2,2':6'2"-terpyridine ligand L is mixed with the copper bromide aqueous solution under ultrasonic conditions to prepare a hydrogel; wherein the ultrasonic power is 300W, the ultrasonic frequency is 40kHz, and the ultrasonic time is 3 minutes.

8. The preparation method according to claim 6, characterized in that: The concentration of the copper bromide aqueous solution is 9.98 μmol / mL to 40.0 μmol / mL.

9. Use of the antibacterial hydrogel according to any one of claims 1 to 5 in the preparation of antibacterial drugs or antibacterial drugs.

10. The use according to claim 9, characterized in that: The antibacterial drug or antibacterial drug is a drug that inhibits the growth of Staphylococcus aureus and Escherichia coli.

Images