Multifunctional antibiotic nano-material, preparation method and application thereof

Abstract

According to the multifunctional antibiotic nano-material and the preparation method provided by the invention, firstly, Fe ions and Car are subjected to primary coordination to form an FC nano-composite, then DFO and the FC nano-composite are subjected to secondary coordination to form a DFC multifunctional nano-material, and due to the fact that Car is high in sterilization efficiency and high in biological safety, the DFC multifunctional nano-material can be used for preparing the antibiotic nano-material. The side chain has carboxyl and can realize in-situ nanocrystallization with iron ions, which is helpful for entering an MRSA biological membrane and protecting antibiotics from being degraded and inactivated by beta-lactamase secreted by MRSA at the same time; besides, due to the strong coordination effect of the DFO and the iron ions and the potential effect of promoting blood vessels and bacterium targeting, the DFO is modified on the iron ion-antibiotic nano-composite, so that the double-function fusion of bacterium targeting and blood vessel promoting can be realized, and the iron ion-antibiotic nano-composite has a better effect of resisting the development of drug-resistant bacteria and has a wide application prospect in the biomedical field.

Description

Technical Field

[0001] The present application relates to the technical field of biomedical materials, and in particular to a multifunctional antibiotic nanomaterial, a preparation method and application thereof. Background Art

[0002] Biofilm infections are very common in chronic wounds and can delay wound healing. Antibiotics are the main choice for treating chronic wound infections. Unfortunately, the misuse of antibiotics has led to the spread of multidrug-resistant (MDR) bacteria, especially multidrug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA). According to statistics, antibiotic-resistant infections directly caused 1.27 million deaths in 2019, and it is estimated that by 2050, the number of deaths caused by drug-resistant infections will reach 10 million per year. Vancomycin is currently the "last resort" for treating multidrug-resistant bacteria MRSA, but vancomycin cannot penetrate biofilms and the emergence of vancomycin-resistant Staphylococcus aureus means that the last line of defense for humans to treat MDR infections is in jeopardy. However, the development of new antibiotics is expensive and time-consuming. Therefore, how to maximize the therapeutic effect while reducing drug resistance by redesigning existing antibiotics has become an important research direction at this stage.

[0003] Researchers have explored a variety of strategies, such as modifying antibiotics with polymers through covalent bonds, encapsulating antibiotics in polymer matrices, and combining antibiotics with enzyme inhibitors. However, these methods have limitations, such as cumbersome processes, inadequate antibiotic release, low targeting efficiency, and possible side effects of enzyme inhibitors. Summary of the invention

[0004] In view of this, it is necessary to provide a multifunctional antibiotic nanomaterial, preparation method and application that can delay bacterial resistance and eradicate biofilm in order to address the technical defects of current antibiotic in situ nanomaterials, which have poor therapeutic effects and are prone to drug resistance.

[0005] To solve the above problems, this application adopts the following technical solutions:

[0006] One of the purposes of this application is to provide a method for preparing a multifunctional antibiotic nanomaterial, comprising the following steps:

[0007] The Fe ions and Car are coordinated once to form FC nanocomposites;

[0008] The DFO and the FC nanocomposite are secondary coordinated to form a DFC multifunctional nanomaterial.

[0009] In some embodiments, the step of coordinating Fe ions and Car to form FC nanocomposites specifically includes the following steps:

[0010] The Fe ions and Car are stirred at room temperature to form Fe-Car nanoparticles, and then the Fe-Car nanoparticles are centrifugally washed and freeze-dried to obtain the FC nanocomposite.

[0011] In some of the embodiments, the volume and molar concentration of the FeCl3.6H2O are the same as those of the Car.

[0012] In some of the embodiments, the Fe ions include FeCl3·6H2O or Fe(NO3)3·9H2O or Fe2(SO4)3·xH2O.

[0013] In some embodiments, the step of performing secondary coordination between DFO and the FC nanocomposite to form the DFC multifunctional nanomaterial specifically includes the following steps:

[0014] Dispersing the DFO and FC nanocomposite in distilled water, ultrasonically treating the nanocomposite, and stirring at room temperature to obtain nanoparticles;

[0015] The nanoparticles are centrifugally washed and freeze-dried to obtain the DFC multifunctional nanomaterial.

[0016] In some of the embodiments, the volumes of the DFO and the Car are the same, and the molar ratio of the DFO and the FC nanoparticles is 1:10.

[0017] The second purpose of the present application is to provide a multifunctional antibiotic nanomaterial, including one prepared by the preparation method.

[0018] The third purpose of the present application is to provide a use of the multifunctional antibiotic nanomaterial in treating drug-resistant bacteria and / or eradicating biofilm.

[0019] This application adopts the above technical solution, and its beneficial effects are as follows:

[0020] The multifunctional antibiotic nanomaterial and preparation method provided in the present application first utilize Fe ions and Car for a primary coordination to form an FC nanocomplex, and then perform a secondary coordination with DFO and the FC nanocomplex to form a DFC multifunctional nanomaterial. Since Car has high bactericidal efficiency and high biosafety, its side chain has a carboxyl group that can be in situ nanosized with iron ions, which helps to enter the MRSA biofilm while protecting the antibiotic from degradation and inactivation by β-lactamase secreted by MRSA. In addition, due to the strong coordination effect of DFO and iron ions and the potential angiogenic and bacterial targeting effects, DFO is modified on the iron ion-antibiotic nanocomplex to achieve the dual functional fusion of bacterial targeting and angiogenicity, which has a good effect in combating the development of drug-resistant bacteria and has broad application prospects in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments of the present application or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 The morphology diagram provided in Example 1 of the present application, wherein: Figure 1 (a) is the SEM image of FC nanocomposite. Figure 1 (b) is the SEM image of DFC multifunctional nanomaterials. Figure 1 (c) is the transmission image of DFC. Figure 1 (d) is the HAADF image of DFC. Figure 1 Middle (ei) is the EDS image of DFC multifunctional nanomaterials;

[0023] Figure 2 This is a structural representation diagram of FC and DFC provided in Example 1 of the present application, wherein: Figure 1 (a), (b) and (c) represent the XRD, IR and XPS spectra of FC and DFC nanomaterials, respectively.

[0024] Figure 3 Schematic diagram of the antibacterial effect of nanomaterials of different concentrations on MRSA and the antibacterial effect of antibiotics vancomycin (VAN) and carbenicillin (Car) provided in Example 1 of the present application.

[0025] Figure 4 Schematic diagram of the bacterial targeting effect of bacteria and rhodamine-labeled nanomaterials provided in Example 1 of the present application.

[0026] Figure 5 This is a schematic diagram of the confocal observation of the effect of nanomaterials in eradicating biofilms and the crystal violet staining observation of the effect of materials in eradicating biofilms provided in Example 1 of the present application.

[0027] Figure 6 This is a schematic diagram of the DFC multifunctional nanomaterial provided in Example 1 of the present application in delaying the progression of MRSA resistance.

[0028] Figure 7 Schematic diagram of the effects of different concentrations of DFC multifunctional nanomaterials on cell migration (a) and ring formation (b) provided in Example 1 of the present application. DETAILED DESCRIPTION

[0029] Embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application.

[0030] In the description of the present application, it should be understood that the terms "upper", "lower", "horizontal", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0031] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0032] In order to make the objectives, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0033] The step flow of the method for preparing an antibiotic in-situ nanomaterial provided in the embodiment of the present application includes the following steps S110 to S120, and the technical solution for its implementation is described in detail below.

[0034] Step S110: Fe ions and Car are coordinated once to form FC nanocomposites.

[0035] In this embodiment, the step of coordinating Fe ions and Car to form FC nanocomposite includes the following steps:

[0036] The Fe ions and Car are stirred at room temperature to form Fe-Car nanoparticles, and then the Fe-Car nanoparticles are centrifugally washed and freeze-dried to obtain the FC nanocomposite.

[0037] In this embodiment, the volume and molar concentration of the FeCl3.6H2O and the Car are the same.

[0038] In this embodiment, the Fe ions include FeCl3·6H2O or Fe(NO3)3·9H2O or Fe2(SO4)3·xH2O.

[0039] It can be understood that carbenicillin (Car) is an antibiotic with a broad-spectrum antibacterial effect and a carboxyl group in the side chain. Car has high bactericidal efficiency and high biosafety. The carboxyl group in its side chain can be nanosized in situ with iron ions, which helps to enter the MRSA biofilm while protecting the antibiotic from degradation and inactivation by β-lactamase secreted by MRSA.

[0040] Step S120: performing secondary coordination on DFO and the FC nanocomposite to form a DFC multifunctional nanomaterial.

[0041] In this embodiment, the step of secondary coordination of DFO and the FC nanocomposite to form the DFC multifunctional nanomaterial specifically includes the following steps: dispersing the DFO and the FC nanocomposite in distilled water, ultrasonically treating the nanocomposite, and stirring at room temperature to obtain nanoparticles; centrifugally washing the nanoparticles and freeze-drying them to obtain the DFC multifunctional nanomaterial.

[0042] In this embodiment, the volumes of the DFO and the Car are the same, and the molar ratio of the DFO and the FC nanoparticles is 1:10.

[0043] It can be understood that due to the strong coordination effect between DFO and iron ions and its potential angiogenic and bacterial targeting effects, modifying DFO on iron ion-antibiotic nanocomplexes can achieve the dual functional fusion of bacterial targeting and angiogenic effects.

[0044] The multifunctional antibiotic nanomaterial and preparation method provided in the above embodiments of the present application utilize Fe ions and Car for a primary coordination to form an FC nanocomposite, and then perform a secondary coordination through DFO and the FC nanocomposite to form a DFC multifunctional nanomaterial, which has a good effect in combating the development of drug-resistant bacteria and has a wide range of applications in the biomedical field.

[0045] The above technical solution of the present application is described in detail below in conjunction with specific embodiments.

[0046] Example

[0047] (1) Preparation, configuration and precautions of each solution and reagent

[0048] a. Ferric chloride solution (concentration: 10mM): weigh 1.3515g of ferric chloride hexahydrate and dissolve it in 500ml of ultrapure water (deionized water is also acceptable, the same below). Note: Ferric chloride absorbs water very seriously, so the surrounding environment should be kept dry during the weighing process, and the weighing should be done quickly. After weighing, seal it and store it in a drying cabinet.

[0049] b. Carbenicillin solution (Car) (concentration: 10mM): weigh 2.001g of carbenicillin sodium and dissolve it in 500ml of ultrapure water (deionized water is also acceptable, the same below). Note: It will lose its effectiveness if stored for a long time, and the usage time should not exceed 1 month.

[0050] c. Desferrioxamine sodium sulfonate (DFO) (1mM): Weigh 0.3284g of DFO and dissolve it in 500ml of ultrapure water (deionized water is also acceptable, the same below). Note: It will lose its effectiveness if left for a long time and should not be used for more than 1 month.

[0051] (2) Preparation of FC nanocomposites

[0052] a. Add 500mL of 10mM FeCl3.6H2O to 500mL of 10mM Car and stir at room temperature for 24 hours at 400rpm. Centrifuge the synthesized Fe-Car (FC) nanomaterial (10000prm, 5min) and wash it with distilled water 5 times. Freeze it in a -20℃ refrigerator overnight and dry it in a freeze dryer for 48h to obtain FC nanocomposite.

[0053] (3) Preparation of DFC multifunctional nanomaterials

[0054] a. Disperse 500mL of 1mM DFO and FC nanocomplex in a molar ratio of 1:10 in distilled water, ultrasonicate for half an hour, and stir at room temperature for 3 hours. Centrifuge the nanoparticles (10000prm, 5min) and wash them five times with distilled water. Freeze them in a -20℃ refrigerator overnight and dry them in a freeze dryer for 48 hours to obtain DFC multifunctional nanomaterials.

[0055] Antibiotic nanomaterials were prepared by the above scheme, and the nanoparticles were characterized by SEM. The results showed that the diameter of the obtained FC and DFC nanomaterials was about 80-100nm ( Figure 1 The transmission results show that the DFC multifunctional nanomaterial is nano-sized ( Figure 1 (c) EDS results show that DFC multifunctional nanomaterials are composed of Fe, C, O, S, and N.

[0056] Further XRD analysis shows that both FC and DFC materials are amorphous and have no obvious diffraction peaks ( Figure 2 In addition, its composition was characterized by infrared. Figure 2 In b, we can see that the DFC multifunctional nanomaterial has a peak at 2875 cm -1 and 1770cm -1 The peaks at are characteristic peaks of DFO and Car. Figure 2(c) It is proved that DFC multifunctional nanomaterials contain Fe, O, N, C, and S elements.

[0057] In order to determine whether the functional nanomaterial (hereinafter referred to as DFC) obtained in the first step of the present invention has antibacterial properties, methicillin-resistant Staphylococcus aureus (MRSA, ATCC43000), a common drug-resistant bacterial species in clinical practice, was selected to detect the antibacterial properties of the particles: 5×10 5 After the MRSA liquid with CFU / ml and the DFC nanomaterials were diluted to a specific concentration, they were spread in a 48-well plate and cultured in a bacterial incubator at 37°C for 24 hours. After shaking on a horizontal shaker for 1 minute, they were transferred to a 96-well plate and the absorbance at 600nm was read to reflect the proliferation of bacteria. In addition, the actual content of antibiotics in the nanomaterial reagents was selected for comparison to determine the changes in their antibacterial properties after nano-sizing.

[0058] from Figure 3 It can be seen from Figure a that as the concentration of DFC multifunctional nanomaterials increases, the antibacterial effect on MRSA becomes more obvious. At 25μg / ml, the antibacterial rate is close to 95%. Then, by comparing the antibacterial effect of FC nanocomplexes and antibiotics with the same content, DFC multifunctional nanomaterials have the best antibacterial effect, and its antibacterial effect is equivalent to that of VAN.

[0059] The targeting effect of nanomaterials on MRSA bacteria was further investigated. RhB-DFC and RhB-FC nanomaterials were obtained by labeling DFC and FC nanomaterials with rhodamine (RhB), and after co-culturing them with MRSA, their bacterial targeting effect was observed under confocal microscope.

[0060] from Figure 4 It can be seen that the nanomaterials labeled with RhB are red, and the MRSA labeled with SYTO-9 are green. After the bacteria are treated with DFC, the materials and bacteria co-localize in fluorescence and appear yellow, indicating that the DFC material has a bacterial targeting effect. After the bacteria are treated with FC, the co-localization of the materials and bacteria is relatively small, indicating that the FC nanocomplex basically has no bacterial targeting effect.

[0061] The material's ability to eradicate MRSA biofilm was further investigated. Figure 5 It can be seen that the control group and Car are basically green fluorescence, indicating that they are all living bacteria, while VAN has a small amount of red fluorescence, which may be due to the limited penetration of antibiotics into the biofilm. The biofilms of the FC and DFC groups both show red fluorescence penetration, especially the DFC group, indicating that the DFC group has a good biofilm eradication effect compared with the FC and antibiotic groups.

[0062] To determine whether antibiotic nanomaterials can delay the progression of drug resistance, we selected Staphylococcus aureus as a model and incubated the nanomaterials and antibiotics in a gradient dilution with the bacteria (5×10 5 CFU / ml) for 24 h, and then the absorbance of bacteria at OD600 was detected. If there was no growth, it was the MIC of the material. Then, 1 / 2 MIC concentration was selected for co-culture with bacteria, and the MIC of the material was re-determined after 1 day of drug stimulation, and the MIC of the material was updated, and repeated 16 times.

[0063] from Figure 6 It can be seen that the MIC of DFC material remains almost unchanged with the increase of stimulation times, while the MIC of Car and VAN increase significantly after multiple stimulations, indicating that DFC can delay the progression of bacterial resistance.

[0064] Based on the above determination of the antibacterial activity of the prepared nanoparticles, their angiogenic effect was evaluated. Given that vascularization plays an important role in the rapid treatment of chronic infections, the effects of nanoparticles on angiogenesis were tested using ring formation and migration experiments. The results showed that DFC nanoparticles can significantly promote the in vitro ring formation effect ( Figure 7 ), indicating that it has a good ability to promote angiogenesis. Therefore, the present invention successfully prepared nanoparticles with antibacterial and regeneration-promoting effects.

[0065] In this embodiment, Fe ions and Car are first coordinated to realize in-situ nano-ization of antibiotics, and then DFO and Fe are coordinated to form DFC multifunctional nanomaterials. In vitro antibacterial experiments verify that DFC multifunctional nanomaterials have good anti-MRSA and MRSA biofilm experiments. Cell experiments verify that the material has good biocompatibility and angiogenesis-promoting effects.

[0066] The above are only preferred embodiments of the present application, and only specifically describe the technical principles of the present application. These descriptions are only for explaining the principles of the present application and cannot be interpreted as limiting the scope of protection of the present application in any way. Based on the explanation here, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application, and other specific implementation methods of the present application that can be associated with the technicians in this field without creative work, should be included in the scope of protection of the present application.

Claims

1. A method for preparing a multifunctional antibiotic nanomaterial, characterized in that: The steps include: The Fe ions and Car are coordinated once to form FC nanocomposites; DFO and the FC nanocomplex undergo secondary coordination to form DFC multifunctional antibiotic nanomaterials.

2. The method for preparing the multifunctional antibiotic nanomaterial according to claim 1, characterized in that: The step of coordinating Fe ions and Car to form FC nanocomposite comprises the following steps: The Fe ions and Car are stirred at room temperature to form Fe-Car nanoparticles, and then the Fe-Car nanoparticles are centrifugally washed and freeze-dried to obtain the FC nanocomposite.

3. The method for preparing the multifunctional antibiotic nanomaterial according to claim 2, characterized in that: The volume and molar concentration of the FeCl3.6H2O are the same as those of the Car.

4. The method for preparing the multifunctional antibiotic nanomaterial according to claim 2, characterized in that: The Fe ions include FeCl3·6H2O or Fe(NO3)3·9H2O or Fe2(SO4)3·xH2O.

5. The method for preparing the multifunctional antibiotic nanomaterial according to claim 3, characterized in that: The step of performing secondary coordination between DFO and the FC nanocomposite to form the DFC multifunctional antibiotic nanomaterial specifically includes the following steps: Dispersing the DFO and FC nanoparticles in distilled water, ultrasonically treating the nanoparticles, and stirring at room temperature to obtain nanoparticles; The nanoparticles are centrifuged, washed, and freeze-dried to obtain the DFC multifunctional antibiotic nanomaterial.

6. The method for preparing the multifunctional antibiotic nanomaterial according to claim 5, characterized in that: The volumes of the DFO and the Car are the same, and the molar ratio of the DFO and the FC nanoparticles is 1:

10.

7. A multifunctional antibiotic nanomaterial, characterized in that: The invention comprises the preparation method according to any one of claims 1 to 5.

8. Use of the multifunctional antibiotic nanomaterial as claimed in claim 7 in treating drug-resistant bacteria and / or eradicating biofilm.

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