A multifunctional antibiotic nanomaterial, preparation method and application thereof

Abstract

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 it 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 its potential angiogenic and bacterial targeting effects, DFO is modified on the iron ion-antibiotic nanocomplex to achieve a 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.

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 applications thereof. Background Art

[0002] Biofilm infections are highly prevalent in chronic wounds and can delay wound healing. Antibiotics are the primary treatment of choice for chronic wound infections. Unfortunately, the overuse of antibiotics has led to the spread of multidrug-resistant (MDR) bacteria, particularly multidrug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA). Vancomycin is currently the "last resort" for treating multidrug-resistant MRSA, but the inability of vancomycin to penetrate biofilms and the emergence of vancomycin-resistant Staphylococcus aureus mean that humanity's last line of defense against MDR infections is in jeopardy. However, the development of new antibiotics is both expensive and time-consuming. Therefore, redesigning existing antibiotics to maximize therapeutic efficacy while reducing drug resistance has become an important research direction at this stage.

[0003] Researchers have explored various strategies, such as modifying antibiotics with polymers through covalent bonds, encapsulating antibiotics in polymer matrices, and combining antibiotics with enzyme inhibitors. However, these approaches have limitations, such as cumbersome processes, inadequate antibiotic release, low targeting efficiency, and potential 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 in situ antibiotic 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 nanocomplexes;

[0008] DFO and the FC nanocomposite undergo secondary coordination to form DFC multifunctional nanomaterials.

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

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

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

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

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

[0014] Dispersing the DFO and FC nanocomplex in distilled water, performing ultrasonic treatment, and then stirring at room temperature to obtain nanoparticles;

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

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

[0017] The second purpose of this application is to provide a multifunctional antibiotic nanomaterial, which is 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 biofilms.

[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 it 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 its potential angiogenic and bacterial targeting effects, DFO is modified on the iron ion-antibiotic nanocomplex to achieve a 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 following briefly introduces the drawings required for use in the embodiments of the present application or the description of the prior art. 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 any creative work.

[0022] Figure 1 This is a 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) are the XRD, IR and XPS spectra of FC and DFC nanomaterials, respectively.

[0024] Figure 3 Schematic diagram of the antibacterial effects of different concentrations of nanomaterials on MRSA and the antibacterial effects 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 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 Schematic diagram of the DFC multifunctional nanomaterial provided in Example 1 of the present application in delaying the progression of MRSA drug 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] The following describes in detail embodiments of the present application, examples of which 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 this 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 this 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 this application.

[0031] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. Throughout the description of this application, "plurality" means two or more, unless otherwise specifically defined.

[0032] In order to make the purpose, technical solutions and advantages of this application more clear, this application is further described in detail below with reference to the accompanying drawings and embodiments.

[0033] The steps of the method for preparing the in-situ nanostructured antibiotic material provided in the embodiment of the present application include 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 an FC nanocomposite.

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

[0036] Fe ions and Car are stirred at room temperature to form Fe-Car nanoparticles, and then the Fe-Car nanoparticles are centrifuged, 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 its 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 it 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; centrifuging and washing the nanoparticles, and then 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 to 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 primary coordination to form an FC nanocomplex, and then perform secondary coordination with DFO and the FC nanocomplex 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 solutions of the present application are described in detail below with reference to 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 will also suffice). Note: Ferric chloride absorbs water strongly, so maintain a dry environment and weigh quickly. After weighing, seal the solution and store it in a desiccator.

[0049] b. Carbenicillin Solution (Car) (Concentration: 10mM): Weigh 2.001g of carbenicillin sodium and dissolve it in 500ml of ultrapure water (deionized water will also suffice). Note: This solution will lose its effectiveness over time and should not be used for more than one month.

[0050] c. Desferrioxamine sodium sulfonate (DFO) (1mM): Weigh 0.3284 g of DFO and dissolve it in 500 ml of ultrapure water (deionized water is also acceptable). Note: This solution will lose its effectiveness if stored for an extended period of time and should not be used for more than one month.

[0051] (2) Preparation of FC nanocomposites

[0052] a. Add 500 mL of 10 mM FeCl₃.6H₂O to 500 mL of 10 mM Car₂ solution and stir at room temperature for 24 hours at 400 rpm. Centrifuge the synthesized Fe-Car (FC) nanomaterial (10,000 rpm, 5 minutes) and wash five times with distilled water. Freeze at -20°C overnight and freeze dry for 48 hours to obtain the FC nanocomposite.

[0053] (3) Preparation of DFC multifunctional nanomaterials

[0054] a. Disperse 500 mL of 1 mM DFO and FC nanocomplex at a molar ratio of 1:10 in distilled water, sonicate for half an hour, and stir at room temperature for 3 hours. The nanoparticles were centrifuged (10,000 rpm, 5 minutes) and washed five times with distilled water. Freeze at -20°C overnight and freeze-dry 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 EDS results show that the DFC multifunctional nanomaterial is 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 nanomaterials have a peak at 2875 cm -1 and 1770cm -1The peaks at are characteristic peaks of DFO and Car. Figure 2 Middle c) proves that DFC multifunctional nanomaterials contain Fe, O, N, C, and S elements.

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

[0058] from Figure 3 As shown in Figure a, the antibacterial effect of the DFC multifunctional nanomaterial against MRSA becomes more pronounced as the concentration increases, reaching nearly 95% at 25µg / ml. Furthermore, when compared to the antibacterial efficacy of the FC nanocomposite and the same antibiotic concentration, the DFC multifunctional nanomaterial exhibits the best antibacterial effect, comparable to that of VAN.

[0059] The targeting effect of the nanomaterials on MRSA bacteria was further investigated. Rhodamine (RhB) was used to label DFC and FC nanomaterials to obtain RhB-DFC and RhB-FC nanomaterials. After co-culture with MRSA, their bacterial targeting effect was observed under confocal microscopy.

[0060] from Figure 4 It can be seen that the RhB-labeled nanomaterial is red, the SYTO-9-labeled MRSA is green, and after the bacteria are treated with DFC, the material and the 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 material and the 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 As can be seen, the control group and Car basically have 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 have red fluorescence penetrating through them, 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 diluted the nanomaterials and antibiotics in a gradient manner with the bacteria (5×10 5 Co-culture with the drug at a concentration of 1 / 2 the MIC (0.05 CFU / ml) for 24 hours. Measure the absorbance at OD600 for the bacterial strain. No bacterial growth is considered the MIC of the material. Subsequently, co-culture the bacteria at a concentration of 1 / 2 the MIC. Re-measure the MIC of the material one day after drug stimulation and update the MIC. Repeat 16 times.

[0063] from Figure 6 It can be seen that the MIC of DFC material remains almost unchanged with the increase in the number of stimulations, while the MICs 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 properties 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 ring formation effect in vitro ( Figure 7 ), indicating that it has a good ability to promote angiogenesis. Therefore, the present invention successfully prepared nanoparticles with antibacterial and pro-regenerative effects.

[0065] This example first utilizes Fe ions and Car for primary coordination to achieve in situ nanofiberization of antibiotics. DFO and Fe then undergo secondary coordination to form a DFC multifunctional nanomaterial. In vitro antibacterial tests validated the DFC multifunctional nanomaterial's ability to resist MRSA and MRSA biofilms. Cell-based experiments also demonstrated the material's excellent biocompatibility and pro-angiogenic properties.

[0066] The above are merely preferred embodiments of the present application and only specifically describe the technical principles of the present application. These descriptions are intended only to explain the principles of the present application and should not be construed in any way as limiting the scope of protection of the present application. Based on the explanations herein, any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present application, as well as other specific implementations of the present application that can be conceived by those skilled in the art without inventive effort, shall be included within 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 ion and Car are coordinated once to form an FC nanocomposite, wherein the Car is carbenicillin; The DFO and the FC nanocomplex are subjected to secondary coordination to form a DFC multifunctional antibiotic nanomaterial; the DFO is sodium deferoxamine sulfonate; The Fe ions include FeCl3·6H2O or Fe(NO3)3·9H2O or Fe2(SO4)3·xH2O; The volumes of the DFO and the Car are the same, and the molar ratio of the DFO to the FC nanocomposite is 1:

10.

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

3. The method for preparing the multifunctional antibiotic nanomaterial according to claim 2, wherein: 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 3, wherein: The step of performing secondary coordination between DFO and the FC nanocomplex to form the DFC multifunctional antibiotic nanomaterial specifically includes the following steps: Dispersing the DFO and FC nanocomplex in distilled water, performing ultrasonic treatment, and then stirring at room temperature to obtain nanoparticles; The nanoparticles are centrifuged, washed, and then freeze-dried to obtain the DFC multifunctional antibiotic nanomaterial.

5. A multifunctional antibiotic nanomaterial, characterized in that: The invention relates to a method for preparing the invention according to any one of claims 1 to 4.

6. Use of the multifunctional antibiotic nanomaterial according to claim 5 in the preparation of drugs for treating drug-resistant bacteria.

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