Preparation and application of targeted magnetic composite nanomaterial with multiple sterilization functions
By preparing targeted magnetic composite nanomaterials and combining them with photothermal and photodynamic therapy, the problems of dispersion and penetration of existing antibacterial materials in the treatment of bacterial infections after mechanical trauma and burns have been solved, achieving efficient removal of bacteria and bioimaging, which has broad prospects for biomedical applications.
Patent Information
- Application Number
- CN202310810884.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-04
- Publication Date
- 2026-01-09
- Estimated Expiration
- 2043-07-04
AI Technical Summary
Existing antibacterial materials, when used to treat mechanical trauma and bacterial infections following burns, suffer from poor dispersibility, inability to completely eliminate bacteria and endotoxins, leading to inflammatory reactions. Furthermore, conventional photothermal and photodynamic therapies have limited range of action and cannot effectively penetrate deep into biological membranes.
A targeted magnetic composite nanomaterial was prepared by loading a multifunctional photosensitive material onto a magnetic material using a one-pot solvothermal method and encapsulating it with a positively charged polymer. This process enables targeted binding to bacteria, catalyzing the production of oxygen from hydrogen peroxide, activating dormant bacteria, and generating thermotherapy and ROS sterilization by combining with near-infrared light.
It achieves targeted sterilization of bacteria, can penetrate deep into biofilms to eliminate bacteria, reduce inflammation caused by endotoxins, has good biocompatibility and multiple bactericidal properties, avoids drug resistance, and is suitable for bioimaging and antibacterial fields.
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Figure CN116870151B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biological nanomaterials, and particularly relates to preparation and application of a targeted magnetic composite nanomaterial with multiple sterilization functions. BACKGROUND
[0002] Mechanical trauma and burn injury are facing serious wound bacterial infection, which seriously endangers people's quality of life and health. However, the current treatment of bacterial infection mainly relies on antibiotics, leading to frequent misuse of antibiotics and the emergence of multiple drug-resistant bacteria, which greatly reduces the treatment effect. Therefore, it is necessary to develop an antibacterial means with fast effect and low side effects. Among them, emerging treatment methods such as photothermal therapy (PTT) and photodynamic therapy (PDT) are widely reported due to their low toxicity and non-resistance.
[0003] In photothermal therapy, the antibacterial agent can increase its temperature under near-infrared light irradiation. This temperature rise can effectively kill bacteria through various thermal effects, including cell membrane rupture, cell emptying, protein / enzyme denaturation, and liquid evaporation. However, high temperature or long-term exposure to high temperature may cause thermal damage to healthy tissues around the microorganism, and even trigger new inflammation. Therefore, combining photothermal therapy with other treatment methods can effectively avoid damage to normal tissues.
[0004] Photodynamic therapy is an emerging treatment method that destroys bacterial cell membranes, DNA, proteins, etc. through active oxygen (ROS) generated by laser irradiation of photosensitizers. Then, excessive ROS may trigger unnecessary inflammatory reactions and hinder wound healing. Therefore, it is urgent to develop a multifunctional material that has multiple antibacterial strategies. Fortunately, carbon dots, metals and metal oxides, organic molecules, etc. materials with multiple antibacterial properties have been developed and widely used, but their poor dispersibility and other weaknesses still limit their application.
[0005] The development of biofilm makes it difficult to completely eliminate bacteria. Bacterial biofilm can achieve drug resistance to antibiotics through a series of mechanisms such as drug efflux, extracellular matrix blockage, dormancy effect, group effect, etc., prevent the release of antibacterial drugs, reduce the treatment effect, prolong the disease, and greatly reduce the effect of common antibacterial nanomaterials. More importantly, bacteria usually release endotoxins after death, which further cause adverse reactions such as inflammation and even shock, so killing and completely removing bacteria is an important development direction in the future. Traditional photothermal therapy, photodynamic therapy, etc. can only kill bacteria through hyperthermia and oxidative stress, but cannot completely remove the inner wall bacteria and dead bacteria, and cannot completely avoid the inflammatory response caused by residual bacteria and endotoxins, delaying the healing speed of the wound. In addition, due to the influence of material dispersion, the range of photothermal therapy and photodynamic therapy is very limited, so it is necessary to develop an antibacterial material with bacterial targeting ability, strong penetration and the ability to activate internal bacteria.
[0006] In recent years, magnetic materials have attracted attention due to their controllable movement under an external magnetic field. Under the intervention of an external magnetic field, magnetic materials can penetrate the deep layer of a biofilm and destroy its dense structure. More importantly, the modification of magnetic materials with positive charges can make full use of electrostatic interaction to combine with bacteria. This targeted magnetic composite nanomaterial with multiple properties has great application prospects. SUMMARY
[0007] In view of the limitations of existing antibacterial materials, the present application designs and prepares a targeted magnetic composite nanomaterial with multiple sterilization functions. The preparation method of the material is simple and does not require expensive costs. The multiple sterilization function targeted magnetic composite nanomaterial prepared has good antibacterial performance and is removable, has good biocompatibility, and can be used in various fields such as antibacterial and biological imaging.
[0008] In order to achieve the above-mentioned application purposes, the application adopts the following technical solutions:
[0009] The first object of the present application is to provide a preparation method of a targeted magnetic composite nanomaterial with multiple sterilization functions, comprising the following steps:
[0010] Step 1: Dissolve the precursor in deionized water, stir to uniformly disperse, and react under heating and pressurization; after the reaction is completed, cool and filter, collect the filtrate, dry, and obtain the multifunctional photosensitive material, which is stored in vacuum;
[0011] Step 2: Dissolve the iron source in ethylene glycol, add anhydrous acetic acid and the multifunctional photosensitive material obtained in step 1, and react under heating and pressurization; after the reaction is completed, cool, wash repeatedly with distilled water and anhydrous ethanol, collect the reaction system using an external magnetic field to obtain the magnetic composite nanomaterial in the reaction system, and dry; the magnetic composite nanomaterial is stored in vacuum.
[0012] Step 3: Dissolve the positively charged polymer in an acetic acid solution, stir until uniform, add the magnetic composite nanomaterial prepared in step 2, stir, and add a sodium hydroxide solution to the mixture until the pH value reaches 7; wash with deionized water, collect the reaction system using an external magnetic field to obtain the targeted magnetic composite nanomaterial, and store the targeted magnetic composite nanomaterial in vacuum.
[0013] Further, the precursor in step 1 is one or more of an antibiotic, an organic substance that is easy to carbonize, a quaternary ammonium salt, a polyamine compound, and a silver ion.
[0014] Further, the multifunctional photosensitive material in step 1 is one or more of a carbon dot, a metal and a metal oxide, and an organic substance.
[0015] As preferred, the dissolving of the precursor in deionized water and the stirring to uniformly disperse the precursor are adding the precursor to water and fully dissolving the precursor in the deionized water by stirring, ultrasonicating, heating, or the like.
[0016] Further, in step 1, the ratio of the precursor to the deionized water is 5-20 mL of deionized water for every 5-50 mg of precursor; the reaction under heating and pressurization is performed at 80-150℃, 0.1-8 MPa, and for 2-8 h; and the filtration is performed using a 0.22 μm filter head.
[0017] Further, step 1 further includes removing unreacted precursors before drying, and preferably, centrifuging the filtrate or dialyzing the filtrate in deionized water to remove the unreacted precursors.
[0018] In a certain specific embodiment, the drying is selected from heating and drying or vacuum freeze-drying.
[0019] As preferred, the dialysis is performed using a dialysis bag with a MWCO of 500-2000 Da.
[0020] The freeze-drying temperature is -60 to -50℃, the vacuum degree is 9-10 Pa, and the processing time is 20-24 h.
[0021] Further, in step 2, the iron source is one or more of ferric chloride, ferric sulfate, ferric nitrate, and potassium ferricyanide.
[0022] Further, in step 2, the ratio of the iron source, ethylene glycol, anhydrous sodium acetate, and the multifunctional photosensitive material is 5-40 mL of ethylene glycol for dissolving 0.2-0.6 g of the iron source, then 1-4 g of anhydrous sodium acetate and 0.01-0.06 g of the multifunctional photosensitive material are added; the heating and pressurizing conditions are 160-220℃, 0.1-8 MPa, and 4-12 h, and the drying is performed at 40-70℃ for 1-4 h.
[0023] Preferably, the iron source is dissolved in ethylene glycol, and then anhydrous sodium acetate and the multifunctional photosensitive material are added, which is to add the iron source into ethylene glycol, and the iron source is fully dispersed in the ethylene glycol by stirring, ultrasonic, heating, or the like, and then the anhydrous sodium acetate and the multifunctional photosensitive material are fully dispersed in the above solution under stirring.
[0024] The external magnetic field is a material that can adsorb magnetic substances, such as a permanent magnet or a charged coil.
[0025] Further, in step 3, the positively charged polymer is selected from a material that can adsorb bacteria by electrostatic action, and preferably, the positively charged polymer is selected from one or a combination of chitosan and quaternary ammonium salt.
[0026] Further, in step 3, the ratio of the positively charged polymer, acetic acid, the magnetic composite nanomaterial, and sodium hydroxide is 20-60 mL of a 0.5-2% volume percentage concentration of acetic acid solution for dissolving 0.1-0.5 g of the positively charged polymer, 1.0-4.0 g of the magnetic composite nanomaterial is added, and a sodium hydroxide solution is added to the mixture until the pH is 7, and preferably, the concentration of the sodium hydroxide solution is 1M, and the stirring time is 20-40 minutes.
[0027] The second object of the present application is to provide a targeted magnetic composite nanomaterial with multiple sterilization functions prepared by the preparation method.
[0028] Preferably, the targeted magnetic composite nanomaterial with multiple sterilization functions has an average particle size of 300-500 nm.
[0029] The targeted magnetic composite nanomaterial with multiple sterilization functions has absorption in the near-infrared region, can bind and target bacteria by electrostatic action, and can catalyze hydrogen peroxide to produce oxygen and move in an orderly manner under the action of an external magnetic field.
[0030] The third object of the present application is to provide the use of the targeted magnetic composite nanomaterial with multiple sterilization functions in the preparation of bacteria-targeted bacterial fluorescent markers and / or antibacterial agents.
[0031] Further, the bacterial-targeted bacterial fluorescent label can show green, blue and red fluorescence under 405, 488 and 543 nm wavelength excitation; the antibacterial agent can kill bacteria after 808 nm laser irradiation; the bacteria are gram-positive bacteria and / or gram-negative bacteria. In the application of the targeted magnetic composite nanomaterial with multiple sterilization functions in bacterial fluorescent labeling, the specific process is as follows: the prepared targeted magnetic composite nanomaterial with multiple sterilization functions is added to bacterial liquid in a good growth state, and cultured in a 37℃ CO2 incubator; the bacterial fluorescence state is observed under 405, 488 and 543 nm excitation using a laser confocal fluorescence microscope, and photographed.
[0032] In the application of the targeted magnetic composite nanomaterial with multiple sterilization functions in antibacterial application, the specific process is as follows: gram-positive bacteria Staphylococcus aureus (S. aureus) and gram-negative bacteria Escherichia coli (E. coli) are used for colony plate experiments. For example, the targeted magnetic composite nanomaterial with multiple sterilization functions is added to bacterial liquid of S. aureus and E. coli respectively, 1.5 W / cm2 808 nm laser irradiation is applied to the mixture for 10 minutes, an appropriate amount of bacterial liquid is coated on the surface of solid culture medium, cultured in a shaking box for 12-24 hours, and then photographed; and an appropriate amount of bacterial liquid is added to the surface of a polysilicon wafer, and the change of the bacterial membrane is observed by scanning electron microscopy (SEM). -2 The multifunctional photosensitive material is used as raw material, a one-pot solvothermal method is used to combine with magnetic substances to form a magnetic composite nanomaterial, and then a positively charged polymer is used to wrap it, so that it has multiple properties.
[0033] Firstly, the external positively charged polymer can target and combine bacteria through electrostatic action, and cause physical damage to the bacterial cell membrane through its long carbon chain structure; secondly, the targeted magnetic composite nanomaterial with multiple sterilization functions can move orderly and controllably under the action of an external magnetic field, can easily penetrate the extracellular matrix of the bacterial membrane and break the internal dense structure, has a higher bacterial load removal capacity than conventional sterilization materials, and effectively reduces inflammation and other adverse reactions caused by endotoxins from dead bacteria; in addition, the targeted magnetic composite nanomaterial with multiple sterilization functions can catalyze hydrogen peroxide to produce oxygen, relieve the hypoxic environment in the deep layer of the bacterial biofilm, activate dormant bacteria to make them sensitive to external stimuli; finally, the multifunctional photosensitive material can widely absorb near-infrared light to produce local hyperthermia and reactive oxygen species (ROS), cause damage to the cell membrane of bacteria, and cause leakage of the contents and damage to DNA, proteins and the like.
[0034] The application of the E. coli and Staphylococcus aureus in the field of bioimaging is explored, and it is found that the targeted magnetic composite nanomaterial with multiple sterilization functions can be gathered at the site of bacterial colonization, and green, blue and red fluorescence can be shown under excitation at 405, 488 and 543 nm, which shows excellent bacterial co-localization and bioimaging ability. Then the application of the E. coli and Staphylococcus aureus in the field of antibiosis is explored, and compared with other antibacterial materials, the targeted magnetic composite nanomaterial with multiple sterilization functions has the ability of targeting bacteria, removing bacterial biofilm, producing oxygen and specific phototherapy sterilization, and has outstanding sterilization performance and does not cause cytotoxicity problem, and can realize multiple sterilization in the field of biological medicine and can be used for bioimaging. The targeted magnetic composite nanomaterial with multiple sterilization functions has good biocompatibility, excellent sterilization ability and does not cause bacterial resistance and other problems, and therefore has a broad application prospect in the fields of bioimaging and antibiosis in the future.
[0035] Compared with the prior art, the application has the following advantages:
[0036] (1) The targeted magnetic composite nanomaterial with multiple sterilization functions prepared by the application is loaded with multifunctional photosensitive materials on the magnetic material through one-pot solvothermal method, and the experimental scheme is simple and convenient for synthesis.
[0037] (2) The targeted magnetic composite nanomaterial with multiple sterilization functions prepared by the application has good biocompatibility and low toxicity, and has great development prospect in the field of biological medicine.
[0038] (3) The targeted magnetic composite nanomaterial with multiple sterilization functions prepared by the application has excellent fluorescence performance and excitation wavelength dependence, and can realize multicolor imaging in the field of bioimaging.
[0039] (4) The targeted magnetic composite nanomaterial with multiple sterilization functions prepared by the application shows multiple sterilization performances such as targeted combination of bacteria, removal of bacterial biofilm, oxygen production and precise phototherapy, and has excellent sterilization effect and does not cause drug resistance.
[0040] Therefore, the photosensitive material with multiple antibacterial performances is combined with the magnetic material and modified to prepare a targeted magnetic composite nanomaterial with multiple sterilization functions. The material has great development prospect and research value in the fields of bioimaging and antibiosis. BRIEF DESCRIPTION OF DRAWINGS
[0041] Figure 1 is a scanning electron microscope image of the targeted magnetic composite nanomaterial with multiple sterilization functions;
[0042] Figure 2This is a Zeta potential diagram of a targeted magnetic composite nanomaterial with multiple bactericidal functions;
[0043] Figure 3 This is the ultraviolet-visible spectrum of a targeted magnetic composite nanomaterial with multiple bactericidal functions;
[0044] Figure 4 This is a temperature change diagram of a targeted magnetic composite nanomaterial with multiple sterilization functions after being irradiated by an 808nm laser.
[0045] Figure 5 The image shows the UV-Vis spectrum of the ROS detection probe diphenylbenzofuran (DPBF) after the targeted magnetic composite nanomaterial with multiple bactericidal functions is irradiated by an 808nm laser.
[0046] Figure 6 The image shows a targeted magnetic composite nanomaterial with multiple bactericidal functions after being co-cultured with hydrogen peroxide.
[0047] Figure 7 This is a graph showing the colony counts of Escherichia coli and Staphylococcus aureus in a targeted magnetic composite nanomaterial with multiple bactericidal functions after irradiation with an 808nm laser.
[0048] Figure 8 These are SEM images of targeted magnetic composite nanomaterials with multiple bactericidal functions treating bacteria under different conditions.
[0049] Figure 9 This is a bacterial imaging image of a targeted magnetic composite nanomaterial with multiple bactericidal functions at 488nm and 543nm.
[0050] Figure 10 This is a graph showing the cytotoxicity of targeted magnetic composite nanomaterials with multiple bactericidal functions on L929 cells under near-infrared light irradiation and without. Detailed Implementation
[0051] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance thereof are within the scope of the present invention. Experimental methods and reagents not specified in the embodiments are performed according to conventional conditions in the art.
[0052] Example 1
[0053] Preparation of targeted magnetic composite nanomaterials with multiple bactericidal functions
[0054] Step 1, 30 mg of indocyanine green (an organic substance that is easy to carbonize) was weighed into a clean and dry beaker, 10 mL of deionized water was added, and it was stirred at a speed of 400 rpm for 2 hours to make it fully dissolved, then the mixture was transferred to a 50 mL Teflon high-pressure reaction kettle, and reacted at 105°C, 2 MPa for 4h. After cooling, a dark green mixture was obtained, which was filtered with a 0.22 μm filter head, and the filtrate was transferred to a dialysis bag (MWCO = 1000 Da) and dialyzed in deionized water for 3 days. Finally, the dispersion in the dialysis bag was stored at -20°C and freeze-dried to obtain a dark green powder, i.e. carbon dots (multifunctional photosensitive material), which was stored in a vacuum tank.
[0055] Step 2, 0.5 g of iron chloride was dissolved in 30 mL of ethylene glycol to form a uniform transparent solution. Then 2 g of anhydrous sodium acetate and 0.06 g of carbon dots were added, and the mixture was thoroughly mixed under magnetic stirring for 30 min to obtain a uniform green solution. The mixture was transferred to a Teflon high-pressure reaction kettle and reacted at 200°C, 2 MPa for 8h. After cooling, it was washed with distilled water and anhydrous ethanol under ultrasonic for 3 times respectively, and the product was collected by external magnetic field and dried at 50°C for 4h and stored in a vacuum tank.
[0056] Step 3, 0.2 g of chitosan was dissolved in 50 mL of acetic acid solution (2%, V / V), and mechanically stirred to mix uniformly. Then 3.0 g of magnetic composite nanomaterials were added, and stirred at a speed of 800 rpm at room temperature for 30 min. 50 mL of 1M sodium hydroxide solution was added to the mixture to adjust the pH to 7, and then washed with deionized water. Finally, the product was collected by external magnetic field and dried at 50°C for 12h and stored in a vacuum tank.
[0057] Example 2
[0058] Preparation of targeted magnetic composite nanomaterials with multiple sterilization functions
[0059] Step 1, 30 mg of indocyanine green (an organic substance that is easy to carbonize) was weighed into a clean and dry beaker, 10 mL of deionized water was added, and it was stirred at a speed of 400 rpm for 2 hours to make it fully dissolved, then the mixture was transferred to a 50 mL Teflon high-pressure reaction kettle, and reacted at 105°C, 2 MPa for 4h. After cooling, a dark green mixture was obtained, which was filtered with a 0.22 μm filter head, and the filtrate was transferred to a dialysis bag (MWCO = 1000 Da) and dialyzed in deionized water for 3 days. Finally, the dispersion in the dialysis bag was stored at -20°C and freeze-dried to obtain a dark green powder, i.e. carbon dots (multifunctional photosensitive material), which was stored in a vacuum tank.
[0060] Step 2, 0.5 g of FeCl3 was dissolved in 30 mL of ethylene glycol to form a uniform transparent solution. Then 2 g of anhydrous sodium acetate and 0.01 g of carbon dots were added, and the mixture was mixed well under magnetic stirring for 30 min to obtain a uniform green solution. The mixture was transferred to a Teflon high-pressure reaction kettle and reacted at 200 °C and 2 MPa for 8 h. After cooling, it was washed with distilled water and anhydrous ethanol under ultrasonic for 3 times respectively, and the product was collected by external magnetic field, dried at 50 °C for 1 h and stored in a vacuum tank.
[0061] Step 3, 0.2 g of chitosan was dissolved in 50 mL of acetic acid solution (2%, V / V), and mechanically stirred to mix well. Then 3.0 g of magnetic composite nanomaterials were added, and the mixture was stirred at 800 rpm and room temperature for 30 min. 50 mL of 1M NaOH solution was added to adjust the pH to 7, and then washed with deionized water. Finally, the product was collected by external magnetic field, dried at 50 °C for 12 h and stored in a vacuum tank.
[0062] Example 3
[0063] Preparation of targeted magnetic composite nanomaterials with multiple sterilization functions
[0064] Step 1, 30 mg of indocyanine green was weighed in a clean and dry beaker, 10 mL of deionized water was added, and the mixture was stirred at 400 rpm for 2 hours to dissolve completely. Then the mixture was transferred to a 50 mL Teflon high-pressure reaction kettle, and reacted at 105 °C and 2 MPa for 4 h. After cooling, a dark green mixture was obtained, which was filtered with a 0.22 μm filter head. The filtrate was transferred to a dialysis bag (MWCO = 1000 Da) and dialyzed in deionized water for 3 days. Finally, the dispersion in the dialysis bag was stored at -20 °C and freeze-dried to obtain a dark green powder, which was carbon dots, and stored in a vacuum tank.
[0065] Step 2, 0.5 g of FeCl3 was dissolved in 30 mL of ethylene glycol to form a uniform transparent solution. Then 2 g of anhydrous sodium acetate and 0.02 g of carbon dots were added, and the mixture was mixed well under magnetic stirring for 30 min to obtain a uniform green solution. The mixture was transferred to a Teflon high-pressure reaction kettle and reacted at 200 °C and 2 MPa for 8 h. After cooling, it was washed with distilled water and anhydrous ethanol under ultrasonic for 3 times respectively, and the product was collected by external magnetic field, dried at 50 °C for 2 h and stored in a vacuum tank.
[0066] Step 3, 0.3 g of chitosan was weighed into 50 mL of acetic acid solution (2%, V / V), and mechanically stirred to mix evenly. Then 3.0 g of magnetic composite nanomaterials was added, and stirred at 800 rpm for 30 min at room temperature. 50 mL of 1 M sodium hydroxide solution was added to the mixture to adjust the pH to 7, and then washed with deionized water. Finally, the product was collected by an external magnetic field, dried at 50°C for 12 h, and stored in a vacuum tank.
[0067] Example 4
[0068] Preparation of targeted magnetic composite nanomaterials with multiple sterilization functions
[0069] Step 1, 30 mg of urea (polyamine compound) was weighed into a clean and dry beaker, 10 mL of deionized water was added, and stirred at 400 rpm for 2 hours to fully dissolve. Then the mixture was transferred to a 50 mL Teflon high-pressure reactor, and reacted at 105°C and 2 MPa for 4 h. After cooling, it was filtered with a 0.22 μm filter head, and the filtrate was transferred to a dialysis bag (MWCO = 1000 Da) and dialyzed in deionized water for 3 days. Finally, the dispersion in the dialysis bag was stored at -20°C and freeze-dried to obtain a powder, i.e. carbon dots, which were stored in a vacuum tank.
[0070] Step 2, 0.5 g of iron chloride was dissolved in 30 mL of ethylene glycol to form a uniform transparent solution. Then 2 g of anhydrous sodium acetate and 0.03 g of carbon dots were added, and the mixture was mixed thoroughly under magnetic stirring for 30 min. The mixture was transferred to a Teflon high-pressure reactor, and reacted at 200°C and 2 MPa for 8 h. After cooling, it was washed with distilled water and anhydrous ethanol under ultrasonic for 3 times, and the product was collected by an external magnetic field, dried at 50°C for 3 h, and stored in a vacuum tank.
[0071] Step 3, 0.3 g of chitosan was weighed into 50 mL of acetic acid solution (2%, V / V), and mechanically stirred to mix evenly. Then 3.0 g of magnetic composite nanomaterials was added, and stirred at 800 rpm for 30 min at room temperature. 50 mL of 1 M sodium hydroxide solution was added to the mixture to adjust the pH to 7, and then washed with deionized water. Finally, the product was collected by an external magnetic field, dried at 50°C for 12 h, and stored in a vacuum tank.
[0072] Example 5
[0073] Preparation of targeted magnetic composite nanomaterials with multiple sterilization functions
[0074] Step 1, 30 mg of penicillin G sodium salt (antibiotic) was weighed into a clean and dry beaker, 10 mL of deionized water was added, and it was stirred at 400 rpm for 2 hours to fully dissolve, then the mixture was transferred to a 50 mL Teflon high-pressure reactor, and reacted at 105°C, 2 MPa for 4h. After cooling, it was filtered with a 0.22 μm filter head, and the filtrate was transferred to a dialysis bag (MWCO = 1000 Da) and dialyzed in deionized water for 3 days. Finally, the dispersion in the dialysis bag was stored at -20°C and freeze-dried to obtain a powder, i.e. carbon dots, which were stored in a vacuum tank.
[0075] Step 2, 0.5 g of iron chloride was weighed and dissolved in 30 mL of ethylene glycol to form a uniform transparent solution. Then 2 g of anhydrous sodium acetate and 0.04 g of carbon dots were added, and the mixture was mixed well under magnetic stirring for 30 min. The mixture was transferred to a Teflon high-pressure reactor and reacted at 200°C, 2 MPa for 8h. After cooling, it was washed with distilled water and anhydrous ethanol under ultrasonic for 3 times respectively, and the product was collected by external magnetic field, dried at 50°C for 4h and stored in a vacuum tank.
[0076] Step 3, 0.2 g of chitosan was dissolved in 50 mL of acetic acid solution (2%, V / V), and mechanically stirred to mix well. Then 3.0 g of magnetic composite nanomaterials were added, and stirred at 800 rpm at room temperature for 30 min. 50 mL of 1M sodium hydroxide solution was added to the mixture to adjust the pH to 7, and then washed with deionized water. Finally, the product was collected by external magnetic field, dried at 50°C for 12h and stored in a vacuum tank.
[0077] Example 6
[0078] Preparation of targeted magnetic composite nanomaterials with multiple sterilization functions
[0079] Step 1, 30 mg of penicillin G sodium salt (antibiotic) was weighed into a clean and dry beaker, 10 mL of deionized water was added, and it was stirred at 400 rpm for 2 hours to fully dissolve, then the mixture was transferred to a 50 mL Teflon high-pressure reactor, and reacted at 105°C, 2 MPa for 4h. After cooling, it was filtered with a 0.22 μm filter head, and the filtrate was transferred to a dialysis bag (MWCO = 1000 Da) and dialyzed in deionized water for 3 days. Finally, the dispersion in the dialysis bag was stored at -20°C and freeze-dried to obtain a powder, i.e. carbon dots, which were stored in a vacuum tank.
[0080] Step 2, 0.5 g of ferric chloride was weighed and dissolved in 30 mL of ethylene glycol to form a uniform transparent solution. Then 2 g of anhydrous sodium acetate and 0.04 g of carbon dots were added, and the mixture was mixed well under magnetic stirring for 30 min. The mixture was transferred to a Teflon high-pressure reaction kettle and reacted at 200 °C and 2 MPa for 8 h. After cooling, it was washed with distilled water and anhydrous ethanol under ultrasonic for 3 times respectively, and the product was collected by external magnetic field, dried at 50 °C for 4 h and stored in a vacuum tank.
[0081] Step 3, 0.2 g of chitosan was weighed and dissolved in 50 mL of acetic acid solution (2%, V / V), and mechanically stirred to mix well. Then 3.0 g of magnetic composite nanomaterials were added, and the mixture was stirred at 800 rpm at room temperature for 30 min. 50 mL of 1M sodium hydroxide solution was added to adjust the pH to 7, and then washed with deionized water. Finally, the product was collected by external magnetic field, dried at 50 °C for 12 h and stored in a vacuum tank.
[0082] Example 7
[0083] Preparation of targeted magnetic composite nanomaterials with multiple sterilization functions
[0084] Step 1, 30 mg of silver diethyldithiocarbamate (silver ion) was weighed in a clean and dry beaker, 10 mL of deionized water was added, and stirred at a speed of 400 rpm for 2 hours to dissolve completely. Then the mixture was transferred to a 50 mL Teflon high-pressure reaction kettle, and reacted at 105 °C and 2 MPa for 4 h. After cooling, it was filtered with a 0.22 μm filter head, and the filtrate was transferred to a dialysis bag (MWCO = 1000 Da) and dialyzed in deionized water for 3 days. Finally, the dispersion in the dialysis bag was stored at -20 °C and freeze-dried to obtain a powder, i.e. carbon dots, which were stored in a vacuum tank.
[0085] Step 2, 0.5 g of ferric chloride was weighed and dissolved in 30 mL of ethylene glycol to form a uniform transparent solution. Then 2 g of anhydrous sodium acetate and 0.04 g of carbon dots were added, and the mixture was mixed well under magnetic stirring for 30 min. The mixture was transferred to a Teflon high-pressure reaction kettle and reacted at 200 °C and 2 MPa for 8 h. After cooling, it was washed with distilled water and anhydrous ethanol under ultrasonic for 3 times respectively, and the product was collected by external magnetic field, dried at 50 °C for 4 h and stored in a vacuum tank.
[0086] Step 3, 0.2 g of chitosan was weighed into 50 mL of acetic acid solution (2%, V / V), and mechanically stirred to mix well. Then 3.0 g of magnetic composite nanomaterials was added, and stirred at 800 rpm for 30 min at room temperature. 50 mL of 1 M sodium hydroxide solution was added to the mixture to adjust the pH to 7, and then washed with deionized water. Finally, the product was collected by an external magnetic field, dried at 50 °C for 12 h, and stored in a vacuum tank.
[0087] Experimental Example 1
[0088] The scanning electron microscope image of the targeted magnetic composite nanomaterial with multiple sterilization functions prepared in Example 1 is shown in FIG. 1, which shows that the targeted magnetic composite nanomaterial with multiple sterilization functions is successfully prepared, and the particle size is about 350 nm. Figure 1
[0089] The zeta potential image of the targeted magnetic composite nanomaterial with multiple sterilization functions prepared in Example 1 is shown in FIG. 2, which shows that the charge potential of the targeted magnetic composite nanomaterial with multiple sterilization functions is 22.77 mV, which is positively charged. Figure 2
[0090] The ultraviolet-visible spectrum of the targeted magnetic composite nanomaterial with multiple sterilization functions prepared in Example 1 is shown in FIG. 3, which shows that the targeted magnetic composite nanomaterial with multiple sterilization functions has a wide absorption in the near-infrared region. Figure 3
[0091] The photothermal performance experiment of the targeted magnetic composite nanomaterial prepared in Example 1: different concentrations of aqueous solution of the targeted magnetic composite nanomaterial with multiple sterilization functions were prepared, and the concentrations were 0, 25, 50, 100, 200, and 400 μg / mL, respectively. The solution was irradiated under 808 nm laser at 1.5 W / cm 2 for 10 min, and the temperature at different times was recorded by an infrared thermal imager. The results show that the temperature change diagram of the targeted magnetic composite nanomaterial with multiple sterilization functions prepared in Example 1 after 808 nm laser irradiation is shown in FIG. 4, which shows that the temperature of the targeted magnetic composite nanomaterial with multiple sterilization functions at a concentration of 400 μg / mL can be increased to 54.1 °C under 808 nm laser irradiation for 10 min, which is significantly higher than the control group, and the photothermal conversion efficiency is about 35.5%. Figure 4
[0092] The photodynamic performance experiment of the targeted magnetic composite nanomaterial prepared in Example 1: an ethanol solution of the targeted magnetic composite nanomaterial with a concentration of 400 μg / mL was prepared, and 50 μL of ROS detection probe diphenyl benzofuran (DPBF) with a concentration of 1 mM was added to the solution. The solution was irradiated under 808 nm laser (1.5 W / cm 2 for 10 min, and the fluorescence intensity of the solution was measured by a fluorescence spectrophotometer. The results show that the fluorescence intensity of the solution is reduced, which indicates that the targeted magnetic composite nanomaterial with multiple sterilization functions has a certain photodynamic effect.2 ) After continuous irradiation, the absorbance at 410 nm was detected every 2 min. The results showed that the UV-Vis spectrum of the ROS detection probe DPBF after the preparation of the targeted magnetic composite nanomaterial with multiple sterilization functions in Example 1 was irradiated by 808 nm laser, as shown in Figure 5 , which had a special absorption at 410 nm, and the absorption intensity decreased once oxidized. The figure showed that the absorption intensity of DPBF gradually decreased with the increase of irradiation time after 808 nm laser irradiation, indicating that the targeted magnetic composite nanomaterial with multiple sterilization functions had excellent photodynamic effect.
[0093] Oxygen production performance experiment of the targeted magnetic composite nanomaterial prepared in Example 1: The solutions of carbon dots, magnetic composite nanomaterial prepared in Example 1, step 2, and targeted magnetic composite nanomaterial with a concentration of 400 μg / mL were prepared respectively, and the concentration of hydrogen peroxide in each group was set to 1 M. The number of bubbles in the centrifuge tube at different times was recorded by taking pictures. The results showed that the pictures of carbon dots, magnetic composite nanomaterial, and targeted magnetic composite nanomaterial after co-culturing with hydrogen peroxide were as shown in Figure 6 , which showed that carbon dots, magnetic composite nanomaterial, and targeted magnetic composite nanomaterial all produced obvious bubbles after 50 min of co-incubation, proving that carbon dots, magnetic composite nanomaterial, and targeted magnetic composite nanomaterial could all catalyze hydrogen peroxide to produce oxygen.
[0094] The number of E. coli and S. aureus colonies after the preparation of the targeted magnetic composite nanomaterial with multiple sterilization functions in Example 1 was irradiated by 808 nm laser, as shown in Figure 7 , which showed that the targeted magnetic composite nanomaterial with multiple sterilization functions could effectively inhibit the growth of bacteria after 808 nm laser irradiation, indicating that the targeted magnetic composite nanomaterial with multiple sterilization functions had excellent multiple sterilization performance.
[0095] Bactericidal experiment of the targeted magnetic composite nanomaterial with multiple sterilization functions prepared in Example 1: The experiment was divided into 7 groups, in which the concentration of hydrogen peroxide was 4 mM, and NIR was 808 nm laser irradiation for 10 min. The targeted magnetic composite nanomaterial with multiple sterilization functions with a concentration of 400 μg / mL was co-incubated with bacteria for 2 hours, then the bacteria were washed with PBS, fixed with 2.5% glutaraldehyde aqueous solution for 12 hours, dehydrated with 10%, 30%, 50%, 70%, and 90% ethanol aqueous solution, and finally dispersed in anhydrous ethanol and dropped on a single crystal silicon wafer for observation under a scanning electron microscope. The SEM images of the bacteria treated under different conditions were as shown in Figure 8As shown in the figure, the results show that pure hydrogen peroxide, 808nm light and the targeted magnetic composite nanomaterials have no obvious damage to the bacterial cell membrane, while the bacterial cell membrane surface of the targeted magnetic composite nanomaterials+NIR group appears to shrink and break, which can cause the leakage of bacterial contents and kill the bacteria. The membrane surface of the targeted magnetic composite nanomaterials+hydrogen peroxide+external magnetic field+NIR treatment group also appears to shrink and break, which shows that the targeted magnetic composite nanomaterials can be used in combination with hydrogen peroxide, external magnetic field and NIR, etc.
[0096] Bacterial targeting experiment of the targeted magnetic composite nanomaterials with multiple sterilization functions prepared in Example 1: 400 μg / mL of the magnetic composite nanomaterials and the targeted magnetic composite nanomaterials with multiple sterilization functions were respectively incubated with bacteria for 12 hours, and then the materials were washed away with PBS and observed under a laser confocal microscope. The bacterial imaging figures at 488nm and 543nm are as shown in the figure Figure 9 As shown in the figure, the results show that the targeted magnetic composite nanomaterials with multiple sterilization functions can effectively gather at the site of bacterial colonization and emit blue and red fluorescence after incubation with bacteria, which indicates that the targeted magnetic composite nanomaterials with multiple sterilization functions have excellent bioimaging ability.
[0097] Biocompatibility experiment of the targeted magnetic composite nanomaterials prepared in Example 1: 200 μL of L929 cells were inoculated in a 96-well plate with a density of 2×10 5 cells / mL, and cultured in a 37℃, 5% CO2 incubator for 24h. Then the culture medium was discarded, and different concentrations of the targeted magnetic composite nanomaterials were incubated for 24h. After co-culture, PBS was washed twice, 200 μL of fresh culture medium containing 20 μL of MTT (5mg / mL) was added to each well, and incubated for 4h. Finally, 100 μL of DMSO was added to dissolve the formic acid crystals. Finally, the absorbance at 570nm was measured using an enzyme marker. The results show that the cytotoxicity of the targeted magnetic composite nanomaterials with multiple sterilization functions prepared in Example 1 to L929 cells with / without near-infrared light is as shown in the figure Figure 10 As can be seen from the figure, the cell activity is still higher than 80% when the targeted magnetic composite nanomaterials and near-infrared light are applied at the same time, which indicates that the targeted magnetic composite nanomaterials have good biocompatibility.
[0098] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.
Claims
1. A method for preparing a targeted magnetic composite nanomaterial with multiple sterilization properties, characterized in that, The method comprises the following steps: Step 1: dissolving the precursor in deionized water, stirring to uniformly disperse, and reacting under heating and pressurization; after the reaction is completed, cooling, filtering, collecting the filtrate, drying, and obtaining the multifunctional photosensitive material; The precursor in step 1 is indocyanine green; The multifunctional photosensitive material in step 1 is carbon dots; In step 1, the ratio of the precursor to deionized water is 5-20 mL of deionized water for every 5-50 mg of precursor; the reaction under heating and pressurization is carried out at 80-150 ℃, 0.1-8 MPa, and 2-8 h; and the filtering is 0.22 μm filter head filtering; Step 2: dissolving the iron source in ethylene glycol, adding anhydrous acetic acid and the multifunctional photosensitive material obtained in step 1, and reacting under heating and pressurization; after the reaction is completed, cooling, washing with distilled water and anhydrous ethanol, collecting the reaction system by using an external magnetic field, and obtaining the magnetic composite nanomaterial in the reaction system and drying; The iron source is one or more of ferric chloride, ferric sulfate, ferric nitrate, and potassium ferricyanide; Step 3: dissolving the positively charged polymer in an acetic acid solution, stirring to be uniform, adding the magnetic composite nanomaterial prepared in step 2, stirring, adding sodium hydroxide solution to the mixture until the pH value reaches 7, washing with deionized water, collecting the reaction system by using an external magnetic field, and obtaining the targeted magnetic composite nanomaterial; and the positively charged polymer is chitosan.
2. The method of claim 1, wherein: Step 1 further comprises removing the unreacted precursor before drying.
3. The method of claim 2, wherein: The filtrate is centrifuged or the filtrate is dialyzed in deionized water to remove the unreacted precursor.
4. The method of claim 1, wherein: In step 2, the ratio of the iron source, ethylene glycol, anhydrous sodium acetate, and the multifunctional photosensitive material is 5-40 mL of ethylene glycol for every 0.2-0.6 g of iron source, then adding 1-4 g of anhydrous sodium acetate and 0.01-0.06 g of the multifunctional photosensitive material; the heating and pressurization condition is 160-220 ℃, 0.1-8 MPa, and 4-12 h; and the drying is drying at 40-70 ℃ for 1-4 h.
5. The method of claim 1, wherein: In step 3, the ratio of the positively charged polymer, acetic acid, magnetic composite nanomaterial, and sodium hydroxide is 20-60 mL of acetic acid solution with a volume percentage concentration of 0.5-2 % for every 0.1-0.5 g of the positively charged polymer, adding 1.0-4.0 g of the magnetic composite nanomaterial, and adding sodium hydroxide solution to the mixture until the pH is 7.
6. The method of claim 5, wherein: In step 3, the concentration of the sodium hydroxide solution is 1 M.
7. The targeted magnetic composite nanomaterial with multiple sterilization functions prepared by the preparation method in any one of claims 1 to 6.
8. The use of the targeted magnetic composite nanomaterial with multiple sterilization functions in claim 7 in the preparation of a bacteria-targeting bacterial fluorescent marker and / or an antibacterial agent.
9. Use according to claim 8, characterized in that, The bacteria-targeting bacterial fluorescent marker can display green, blue, and red fluorescence under excitation at 405, 488, and 543 nm wavelengths; the antibacterial agent can kill bacteria after 808 nm laser irradiation; and the bacteria are gram-positive bacteria and / or gram-negative bacteria.