Copper-cefazolin nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicine synthesis, in particular to copper-cefazolin nanoparticles and a preparation method and application thereof. The copper-cefazolin nanoparticle provided by the invention comprises copper ions and cefazolin coordinated with the copper ions. The copper-cefazolin nano-particles provided by the invention have a relatively strong killing effect on bacteria (staphylococcus aureus), ROS can be generated under an acidic condition to change the permeability of bacterial cell membranes, and meanwhile, the copper-cefazolin nano-particles flow into the bacteria and down-regulate copper ion transporters, so that the copper-cefazolin nano-particles have a long-acting, stable and remarkable antibacterial effect. In addition, the copper-cefazolin nanoparticles can down-regulate inflammatory cytokines, activate peripheral nerves and promote vascular remodeling and hair follicle regeneration under a weakly alkaline condition, and have a good treatment effect on infectious wounds.

Description

Technical Field

[0001] The invention relates to the technical field of drug synthesis, and in particular to copper-cefazoline nanoparticles and a preparation method and application thereof. Background Art

[0002] Bacterial infection is a key factor causing human disease and death. The advent of the antibiotic era has enabled effective treatment of infectious diseases in the past few decades, but with the abuse of antibiotics, multiple drug-resistant bacteria have emerged rapidly. The rapid development of nanotechnology provides a promising strategy to combat drug-resistant bacteria. Compared with traditional antibiotics, nanomaterials allow rational engineering design, such as size control, surface modification, crystal changes, and stimuli-responsive functionalization, to obtain different interactions with bacterial cells, and can physically break the bacterial membrane barrier and release active ingredients. These interactions often lead to unique killing mechanisms and extraordinary antibacterial properties, making it difficult for bacteria to develop drug resistance. They have now become a promising class of antibacterial drugs. Metal-organic nanoparticles are supramolecular materials composed of metal ions and organic ligands, and have been widely used in the biomedical field. Compared with inorganic nanoparticles, nanoparticles formed by the complexation of natural antibacterial components with metal ions have attracted increasing attention due to their long-term, stable antibacterial behavior and extremely low cytotoxicity.

[0003] Cefazolin is the first choice drug for preventing Staphylococcus aureus infection, with stable therapeutic effect and clear mechanism of action. However, drug-resistant bacteria can greatly reduce the antibacterial effect of antibiotics by reducing drug permeability, active transport of antibiotics, bacterial target change and target protection of antibiotics, inactivation and modification of drugs, and generation of target bypass. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a copper-cefazolin nanoparticle and a preparation method and application thereof. The copper-cefazolin nanoparticle provided by the present invention has a good antibacterial (Staphylococcus aureus) effect.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a copper-cefazolin nanoparticle, comprising copper ions and cefazolin coordinated with the copper ions.

[0007] Preferably, the molar ratio of the copper ion to cefazolin is 1:0.9-2.

[0008] The present invention also provides a method for preparing the copper-cefazolin nanoparticles described in the above technical solution, comprising the following steps:

[0009] The copper salt solution and the cefazolin solution are mixed, coordinated and then separated into solid and liquid, and the obtained solid phase is dried to obtain copper-cefazolin nanoparticles.

[0010] Preferably, the copper salt includes one or more of copper chloride, copper chloride hydrate, copper acetate, copper acetate hydrate, copper sulfate, copper sulfate hydrate, copper nitrate and copper nitrate hydrate.

[0011] Preferably, the concentration of the copper salt solution is 0.01-100 mg / mL; the concentration of the cefazolin solution is 0.01-50 mg / mL.

[0012] Preferably, the solvents of the copper salt solution and the cefazolin solution are independently at least one of water, ethanol, methanol, acetone, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylformamide, dichloromethane, chloroform, ethylene glycol, glycerol, isopropanol and polyethylene glycol 200.

[0013] Preferably, the coordination is performed under vortex conditions.

[0014] Preferably, the drying is freeze drying; the freeze drying temperature is -80 to -40°C, and the freeze drying time is 1 to 200 hours.

[0015] The present invention also provides the use of the copper-cefazolin nanoparticles described in the above technical solution or the copper-cefazolin nanoparticles prepared by the above preparation method in antibacterial drugs, antitumor drugs or wound repair drugs.

[0016] Preferably, the antibacterial drug includes an anti-Staphylococcus aureus drug; the anti-tumor drug includes an anti-intratumoral flora drug, a copper death inducer, a cell apoptosis inducer or a cytotoxic drug.

[0017] The present invention provides a copper-cefazolin nanoparticle, comprising copper ions and cefazolin coordinated with the copper ions. The copper-cefazolin nanoparticle provided by the present invention has a strong killing effect on bacteria (Staphylococcus aureus), and because it can produce ROS under acidic conditions to change the permeability of bacterial cell membranes, and the copper-cefazolin nanoparticles flow into bacteria and downregulate copper ion transporters, it has a long-lasting, stable and significant antibacterial effect. In addition, the copper-cefazolin nanoparticles can downregulate inflammatory cytokines under weakly alkaline conditions, activate peripheral nerves, promote vascular reconstruction and hair follicle regeneration, and have a good therapeutic effect on infectious wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a photo of the freeze-dried copper-cefazolin nanoparticles prepared in Example 1;

[0019] Figure 2This is a photograph of the copper-cefazolin nanoparticles prepared in Example 1 in an aqueous solution (concentration of 8 μM);

[0020] Figure 3 TEM image of copper-cefazolin nanoparticles prepared in Example 1;

[0021] Figure 4 FTIR image of copper-cefazolin nanoparticles prepared in Example 1;

[0022] Figure 5 is the XRD image of the copper-cefazolin nanoparticles prepared in Example 1;

[0023] Figure 6 The XPS images of the copper-cefazolin nanoparticles prepared in Example 1, wherein: a is the full spectrum of Cu-CZO, b is the fine spectrum of Cu element, c is the fine spectrum of O element, and d is the fine spectrum of S element;

[0024] Figure 7 The pictures are enzyme-like catalytic activity pictures of the copper-cefazolin nanoparticles prepared in Example 1, wherein: a is a picture of the copper-cefazolin nanoparticles scavenging free radicals, and b is a picture of the copper-cefazolin nanoparticles catalyzing H2O2 to generate ROS;

[0025] Figure 8 The digital photos and bacterial growth curves of the copper-cefazolin nanoparticle coating prepared in Example 1, wherein: a is the anti-MRSA bacterial coating data of copper-cefazolin nanoparticle aqueous dispersions of different concentrations, and b is the anti-MRSA bacterial growth curve of copper-cefazolin nanoparticle aqueous dispersions of different concentrations;

[0026] Fig. 9 This is a scanning electron microscope image of the copper-cefazolin nanoparticles prepared in Example 1 killing MRSA;

[0027] Fig.10 The pictures are of the copper-cefazolin nanoparticles prepared in Example 1 promoting cell migration: a is a picture of a cell Transwell experiment, and b is a picture of a cell scratch experiment;

[0028] Fig.11 This is the test result of the copper-cefazolin nanoparticles prepared in Example 1 by plasma inductively coupled plasma (ICP): wherein: a is the ICP standard curve, and b is the quantitative test result of the copper element. DETAILED DESCRIPTION

[0029] The invention provides a copper-cefazolin nanoparticle, comprising copper ions and cefazolin coordinated with the copper ions.

[0030] As an embodiment, the molar ratio of the copper ion to cefazolin is 1:0.9-2. In a specific embodiment, the molar ratio of the copper ion to cefazolin is 1:1.

[0031] The copper-cefazolin nanoparticles provided by the present invention have good water dispersibility and can be dispersed in liquids such as water, physiological saline, PBS buffer, cell culture medium, etc. when used, and can also be dispersed in hydrogels such as sodium alginate hydrogel, F127 hydrogel, guar gum, etc.

[0032] The present invention also provides a method for preparing the copper-cefazolin nanoparticles described in the above technical solution, comprising the following steps:

[0033] The copper salt solution and the cefazolin solution are mixed, coordinated and then separated into solid and liquid, and the obtained solid phase is dried to obtain copper-cefazolin nanoparticles.

[0034] As an embodiment, the copper salt includes one or more of copper chloride, copper chloride hydrate, copper acetate, copper acetate hydrate, copper sulfate, copper sulfate hydrate, copper nitrate and copper nitrate hydrate. In a specific embodiment, the copper salt can be copper chloride.

[0035] As an embodiment, the solvents of the copper salt solution and the cefazolin solution are independently at least one of water, ethanol, methanol, acetone, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylformamide, dichloromethane, chloroform, ethylene glycol, glycerol, isopropanol and polyethylene glycol 200.

[0036] As an embodiment, the concentration of the copper salt solution is 0.01 to 100 mg / mL, and in a specific embodiment, the concentration of the copper salt solution can be 4 mg / mL. As an embodiment, the concentration of the cefazolin solution is 0.01 to 50 mg / mL, and in a specific embodiment, the concentration of the copper salt solution can be 1 mg / mL.

[0037] As an embodiment, the coordination is carried out under vortex conditions.

[0038] As an embodiment, the solid-liquid separation method is centrifugation, the centrifugal speed is 10000 rpm, and the time is 10 minutes.

[0039] As an embodiment, the drying is freeze drying; the freeze drying temperature is -80 to -40°C, and the freeze drying time is 1 to 200 hours.

[0040] The preparation method of the invention is simple and can be easily industrialized.

[0041] Application of the copper-cefazolin nanoparticles of the present invention or the copper-cefazolin nanoparticles prepared by the above-mentioned preparation method in antibacterial drugs, antitumor drugs or wound repair drugs.

[0042] As an embodiment, the antibacterial drug includes an anti-Staphylococcus aureus (MRSA) drug.

[0043] The copper-cefazolin nanoparticles provided by the present invention improve the antibacterial effect by combining multiple killing mechanisms during the antibacterial process, and have high antibacterial activity at an extremely low dose (the concentration of the copper-cefazolin nanoparticle working solution is 2 μM), thereby improving biological safety, and can also inhibit inflammatory reactions, promote tissue structure regeneration, and further promote wound healing. Therefore, the copper-cefazolin nanoparticles of the present invention have broad application prospects in the preparation of antibacterial and wound repair drugs.

[0044] As an embodiment, the anti-tumor drug includes an anti-intratumoral flora drug, a copper death inducer, a cell apoptosis inducer or a cytotoxic drug.

[0045] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] Example 1

[0047] Dissolve 10 mg of cefazolin in 10 mL of deionized water to obtain a cefazolin solution;

[0048] Dissolve 40 mg of copper chloride in 10 mL of deionized water to obtain a copper chloride solution;

[0049] The cefazolin solution and the copper chloride solution were mixed and vortexed at room temperature, the mixed solution was centrifuged at 10000 rpm for 10 min, the supernatant was discarded, and the precipitate was left. The precipitate was washed three times with deionized water and freeze-dried (drying temperature was -60°C for 24 h) to obtain copper-cefazolin nanoparticles (the molar ratio of copper ions to cefazolin was 1:1).

[0050] Figure 1 This is a photo of the copper-cefazolin nanoparticles prepared in Example 1. As can be seen from the photo, the copper-cefazolin nanoparticles are in the form of green powder.

[0051] Figure 2 This is a photo of the copper-cefazolin nanoparticles prepared in Example 1 in an aqueous solution. It can be seen from the photo that the copper-cefazolin nanoparticles are evenly dispersed (good dispersibility) in the aqueous solution and are light green.

[0052] Figure 3This is a TEM image of the copper-cefazolin nanoparticles prepared in Example 1. It can be seen from the figure that the copper-cefazolin nanoparticles are spherical, with a diameter of about 10 nm and a uniform morphology.

[0053] Figure 4 The Fourier transform infrared (FTIR) spectrum of the copper-cefazolin nanoparticles prepared in Example 1 shows that the -1 、1240cm -1 and 1680cm -1 The absorption bands at the two locations are the stretching vibrations of CO, CC and C=N.

[0054] Figure 5 This is an XRD picture of the copper-cefazolin nanoparticles prepared in Example 1. It can be seen from the figure that the copper-cefazolin nanoparticles are amorphous nanoparticles containing cefazolin.

[0055] Figure 6 This is an XPS picture of the copper-cefazolin nanoparticles prepared in Example 1. It can be seen from the figure that the copper-cefazolin nanoparticles are composed of Cu, O, N, C, and S. 2+ The peaks at 933 eV and 953 eV are the spin-orbit doublets of 2p3 / 2 and 2p1 / 2, and the peaks at 532 eV and 162 eV are the coordination bonds between Cu-O (O 1s signal) and Cu-S (S2p signal), respectively.

[0056] Figure 7 This is a picture of the enzyme-like catalytic activity of the copper-cefazolin nanoparticles prepared in Example 1. Under weak alkaline conditions, the copper-cefazolin nanoparticles can remove DPPH* and ABTS + *, with UV absorption peaks at 519nm and 734nm respectively. Under acidic conditions, copper-cefazolin nanoparticles can catalyze H2O2 to generate ROS. The OPD reaction system is yellow with a UV absorption peak at 430nm, and the TMB reaction system is blue with a UV absorption peak at 652nm.

[0057] Figure 8 The bacterial plate photo and bacterial growth curve after the copper-cefazolin nanoparticles prepared in Example 1 were used to treat MRSA. A certain volume of MRSA bacterial suspension was taken, and the OD value was adjusted to about 0.6 (1.0×107 CFU / mL). The gradient concentration of copper-cefazolin nanoparticle working solution was prepared, and 20 μL of MRSA bacterial suspension was added to the 96-well plate. Then, 180 μL of the corresponding concentration of copper-cefazolin nanoparticle working solution was added to each well, and the plates were incubated at 37°C for 4 h. The incubated bacterial suspension was diluted by 10 3Times, take 20μL of the diluted bacterial suspension and drop it onto the broth agar plate, spread it evenly with glass beads and place it in a 37℃ incubator for 12h, then take out the agar plate and take pictures. When the concentration of the copper-cefazolin nanoparticle working solution reaches 2μM, the MRSA activity is negligible. Incubate MRSA in a 96-well plate in the same way, and continuously monitor the absorbance of the bacterial suspension for 12h with 1h as the time node. When the concentration of the copper-cefazolin nanoparticle working solution reaches 2μM, the MRSA growth rate is slow.

[0058] Fig. 9 The scanning electron microscope picture of the copper-cefazolin nanoparticles prepared in Example 1 killing MRSA. In order to perform morphological analysis, the bacteria treated with copper-cefazolin nanoparticles were collected by centrifugation, fixed with 2.5% glutaraldehyde, incubated at 4°C for 12h, and the bacteria were centrifuged to remove glutaraldehyde. The bacteria were treated with a series of ethanol (30, 50, 70, 80, 90, 95 and 100%) for 10 minutes in sequence. Then, the bacteria were drop-coated on a silicon wafer and freeze-dried. The morphology of the bacteria was observed using a field emission scanning electron microscope. Scanning electron microscopy observed that the cell wall and cell membrane structure of MRSA were greatly destroyed.

[0059] Fig.10 This is a picture of the copper-cefazolin nanoparticles prepared in Example 1 promoting cell migration. The cell migration rate affects the wound healing rate. The Transwell cell migration experiment and the cell scratch experiment prove that the copper-cefazolin nanoparticles have a good effect in promoting cell migration. Therefore, it can be concluded that the copper-cefazolin nanoparticles have a good effect in promoting wound healing.

[0060] Fig.11 The copper-cephalosporin nanoparticles prepared in Example 1 were tested by plasma inductively coupled plasma (ICP). The standard curve R 2 =0.99931, which has a good correlation. The quantitative results of copper element are 11.9197-12.4914 mg / L. It is known that the relative atomic mass of copper element is 63.546 and the molecular weight of cefazolin is 476.489. Based on the above conditions, it can be inferred that the molar ratio of copper ions and cefazolin in copper-cefazolin nanoparticles is 1:1.

[0061] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A copper-cefazolin nanoparticle, characterized in that: It includes copper ions and cefazolin coordinated with copper ions.

2. The copper-cefazolin nanoparticles according to claim 1, characterized in that The molar ratio of the copper ion to cefazolin is 1:0.9-2.

3. The method for preparing the copper-cefazolin nanoparticles according to claim 1 or 2, characterized in that: The following steps are involved: The copper salt solution and the cefazolin solution are mixed, coordinated and then separated into solid and liquid, and the obtained solid phase is dried to obtain copper-cefazolin nanoparticles.

4. The preparation method according to claim 3, characterized in that: The copper salt includes one or more of copper chloride, copper chloride hydrate, copper acetate, copper acetate hydrate, copper sulfate, copper sulfate hydrate, copper nitrate and copper nitrate hydrate.

5. The preparation method according to claim 3, characterized in that: The concentration of the copper salt solution is 0.01-100 mg / mL; the concentration of the cefazolin solution is 0.01-50 mg / mL.

6. The preparation method according to claim 3 or 5, characterized in that: The solvents of the copper salt solution and the cefazolin solution are independently at least one of water, ethanol, methanol, acetone, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylformamide, dichloromethane, chloroform, ethylene glycol, glycerol, isopropanol and polyethylene glycol 200.

7. The preparation method according to claim 3, characterized in that: The coordination was performed under vortex conditions.

8. The preparation method according to claim 3, characterized in that: The drying is freeze drying; the freeze drying temperature is -80 to -40°C, and the freeze drying time is 1 to 200 hours.

9. Use of the copper-cefazolin nanoparticles according to claim 1 or 2 or the copper-cefazolin nanoparticles prepared by the preparation method according to any one of claims 3 to 8 in antibacterial drugs, antitumor drugs or wound repair drugs.

10. The use according to claim 9, characterized in that The antibacterial drugs include anti-Staphylococcus aureus drugs; The anti-tumor drugs include anti-intratumoral flora drugs, copper death inducers, cell apoptosis inducers or cytotoxic drugs.

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