A nano magnetic antibacterial agent, its preparation method, and an antibacterial method

Abstract

The present invention provides a nano magnetic antibacterial agent, a preparation method thereof, and an antibacterial method. In the preparation method provided by the present invention, ferric trichloride, sodium citrate, urea, polyacrylamide and water are mixed as the internal phase, and span 80 and mineral oil are mixed as the external phase, and porous nano-ferroferric oxide is prepared by a microfluidic process and a hydrothermal method; then, silver nitrate is in-situ reduced thereon so that silver nanoparticles are loaded on the surface of the porous nano-ferroferric oxide; and finally, surface coating is achieved with chitosan to obtain the nano magnetic antibacterial agent. The nano magnetic antibacterial agent prepared by the present invention has multiple antibacterial effects, does not rely on antibiotics, does not generate drug resistance, can kill drug-resistant bacteria, and provides a new strategy for the treatment of drug-resistant bacterial infections.

Description

Technical Field

[0001] The present invention relates to the field of bio-nanomaterials, and particularly to a nano-magnetic antibacterial agent, a preparation method thereof, and an antibacterial method. Background Art

[0002] With the long-term and extensive use of antibiotics, bacteria mutate to produce drug resistance, and this drug-resistant characteristic will be inherited by bacteria and passed on to the next generation. If this situation deteriorates, super bacteria will be produced, which are difficult to be killed by existing antibiotics. Therefore, how to effectively treat drug-resistant bacteria without generating drug resistance will be a severe challenge faced globally.

[0003] In recent years, photothermal therapy, magnetic hyperthermia, and photodynamic therapy have been introduced to combat drug-resistant bacteria. Because of their characteristics such as high-efficiency sterilization, not relying on antibiotics, and not generating drug resistance, they are considered effective antibacterial methods against super drug-resistant bacteria. In the prior art, there are many substances with antibacterial effects, but it is very difficult to actually cooperate with the above-mentioned therapies, achieve high-efficiency sterilization, and not generate drug resistance, and there are still great technical challenges. Summary of the Invention

[0004] In view of this, the present invention provides a nano-magnetic antibacterial agent, a preparation method thereof, and an antibacterial method. The nano-magnetic antibacterial agent provided by the present invention can achieve high-efficiency antibacterial, does not rely on antibiotics, and does not generate drug resistance.

[0005] The present invention provides a preparation method of a nano-magnetic antibacterial agent, including the following steps:

[0006] A) Prepare porous nano-ferroferric oxide:

[0007] a1. Mix ferric chloride, sodium citrate, urea, polyacrylamide, and water to obtain an inner phase;

[0008] a2. Mix span 80 and mineral oil to obtain an outer phase;

[0009] a3. Introduce the inner phase and the outer phase into a microfluidic device, and use the microfluidic process to obtain pre-polymer emulsion droplets;

[0010] a4. Perform heat treatment on the pre-polymer emulsion droplets to obtain porous nano-ferroferric oxide;

[0011] Among them, there is no order limit for step a1 and step a2;

[0012] B) Prepare porous nano-ferroferric oxide loaded with silver nanoparticles:

[0013] b1. Mix the porous nano-ferroferric oxide with silver nitrate, water, and formaldehyde solution to obtain a first mixed solution;

[0014] b2. Centrifuge the first mixture to collect the product, wash and dry it to obtain porous nano-magnetite loaded with silver nanoparticles;

[0015] C) Prepare chitosan-modified antibacterial agent:

[0016] c1. Mix the porous nano-magnetite loaded with silver nanoparticles with the chitosan solution to obtain a second mixture;

[0017] The chitosan solution is a solution obtained by dissolving chitosan in an acetic acid solution;

[0018] c2. Centrifuge the second mixture to collect the product and wash it to obtain a chitosan-modified nano-magnetic antibacterial agent.

[0019] Preferably, in step a3, in the microfluidic process, the inner phase flow rate is controlled at 5 - 10 μL / min, and the outer phase flow rate is controlled at 100 - 120 μL / min;

[0020] In step a4, the temperature of the heat treatment is 200 °C, and the time is 6 - 10 h.

[0021] Preferably, in step a3, in the microfluidic process, the inner phase flow rate is controlled at 10 μL / min, and the outer phase flow rate is controlled at 100 μL / min;

[0022] In step a4, the temperature of the heat treatment is 200 °C, and the time is 10 h.

[0023] Preferably, in step a1:

[0024] The ferric chloride is ferric chloride hexahydrate, and the sodium citrate is anhydrous sodium citrate;

[0025] The mass ratio of ferric chloride hexahydrate to anhydrous sodium citrate is 0.36:(0.2 - 0.5);

[0026] The mass ratio of ferric chloride hexahydrate to urea is 0.36:(0.1 - 0.5);

[0027] The mass ratio of ferric chloride hexahydrate to polyacrylamide is 0.36:(0.1 - 0.2);

[0028] The dosage ratio of ferric chloride hexahydrate to water is 0.36 g:(10 - 30) mL;

[0029] In step a2:

[0030] The mass ratio of span 80 to mineral oil is (0.1 - 0.5):1.

[0031] Preferably, in step b1:

[0032] The dosage ratio of silver nitrate to water is 0.06 g:(5 - 20) mL;

[0033] The mass ratio of silver nitrate to porous nano - magnetite is 0.06 g:(10 - 30) mg;

[0034] The dosage ratio of silver nitrate to formaldehyde solution is 0.06 g:(1 - 5) mL;

[0035] The mass concentration of the formaldehyde solution is 18% - 37%;

[0036] Step b1 specifically includes:

[0037] First, dissolve silver nitrate in water, stir evenly, then add porous nano - magnetite and stir evenly, and finally add formaldehyde solution and continue to stir to obtain a first mixed solution.

[0038] Preferably, in step c1:

[0039] The chitosan solution is prepared by the following method: mix chitosan with acetic acid solution to obtain a chitosan solution;

[0040] The dosage ratio of chitosan to acetic acid solution is 1 g:(90 - 110) mL;

[0041] The mass concentration of the acetic acid solution is 2% - 10%;

[0042] The dosage ratio of the porous nano - magnetite loaded with silver nanoparticles to the chitosan solution is 30 mg:(15 - 20) mL.

[0043] The present invention also provides a nano - magnetic antibacterial agent prepared by the preparation method described in the above technical solution.

[0044] The present invention also provides an antibacterial method, wherein the antibacterial agent used is the antibacterial agent described in the above technical solution.

[0045] Preferably, it is carried out under the irradiation of a laser.

[0046] Preferably, the laser is a 635 nm laser; the laser power density of the laser irradiation is 1.5 W / cm 2 .

[0047] The present invention provides a preparation method of a nano-magnetic antibacterial agent. First, porous nano-ferroferric oxide is prepared by a microfluidic process. During this process, some factors are controlled, such as introducing polyacrylamide into the inner phase, using a specific combination of Span 80 and mineral oil in the outer phase, controlling the flow rates of the inner and outer phases in the microfluidic process, and the heat treatment process conditions, so as to obtain porous nano-ferroferric oxide with a particle size of about 2 μm, a porous structure, a large specific surface area, and a uniform and stable structure. Then, the porous nano-ferroferric oxide and silver nitrate are dissolved in water, and silver nanoparticles-loaded porous nano-ferroferric oxide is obtained by adding formaldehyde; finally, the material obtained above is added to the prepared chitosan solution, stirred at room temperature for a certain time, and a weak intermolecular interaction is formed between chitosan and the silver nanoparticles-loaded porous nano-ferroferric oxide to form a wrapping, so that chitosan forms an outer wrapping layer on the surface of the nanoparticles. Then, the precipitate is collected by centrifugation to obtain the nano-magnetic antibacterial agent of chitosan-wrapped silver nanoparticles-loaded porous nano-ferroferric oxide. The present invention prepares porous and uniform nano-ferroferric oxide with a stable structure through a certain process, and then in-situ reduces and loads silver nanoparticles, realizing uniform loading of silver nanoparticles and loading more silver nanoparticles, which can better sterilize. At the same time, it further includes a cationic chitosan layer, and silver ions and chitosan play a synergistic role on the porous ferroferric oxide, which can further improve the bactericidal efficiency against Gram-negative bacteria; moreover, the porous ferroferric oxide has good photothermal effect and magnetothermal effect, which can increase the temperature to cause damage and lysis of the bacterial outer membrane, and finally eliminate bacteria. The components in the above antibacterial agent synergistically antibacterial, greatly improving the antibacterial effect and not generating drug resistance.

[0048] The test results show that the sterilization rate of the antibacterial agent provided by the present invention reaches more than 91%, showing excellent antibacterial properties; moreover, there is no antibiotic in the antibacterial agent of the present invention, and it belongs to physical killing and does not generate drug resistance. Description of the Drawings

[0049] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0050] Figure 1 It is the infrared spectrogram of the product of step A), the product of step B), and the product of step C) in Example 1;

[0051] Figure 2 It is the SEM diagram of the chitosan-modified antibacterial agent product obtained in Example 1;

[0052] Figure 3It is the antibacterial property test effect diagram of chitosan and the antibacterial agent modified by the chitosan obtained in Example 1; among them, Figure 3 (A) is the antibacterial property test effect diagram of chitosan, Figure 3 (B) is the antibacterial property test effect diagram of the antibacterial agent modified by the chitosan obtained in Example 1. Specific implementation mode

[0053] The present invention provides a preparation method of a nano-magnetic antibacterial agent, which comprises the following steps:

[0054] A) Prepare porous nano-ferroferric oxide:

[0055] a1. Mix ferric chloride, sodium citrate, urea, polyacrylamide and water to obtain an inner phase;

[0056] a2. Mix Span 80 and mineral oil to obtain an outer phase;

[0057] a3. Introduce the inner phase and the outer phase into a microfluidic device, and use the microfluidic process to prepare pre-polymer emulsion droplets;

[0058] a4. Perform heat treatment on the pre-polymer emulsion droplets to obtain porous nano-ferroferric oxide;

[0059] Among them, there is no order limit for step a1 and step a2;

[0060] B) Prepare porous nano-ferroferric oxide loaded with silver nanoparticles:

[0061] b1. Mix the porous nano-ferroferric oxide with silver nitrate, water and formaldehyde solution to obtain a first mixed solution;

[0062] b2. Centrifuge the first mixed solution to collect the product, wash and dry it to obtain porous nano-ferroferric oxide loaded with silver nanoparticles;

[0063] C) Prepare an antibacterial agent modified by chitosan:

[0064] c1. Mix the porous nano-ferroferric oxide loaded with silver nanoparticles with a chitosan solution to obtain a second mixed solution;

[0065] The chitosan solution is a solution of chitosan dissolved in acetic acid;

[0066] c2. Centrifuge the second mixed solution to collect the product and wash it to obtain a nano-magnetic antibacterial agent modified by chitosan.

[0067] The preparation method provided by the present invention first prepares porous nano-ferroferric oxide through microfluidic technology and hydrothermal method, and in-situ reduces silver nitrate on it to load silver nanoparticles on the surface of the porous nano-ferroferric oxide; finally, surface coating is achieved with chitosan to obtain a nano-magnetic antibacterial agent. The nano-magnetic antibacterial agent prepared by the present invention has multiple antibacterial effects, does not rely on antibiotics, does not produce drug resistance, can kill drug-resistant bacteria, and provides a new strategy for the treatment of drug-resistant bacterial infections.

[0068] Regarding step A) :

[0069] In the present invention, step A) is to prepare porous nano-ferroferric oxide, which specifically includes four steps a1 to a4.

[0070] [Regarding step a1]:

[0071] a1. Mix ferric chloride, sodium citrate, urea, polyacrylamide and water to obtain the internal phase.

[0072] In the present invention, the ferric chloride may be ferric chloride hexahydrate, and the sodium citrate may be anhydrous sodium citrate. The mass ratio of ferric chloride hexahydrate to anhydrous sodium citrate is preferably 0.36:(0.2 - 0.5), specifically 0.36:0.20, 0.36:0.30, 0.36:0.40, 0.36:0.412, 0.36:0.50, and more preferably 0.36:0.412. The mass ratio of ferric chloride hexahydrate to urea is preferably 0.36:(0.1 - 0.5), specifically 0.36:0.10, 0.36:0.18, 0.36:0.20, 0.36:0.30, 0.36:0.40, 0.36:0.50, and more preferably 0.36:0.18.

[0073] In the present invention, the mass ratio of ferric chloride hexahydrate to polyacrylamide is preferably 0.36:(0.1 - 0.2), specifically 0.36:0.1, 0.36:0.2. The introduction of polyacrylamide in the present invention will not hinder the formation of ferroferric oxide, and can improve the dispersion of ferroferric oxide and prevent its aggregation, which is beneficial to obtaining porous and uniform nano-ferroferric oxide.

[0074] In the present invention, the dosage ratio of ferric chloride hexahydrate to water is preferably 0.36 g:(10 - 30) mL, specifically 0.36 g:10 mL, 0.36 g:18 mL, 0.36 g:20 mL, 0.36 g:30 mL, and more preferably 0.36 g:18 mL.

[0075] In the present invention, the method of mixing ferric chloride hexahydrate, sodium citrate, urea, polyacrylamide and water is preferably stirring and mixing. The rate of the stirring and mixing is preferably 200 - 450 rpm. The time of the stirring and mixing is preferably 10 - 20 h, more preferably 12 h. After the above mixing treatment, a clear and transparent solution is obtained, which is the internal phase (or dispersed phase).

[0076] [Regarding step a2]:

[0077] a2. Mix Span 80 and mineral oil to obtain the external phase.

[0078] In the present invention, using a specific combination of Span 80 and mineral oil as the external phase is beneficial to improving the product effect. If other surfactants (such as Tween 80, etc.) and other oil substances (such as dimethyl silicone oil, fluorinated oil, etc.) are used in combination, the product preparation effect is poor, and the antibacterial performance of the product is also poor. In some embodiments of the present invention, the CAS number of the mineral oil is 8042 - 47 - 5, the product number is P104803, and it is provided by Shanghai Aladdin Biochemical Technology Co., Ltd.

[0079] In the present invention, the mass ratio of Span 80 to mineral oil is preferably (0.1 - 0.5):1, specifically 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1.

[0080] The present invention has no special limitation on the method of mixing Span 80 and mineral oil, as long as the two can be mixed evenly, such as stirring and mixing. After mixing, the external phase (or continuous phase) is obtained.

[0081] [Regarding step a3]:

[0082] a3. Introduce the internal phase and the external phase into a microfluidic device, and use the microfluidic process to prepare pre-polymerized emulsion droplets.

[0083] In the present invention, in the microfluidic process, the flow rate of the internal phase is preferably controlled at 5 - 10 μL / min, specifically 5 μL / min, 6 μL / min, 7 μL / min, 8 μL / min, 9 μL / min, 10 μL / min, and most preferably 10 μL / min. The flow rate of the external phase is preferably controlled at 100 - 120 μL / min, specifically 100 μL / min, 105 μL / min, 110 μL / min, 115 μL / min, 120 μL / min, and most preferably 100 μL / min. Through the microfluidic process, the internal phase and the external phase are made into pre-polymerized emulsion droplets.

[0084] [Regarding step a4]:

[0085] a4. Perform heat treatment on the pre-polymerized emulsion droplets to obtain porous nano-ferroferric oxide.

[0086] After obtaining the prepolymer emulsion droplets in step a3, the present invention transfers them to an autoclave for heat treatment. In the present invention, the temperature of the heat treatment is preferably 200 °C. The heat preservation time of the heat treatment is preferably 6-10 h, specifically 6 h, 7 h, 8 h, 9 h, 10 h, and most preferably 10 h. Through the above heat treatment, the materials react with each other to form porous nano-ferroferric oxide.

[0087] In the present invention, after the above heat treatment, it is preferably followed by the following post-treatment: cooling, washing, and drying. Among them, the cooling is preferably cooling to room temperature, and the washing is preferably repeatedly washing with anhydrous ethanol and water respectively. The drying temperature is preferably 30-60 °C. After the above post-treatment, porous nano-ferroferric oxide is obtained.

[0088] Compared with other preparation methods of ferroferric oxide, the present invention is prepared based on a microfluidic process, and some factors are controlled during the process, such as introducing polyacrylamide into the inner phase, using a specific combination of Span 80 and mineral oil in the outer phase, controlling the flow rates of the inner and outer phases in the microfluidic process link, and the heat treatment process conditions, so as to obtain porous nano-ferroferric oxide with a particle size of about 2 μm, a porous structure, a large specific surface area, and a uniform and stable structure, which is beneficial to uniformly loading silver nanoparticles and loading more silver nanoparticles in the subsequent process, not only achieving better photothermal and magnetic thermal effects, but also enabling the silver particles to better exert their antibacterial properties. Among them, in the above steps a3-a4, when the flow rates of the inner and outer phases are controlled at 10 μL / min and 100 μL / min and the heat preservation time of the heat treatment is 10 h, porous nano-ferroferric oxide with the best structure can be obtained, and the antibacterial effect of the final antibacterial agent product can reach the best.

[0089] Regarding step B) :

[0090] In the present invention, step B) is to prepare porous nano-ferroferric oxide loaded with silver nanoparticles, specifically including two steps b1-b2.

[0091] [Regarding step b1]:

[0092] b1. Mix the porous nano-ferroferric oxide with silver nitrate, water, and formaldehyde solution to obtain a first mixed solution.

[0093] In the present invention, the order of mixing the above materials is preferably as follows: First, dissolve silver nitrate in water, stir evenly, then add porous nano-ferroferric oxide and stir evenly, and finally add formaldehyde solution and continue to stir to obtain a first mixed solution. Among them, the stirring time for the first time is preferably 10 min. The stirring time after adding porous nano-ferroferric oxide is preferably 20 min. The temperature condition for stirring after adding the formaldehyde solution is preferably 25 - 35 °C, specifically 25 °C, 30 °C, 35 °C; the stirring time after adding the formaldehyde solution is preferably 1 - 2 h, specifically 1 h, 2 h. During the above mixing process, silver nitrate is in-situ reduced to silver nanoparticles under the action of formaldehyde.

[0094] In the present invention, the dosage ratio of silver nitrate to water is preferably 0.06 g:(5 - 20) mL, specifically 0.06 g:5 mL, 0.06 g:10 mL, 0.06 g:15 mL, 0.06 g:20 mL. The water is preferably deionized water. The mass ratio of silver nitrate to porous nano-ferroferric oxide is preferably 0.06 g:(10 - 30) mg, specifically 0.06 g:10 mg, 0.06 g:15 mg, 0.06 g:20 mg, 0.06 g:25 mg, 0.06 g:30 mg. The dosage ratio of silver nitrate to formaldehyde solution is preferably 0.06 g:(1 - 5) mL, specifically 0.06 g:1 mL, 0.06 g:2 mL, 0.06 g:3 mL, 0.06 g:4 mL, 0.06 g:5 mL. The formaldehyde solution is an aqueous formaldehyde solution, and its mass concentration is preferably 18% - 37%, specifically 18%, 20%, 25%, 30%, 35%, 37%.

[0095] [Regarding step b2]:

[0096] b2. Centrifuge the first mixed solution to collect the product, wash and dry it to obtain porous nano-ferroferric oxide loaded with silver nanoparticles.

[0097] In the present invention, first centrifuge the first mixed solution obtained in step b1 to collect the product, and then wash the collected material. The washing is preferably carried out by repeatedly washing with ethanol and water. After washing, dry it. The drying temperature is preferably 30 - 60 °C. After the above treatment, porous nano-ferroferric oxide loaded with silver nanoparticles is obtained, wherein the silver nanoparticles are uniformly loaded on the porous ferroferric oxide.

[0098] Regarding step C) :

[0099] In the present invention, step C) is to modify the chitosan coating layer, which specifically includes two steps, step c1 - c2.

[0100] [Regarding step c1]:

[0101] c1. Mix the porous nano - magnetite loaded with silver nanoparticles with the chitosan solution to obtain a second mixture.

[0102] In the present invention, the chitosan solution is a solution obtained by dissolving chitosan in an acetic acid solution. The chitosan solution can be prepared by the following method: Stir and mix chitosan and the acetic acid solution evenly to obtain a chitosan solution. Among them, the acetic acid solution is an aqueous acetic acid solution; the mass concentration of the acetic acid solution used is preferably 2% - 10%. The dosage ratio of chitosan to the acetic acid solution is preferably 1 g:(90 - 110) mL, specifically, it can be 1 g:90 mL, 1 g:95 mL, 1 g:100 mL, 1 g:105 mL, 1 g:110 mL. The conditions for the stirring and mixing have no special restrictions and can be carried out at room temperature, which can be 20 - 30 °C. After being mixed evenly, a uniformly viscous chitosan solution is obtained.

[0103] In the present invention, the dosage ratio of the porous nano - magnetite loaded with silver nanoparticles to the chitosan solution is preferably 30 mg:(15 - 20) mL, specifically, it can be 30 mg:15 mL, 30 mg:16 mL, 30 mg:17 mL, 30 mg:18 mL, 30 mg:19 mL, 30 mg:25 mL. By controlling the ratio of chitosan to acetic acid and the ratio of the porous nano - magnetite loaded with silver nanoparticles to the chitosan solution within the above ranges in the present invention, the antibacterial agent can achieve the best bactericidal effect.

[0104] In the present invention, the method of mixing the porous nano - magnetite loaded with silver nanoparticles with the chitosan solution is preferably stirring and mixing. The stirring rate is preferably 200 - 450 rpm; the temperature condition for the stirring and mixing is room temperature, which can be 20 - 30 °C; the stirring time is preferably 10 - 20 h, more preferably 24 h. After the above mixing, a uniform second mixture is obtained.

[0105] [Regarding step c2]:

[0106] c2. Centrifuge and separate the second mixture to collect the product and wash it to obtain a chitosan - modified nano - magnetic antibacterial agent.

[0107] In the present invention, after obtaining the second mixture in step c1, centrifuge and separate it to collect the precipitate, and then wash it. The washing is preferably carried out by repeatedly washing with an acetic acid solution and water respectively. Among them, the acetic acid solution is an aqueous acetic acid solution; the mass concentration of the acetic acid solution is preferably 1%. After the above washing, drying is preferably carried out. The drying temperature is preferably 30 - 60 °C. After the above treatment, a chitosan - modified nano - magnetic antibacterial agent is obtained.

[0108] In step C above, the porous nano-ferroferric oxide loaded with silver nanoparticles is mixed with the chitosan solution and stirred at room temperature for more than 24 h to form weak intermolecular interactions between chitosan and the porous nano-ferroferric oxide loaded with silver nanoparticles, thereby forming a coating, so that chitosan forms an outer coating layer on the surface of the nanoparticles. Then, after centrifugal separation, the chitosan molecules not coated on the surface of the nanoparticles are washed away, thereby obtaining the nano-magnetic antibacterial agent modified with chitosan coating.

[0109] The present invention also provides a nano-magnetic antibacterial agent prepared by the preparation method described in the above technical solution.

[0110] The present invention also provides an antibacterial method, wherein the antibacterial agent used is the nano-magnetic antibacterial agent described in the above technical solution. In the present invention, in the above antibacterial method, it is preferably carried out under the irradiation of a laser. The laser is preferably a 635 nm laser (i.e., the laser wavelength is 635 nm). The laser power density of the laser irradiation is preferably 1.5 W / cm 2 。

[0111] The present invention provides a preparation method of a nano-magnetic antibacterial agent. First, porous nano-ferroferric oxide is prepared by a microfluidic process. In this process, some factors are controlled, such as introducing polyacrylamide into the inner phase, using a specific combination of Span 80 and mineral oil in the outer phase, controlling the flow rates of the inner and outer phases in the microfluidic process, and the heat treatment process conditions, so as to obtain porous nano-ferroferric oxide with a particle size of about 2 μm, a porous structure, a large specific surface area, and a uniform and stable structure. Then, the porous nano-ferroferric oxide and silver nitrate are dissolved in water, and porous nano-ferroferric oxide loaded with silver nanoparticles is obtained by adding formaldehyde; finally, the material obtained above is added to the prepared chitosan solution and stirred at room temperature for a certain time to form weak intermolecular interactions between chitosan and the porous nano-ferroferric oxide loaded with silver nanoparticles, thereby forming a coating, so that chitosan forms an outer coating layer on the surface of the nanoparticles. Then, the precipitate is collected by centrifugal separation to obtain the nano-magnetic antibacterial agent, namely, porous nano-ferroferric oxide loaded with silver nanoparticles wrapped with chitosan. The present invention prepares porous and uniform nano-ferroferric oxide with a stable structure through a certain process, and then in-situ reduces and loads silver nanoparticles, realizing uniform loading of silver nanoparticles and loading more silver nanoparticles, which can better sterilize. At the same time, it further includes a cationic chitosan layer, and silver ions and chitosan play a synergistic role on the porous ferroferric oxide, which can further improve the bactericidal efficiency against Gram-negative bacteria; moreover, the porous ferroferric oxide has good photothermal and magnetothermal effects, which can increase the temperature to cause damage and lysis of the bacterial outer membrane and finally eliminate bacteria. The components in the above antibacterial agent synergistically antibacterial, greatly improving the antibacterial effect and not generating drug resistance.

[0112] The test results show that the sterilization rate of the antibacterial agent provided by the present invention reaches over 91%, showing excellent antibacterial properties; moreover, there is no antibiotic in the antibacterial agent of the present invention, which belongs to physical killing and will not produce drug resistance.

[0113] To further understand the present invention, the preferred embodiments of the present invention will be described below in conjunction with examples. However, it should be understood that these descriptions are only for further explaining the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0114] In the following examples, unless otherwise specified, the raw materials or reagents used are commercially available products. Among them, the mineral oil used has a CAS number of 8042-47-5 and a product number of P104803, and is provided by Shanghai Aladdin Biochemical Technology Co., Ltd.

[0115] Example 1

[0116] A) Preparation of porous nano-ferroferric oxide:

[0117] a1. Dissolve FeCl3·6H2O (0.36 g), anhydrous sodium citrate (0.412 g), urea (0.18 g), and polyacrylamide (0.1 g) in 18 mL of water and stir at 350 rpm for 12 h until the solution is clear and transparent to obtain the inner phase;

[0118] a2. Stir and mix Span 80 and mineral oil evenly at a mass ratio of 0.3:1 to obtain the outer phase;

[0119] a3. Introduce the inner phase and the outer phase into a microfluidic device, control the inner phase flow rate to be 10 μL / min and the outer phase flow rate to be 100 μL / min to prepare pre-polymerized emulsion droplets;

[0120] a4. Transfer the pre-polymerized emulsion droplets to a stainless steel autoclave with a polytetrafluoroethylene lining, keep it at 200 °C for 10 h; then cool to room temperature, wash repeatedly with anhydrous ethanol and water, and dry to obtain porous nano-ferroferric oxide.

[0121] B) Preparation of porous nano-ferroferric oxide loaded with silver nanoparticles:

[0122] b1. Take 0.06 g of silver nitrate and add it to 10 mL of deionized water, stir for 10 min; then, add 20 mg of the porous nano-ferroferric oxide obtained in step A), stir for 20 min; afterwards, add 3 mL of formaldehyde solution (mass concentration 30%) to the system and stir at 30 °C for 1.5 h to obtain the first mixture.

[0123] b2. Centrifuge the first mixture to collect the product, then wash repeatedly with ethanol and water, and finally dry to obtain porous nano-ferroferric oxide loaded with silver nanoparticles;

[0124] C) Preparation of chitosan-modified antibacterial agent:

[0125] c1. Take 1 g of chitosan and add it to 100 mL of acetic acid solution (mass concentration 5%). Stir at room temperature of 25 °C for 6 h to obtain a uniform and viscous chitosan solution. Then, take 30 mg of the porous nano-magnetite loaded with silver nanoparticles obtained in step B) and add it to 15 mL of the chitosan solution. Stir at room temperature of 25 °C for 24 h to obtain a second mixture;

[0126] c2. Centrifuge the above second mixture to collect the precipitate, and then wash it repeatedly with acetic acid solution (mass concentration 1 wt%) and water, and finally dry it to obtain a chitosan-modified nano-magnetic antibacterial agent.

[0127] Example 2

[0128] Implemented according to Example 1, the differences are as follows: in step a1, the dosage of polyacrylamide is 0.2 g; in step a2, the mass ratio of span 80 to mineral oil is 0.1:1; in step b1, the dosage of deionized water is 5 mL, the dosage of porous nano-magnetite is 10 mg, and the dosage of formaldehyde solution is 1 mL; in step c1, the dosage of acetic acid solution is 90 mL and the dosage of chitosan solution is 20 mL.

[0129] Example 3

[0130] Implemented according to Example 1, the differences are as follows: in step a2, the mass ratio of span 80 to mineral oil is 0.5:1; in step b1, the dosage of deionized water is 20 mL, the dosage of porous nano-magnetite is 30 mg, and the dosage of formaldehyde solution is 5 mL; in step c1, the dosage of acetic acid solution is 110 mL and the dosage of chitosan solution is 20 mL.

[0131] Example 4

[0132] Implemented according to Example 1, the differences are as follows: in step a3, the internal phase flow rate is controlled at 5 μL / min and the external phase flow rate is controlled at 120 μL / min; in step a4, the heat preservation time is 6 h.

[0133] Comparative Example 1

[0134] Implemented according to Example 4, the differences are as follows: in step a3, the internal phase flow rate is controlled at 3 μL / min and the external phase flow rate is controlled at 150 μL / min; in step a4, the heat preservation time is 3 h.

[0135] Comparative Example 2

[0136] Implemented according to Example 4, the differences are as follows: in step a3, the internal phase flow rate is controlled at 20 μL / min and the external phase flow rate is controlled at 80 μL / min; in step a4, the heat preservation time is 14 h.

[0137] Comparative Example 3

[0138] It was carried out according to Example 4, except that: span 80 in step a2 was replaced with tween 80, and mineral oil was replaced with dimethyl silicone oil.

[0139] Comparative Example 4

[0140] It was carried out according to Example 4, except that: step C) was not carried out, and only steps A) to B) were carried out to obtain porous nano-ferroferric oxide loaded with silver nanoparticles (denoted as Fe3O4@Ag).

[0141] Comparative Example 5

[0142] It was carried out according to Example 4, except that: step B) was not carried out, and only steps A) and C) were carried out to obtain a composite of chitosan and porous nano-ferroferric oxide (denoted as Fe3O4@CTS).

[0143] Comparative Example 6

[0144] The products obtained in Comparative Example 4 and Comparative Example 5 were mixed evenly. When mixing, the mass ratio of the two was based on the dosage of silver nitrate and chitosan in Example 1 to obtain a mixture (denoted as Fe3O4@Ag-Fe3O4@Ag).

[0145] Test Example: Product Testing

[0146] 1. Infrared Test

[0147] Infrared analysis was respectively carried out on the porous nano-ferroferric oxide obtained in step A) of Example 1, the porous nano-ferroferric oxide loaded with silver nanoparticles obtained in step B), and the chitosan-modified antibacterial agent obtained in step C). The results are shown in Figure 1 .

[0148] 2. SEM Characterization

[0149] SEM characterization was carried out on the chitosan-modified antibacterial agent product obtained in step C) of Example 1. The results are shown in Figure 2 , and it can be seen that the surface of the antibacterial agent is rough and has a porous structure.

[0150] 3. Antibacterial Property Test

[0151] Testing process: First, it was evaluated by the method of growing single colonies of Escherichia coli on an agar plate. Escherichia coli was cultured until the logarithmic growth phase. The pre-cultured original bacterial solution was taken and diluted to 106 CFU / mL. 100 μL of the above-diluted bacterial solution was added to a 96-well plate, and then 100 μL of PBS solutions containing different concentration gradients of the antibacterial agent were added respectively. After light (ultraviolet light) and dark treatments, the 96-well plate was placed in a 37 °C incubator for static co-culture. The treated Escherichia coli was taken out, and 100 μL of the bacterial solution was diluted 10 3 times with PBS. Finally, 100 μL of the diluted bacterial solution was evenly spread on the agar plate, and the culture dish was sealed with a sealing film. Finally, the agar plate was placed upside down in a 37 °C incubator for static culture for about 18 - 24 h. The morphology of Escherichia coli was observed at any time. After the colonies grew to an ideal size, the number of colonies was counted, and the morphology of the colonies on the agar plate was photographed.

[0152] Using chitosan alone as the antibacterial agent and the chitosan-modified antibacterial agent product obtained in Example 1 as the antibacterial agent, the above testing process was carried out for testing. The results are shown in Figure 3 . It can be seen that under the same conditions, the chitosan-modified antibacterial agent has excellent bactericidal performance against bacteria, while the bactericidal effect of chitosan alone is average and cannot kill all bacteria, thus proving that the chitosan-modified antibacterial agent can significantly improve the antibacterial efficacy of the material.

[0153] At the same time, antibacterial tests were carried out on other examples and comparative examples. The results are shown in Table 1.

[0154] Table 1: Test results of each example and comparative example

[0155] Types of antibacterial agents Sterilization rate (%) Example 1 96 Example 2 92 Example 3 93 Example 4 91 Comparative example 1 83 Comparative example 2 85 Comparative example 3 84 Comparative example 4 81 Comparative example 5 74 Comparative example 6 82

[0156] It can be seen that the sterilization rates of Examples 1 to 4 of the present invention reach over 91%, showing excellent antibacterial properties and no drug resistance; while the sterilization rates of Comparative Examples 1 to 6 are significantly reduced. Compared with the examples, the effects of Comparative Examples 1 to 2 become worse, proving that controlling certain internal and external phase flow rates and heat treatment process conditions in step A) of the present invention is beneficial to improving the antibacterial effect of the product. Compared with the examples, the effect of Comparative Example 3 becomes worse, proving that using a specific combination of Span 80 and mineral oil as the external phase in the present invention is beneficial to improving the antibacterial effect of the product. Compared with the examples, the effects of Comparative Examples 4 to 5 become worse, that is, the antibacterial agent of iron tetroxide loaded with silver and chitosan obtained in the present invention has a better effect than any one of silver or chitosan singly loaded on porous nano-iron tetroxide, proving that in the composite product obtained in the present invention, silver nanoparticles and chitosan play a synergistic antibacterial role on porous nano-iron tetroxide, significantly improving the antibacterial effect of the product. Compared with the examples, the effect of Comparative Example 6 becomes worse. It can be seen that although the composition of the overall product of Comparative Example 6 is the same as that of Example 1, only in Comparative Example 6, silver nanoparticles and chitosan are separately loaded on iron tetroxide, while in the present invention, silver nanoparticles and chitosan are co-loaded on iron tetroxide, but the effect is significantly improved. This further proves that silver nanoparticles and chitosan co-loaded on porous nano-iron tetroxide play a synergistic antibacterial role, thereby improving the product effect. Good effects are achieved in Examples 1 to 4. Among them, compared with Example 4, the effect of Example 1 is further significantly improved, proving that when the internal and external phase flow rates are controlled at 10 μL / min and 100 μL / min and the heat treatment holding time is 10 h in step A) of the present invention, the product can reach the best effect.

[0157] It should be noted that the definition of the microfluidic process in the art is as follows: In the field of microfluidics, the flow focusing method is a commonly used method. Three flow paths focus on a pipe, and the dispersed phase and the continuous phase converge at the cross-shaped tube. The symmetrically flowing phases above and below simultaneously squeeze the dispersed phase to break it, thereby forming emulsion droplets. The definition of emulsion droplets in the art is as follows: Generating emulsion droplets on a microfluidic chip is a process in which one-phase fluid is dispersed in another immiscible or partially miscible fluid. For two immiscible liquids, one is used as the continuous phase and the other is used as the dispersed phase, and the dispersed phase is dispersed in the continuous phase to form emulsion droplets. Therefore, the definition of the microfluidic process and emulsion droplets belongs to the common knowledge in the art and will not be elaborated here.

[0158] In this article, specific examples are used to illustrate the principles and implementation modes of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention, including the best mode, and also enables any person skilled in the art to practice the present invention, including manufacturing and using any device or system, and implementing any combined method. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principles of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention. The protection scope of the present invention patent is defined by the claims and may include other embodiments that can be conceived by those skilled in the art. If these other embodiments have structural elements similar to the literal description of the claims, or if they include equivalent structural elements that have no substantial difference from the literal description of the claims, then these other embodiments should also be included within the scope of the claims.

Claims

1. A preparation method of a nano magnetic antibacterial agent, characterized in that, It includes the following steps: A) Preparation of porous nano-ferroferric oxide: a1. Mix ferric chloride, sodium citrate, urea, polyacrylamide and water to obtain the internal phase; a2. Mix span 80 and mineral oil to obtain the external phase; a3. Introduce the internal phase and the external phase into a microfluidic device, and use the microfluidic process to prepare pre-polymer emulsion droplets; Among them, the flow rate of the internal phase is controlled at 5-10 μL / min, and the flow rate of the external phase is controlled at 100-120 μL / min; a4. Perform heat treatment on the pre-polymer emulsion droplets to obtain porous nano-ferroferric oxide; The temperature of the heat treatment is 200 °C, and the time is 6-10 h; Among them, there is no order restriction on step a1 and step a2; B) Preparation of porous nano-ferroferric oxide loaded with silver nanoparticles: b1. Mix the porous nano-ferroferric oxide with silver nitrate, water and formaldehyde solution to obtain a first mixed solution; b2. Centrifuge, collect, wash and dry the product of the first mixed solution to obtain porous nano-ferroferric oxide loaded with silver nanoparticles; C) Preparation of chitosan-modified antibacterial agent: c1. Mix the porous nano-ferroferric oxide loaded with silver nanoparticles with chitosan solution to obtain a second mixed solution; The chitosan solution is a solution obtained by dissolving chitosan in acetic acid solution; c2. Centrifuge and collect the product and wash the second mixed solution to obtain a chitosan-modified nano-magnetic antibacterial agent; The nano-magnetic antibacterial agent is a Gram-negative bacteria antibacterial agent.

2. The preparation method according to claim 1, wherein In step a3, in the microfluidic process, the flow rate of the internal phase is controlled at 10 μL / min, and the flow rate of the external phase is controlled at 100 μL / min; In step a4, the temperature of the heat treatment is 200 °C, and the time is 10 h.

3. The preparation method according to claim 1, characterized in that, In step a1: The ferric chloride is ferric chloride hexahydrate, and the sodium citrate is anhydrous sodium citrate; The mass ratio of ferric chloride hexahydrate to anhydrous sodium citrate is 0.36:(0.2-0.5); The mass ratio of ferric chloride hexahydrate to urea is 0.36:(0.1-0.5); The mass ratio of ferric chloride hexahydrate to polyacrylamide is 0.36:(0.1-0.2); The dosage ratio of ferric chloride hexahydrate to water is 0.36 g:(10-30) mL; In step a2: The mass ratio of span 80 to mineral oil is (0.1-0.5):

1.

4. The preparation method according to claim 1, characterized in that, In step b1: The dosage ratio of silver nitrate to water is 0.06 g:(5-20) mL; The mass ratio of silver nitrate to porous nano-ferroferric oxide is 0.06 g:(10-30) mg; The dosage ratio of silver nitrate to formaldehyde solution is 0.06 g:(1-5) mL; The mass concentration of the formaldehyde solution is 18%-37%; Step b1 specifically includes: First, dissolve silver nitrate in water, stir evenly, then add porous nano-ferroferric oxide and stir evenly, and finally add formaldehyde solution and continue to stir to obtain a first mixed solution.

5. The preparation method according to claim 1, characterized in that, In step c1: The chitosan solution is prepared by the following method: Mix chitosan with acetic acid solution to obtain a chitosan solution; The dosage ratio of chitosan to acetic acid solution is 1 g:(90-110) mL; The mass concentration of the acetic acid solution is 2% to 10%; The dosage ratio of the porous nano-ferroferric oxide loaded with silver nanoparticles to the chitosan solution is 30 mg:(15 - 20) mL.

6. A nano-magnetic antibacterial agent prepared by the preparation method according to any one of claims 1 to 5.

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