Manganese-calcium-doped mesoporous silica particle antibacterial agent as well as preparation method and application thereof

Abstract

The invention discloses a manganese-calcium-doped mesoporous silica particle antibacterial agent and a preparation method and application thereof, the antibacterial agent takes mesoporous silica as a carrier, and manganese ions and calcium ions are loaded in pores of the mesoporous silica. The antibacterial agent can be applied to the field of various filtering materials, the antibacterial effect of the filtering material combined with the antibacterial agent on common bacteria in air is tested through biological inhibition zones and ultraviolet absorption tests, and the result shows that the filtering material combined with the antibacterial agent has high inhibition capacity on the bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of antibacterial agent preparation, and specifically relates to a manganese-calcium doped mesoporous silica particle antibacterial agent and a preparation method and application thereof. Background Art

[0002] In recent years, with the development of the national economy and the improvement of people's living standards, the problem of air pollution has attracted increasing public attention, especially the air pollution in public places such as hospitals, canteens and offices. In the past, pathogenic microorganisms in the air were mainly disinfected by ultraviolet, ozone and peracetic acid spray, but they are very harmful to the human body and the sterilization efficiency is not high.

[0003] Common antimicrobial agents include TiO2 and CuO particles. TiO2 is non-toxic, biocompatible and highly stable, so it is often used in textile materials, but its antimicrobial properties can only be manifested under ultraviolet irradiation; CuO particles are easily mixed with polymers, have relatively stable physical and chemical properties, and have very good photocatalytic properties under visible light irradiation. They can be used to treat pollutants in water and as antimicrobial agents in textiles, but there is a weakness when CuO nanoparticles are loaded onto cotton fabrics as antimicrobial agents: CuO nanoparticles have only weak binding force on the surface of textile fibers. Silver is the most common antimicrobial metal-based ion and is also widely used, but the high specific surface area and high surface energy of nanosilver and the instability of the size of silver nanoparticles will cause them to agglomerate into large nanoparticles during the loading process, so that nanosilver can be easily washed off the fiber surface of the filter material, resulting in the loss of its antimicrobial activity.

[0004] Mesoporous materials refer to porous materials with pore sizes ranging from 2 to 50 nm. Common mesoporous materials include MCM-41, SBA-15, and FDU-15. Mesoporous materials have a relatively high specific surface area and a large pore size, which can load and slowly release antibacterial materials well to achieve efficient antibacterial effects. Mesoporous silica is a porous material with a high surface area and a controllable pore structure. Due to its special pore structure, mesoporous silica has received extensive attention in the fields of drug delivery, biomedicine, and antibacterial. Its large pore structure can be used to carry drug substances, and the pore surface can be used to adsorb antibacterial drugs, thereby achieving controlled release of drugs and enhancing antibacterial properties. How to develop an antibacterial agent with excellent antibacterial activity, antibacterial durability, and stability is a technical problem that needs to be solved urgently. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a manganese-calcium doped mesoporous silica particle antibacterial agent, wherein the antibacterial agent uses mesoporous silica as a carrier, and manganese ions and calcium ions are loaded in the pores of the mesoporous silica.

[0006] According to an embodiment of the present invention, the particle size of the mesoporous silica is 80-150 nm.

[0007] According to an embodiment of the present invention, the particle size of the antibacterial agent is 100-230 nm, preferably 100-180 nm.

[0008] According to an embodiment of the present invention, the loading amount of the manganese ions on the carrier is 12-20wt%, preferably 14-18wt%, and the loading amount of the calcium ions on the carrier is 1-6wt%, preferably 2-4wt%.

[0009] The present invention provides a method for preparing the above-mentioned manganese-calcium doped mesoporous silica granule antibacterial agent, the method comprising:

[0010] The antibacterial agent is prepared by mixing a precursor containing manganese ions, a precursor containing calcium ions, a reducing agent and mesoporous silica.

[0011] According to an embodiment of the present invention, the precursor containing manganese ions is, for example, MnCl2.

[0012] According to an embodiment of the present invention, the precursor containing calcium ions is, for example, CaCl2.

[0013] According to an embodiment of the present invention, the reducing agent is selected from hexamethylenetetramine.

[0014] According to an embodiment of the present invention, the mass ratio of the precursor containing manganese ions, the precursor containing calcium ions, the reducing agent and the mesoporous silica is 0.1-3:0.05-2:1:1-5, specifically 0.8:0.2:1:2.

[0015] According to an embodiment of the present invention, the antibacterial agent is used to fight against Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, hemolytic cocci and Corynebacterium diphtheriae and the like.

[0016] According to an embodiment of the present invention, the preparation of the mesoporous silica is a conventional technique in the art. For example, the preparation method of the mesoporous silica is as follows:

[0017] In an alkaline system, a certain proportion of ethanol-water solution is added to a round-bottom flask, the upper layer is cyclohexane (oily), TEOS ethyl silicate or TMOS methyl orthosilicate is used as a silicon source, and a cationic surfactant (usually a quaternary ammonium salt, such as hexadecyltrimethylammonium bromide) is used as a structure-directing agent (template). A hydrothermal reaction is carried out at 90°C to prepare mesoporous silica.

[0018] According to an embodiment of the present invention, the method further comprises freeze-drying the product.

[0019] According to an embodiment of the present invention, the mixing temperature is 60-120°C, such as 80-100°C.

[0020] According to an embodiment of the present invention, the mixing time is 30 minutes to 24 hours.

[0021] The present invention also provides the use of the manganese and calcium doped mesoporous silica particle antibacterial agent in filter materials, for example, in medical filter materials.

[0022] According to an embodiment of the present invention, the filter material is selected from cotton fabrics (eg polyethylene terephthalate (PET)).

[0023] Beneficial effects of the present invention:

[0024] 1. The antibacterial agent of the present invention is prepared by doping manganese ions and calcium ions into mesoporous silica materials.

[0025] 2. The antibacterial agent of the present invention can be applied to various filter material fields. The antibacterial effect of the filter material combined with the antibacterial agent on common bacteria in the air was tested through biological inhibition zone and ultraviolet absorption tests. The results show that the filter material combined with the antibacterial agent has a strong inhibitory ability against bacteria.

[0026] 3. The preparation method of the antibacterial agent of the present invention is simple and can be mass-produced. After being combined with the filter material, it can be released by visible light stimulation to achieve the antibacterial effect.

[0027] 4. The antibacterial agent of the present invention uses mesoporous silica material (SiO2) as a template, avoiding the common antibacterial materials that are prone to agglomeration and poor binding force on the filter material. Nano-sized SiO2 particles can form covalent bonds with the surface of the filter material (such as cotton fabric), thereby improving the softness and flexibility of the filter material. Manganese can produce ROS through a "Fenton-like" reaction, and can also achieve antibacterial properties under visible light, and the antibacterial effect is excellent. Calcium can cause bacterial calcium death and can play a role in hemostasis. Manganese and calcium can achieve antibacterial effects under visible light, achieving effective and lasting antibacterial effects. Mesoporous silica particles with uniform diameter distribution can be evenly dispersed on the surface of the filter material. The actual antibacterial effect is tested by the inhibition zone experiment and the colony counting method. After adding the antibacterial agent of the present invention to the filter material, the resulting filter material shows a strong and lasting antibacterial effect on common bacteria in the air, including but not limited to Escherichia coli and Staphylococcus aureus.

[0028] 5. The present invention uses an antibacterial agent for the filter material. After the antibacterial agent is combined with the filter material, the antibacterial activity, antibacterial durability and stability of the filter material are further improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The transmission electron microscope (TEM) (left) and scanning electron microscope (SEM) (right) images of the antibacterial agent in Example 1 are shown.

[0030] Figure 2 This is the EDS spectrum image of the antibacterial agent in Example 1.

[0031] Figure 3 This is the UV absorbance experimental graph of bacteria at different times (0, 4h, 8h, 12h and 24h) when the antibacterial agent concentration is 200μg / mL (1 is Escherichia coli, 2 is Staphylococcus aureus).

[0032] Figure 4 is the plate count result in the test case.

[0033] Figure 5 It is the result of the inhibition zone experiment of the filter material in the test case.

[0034] Figure 6 is a SEM image of the filter material after incubation with bacteria in the test case.

[0035] Figure 7 This is the antibacterial effect (inhibition zone test) of the filter material in the test example on Escherichia coli (left) and Staphylococcus aureus (right). DETAILED DESCRIPTION

[0036] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0037] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0038] Example 1

[0039] A method for preparing an antibacterial agent, specifically comprising:

[0040] Mesoporous silica nanospheres with a particle size of 80-150 nm are added to a round-bottom flask, MnCl2 and CaCl2 are added after 30 minutes, and hexamethylenetetramine is added after 30 minutes. The mixture is reacted at 90° C. overnight, and finally freeze-dried to obtain the antibacterial agent, wherein the mass ratio of MnCl2, CaCl2, hexamethylenetetramine and silica nanospheres is 0.8:0.2:1:2.

[0041] Test Case

[0042] 1. EDS Mapping, SEM and TEM were used to characterize the composition and morphology of the antibacterial agent in Example 1.

[0043] 2. Antibacterial test

[0044] 2.1 Preparation of solid culture medium is completed as follows:

[0045] Mix ultrapure water, sodium chloride, yeast powder, peptone and agar powder evenly, cover with a breathable film and tin foil, put into a high-pressure sterilizer for sterilization for 2 hours, and take out when the temperature drops to 50°C for use.

[0046] Prepare a plate with a diameter of 10 cm, place it in a UV clean bench for sterilization for 10 minutes, and pour the prepared culture medium into the plate to a thickness of about 5 mm.

[0047] 2.2 UV absorbance experiment:

[0048] The antibacterial agent solutions of Example 1 with different mass concentrations (the antibacterial agent contents were 1.0%wt, 2.0%wt, 3.0%wt, 4.0%wt and 5.0%wt, respectively) and a blank control group were prepared. The filter material (cotton fabric (polyethylene terephthalate (PET))) was cut into small discs with a diameter of 6 mm. 5 mL of activated bacterial solution (0.5 Mcfarland turbidity standard: concentration of 1.5×10 8 CFU / mL), place the filter material in different mass concentrations of antibacterial solution to mix them evenly, and after they are completely dried, shake the bacterial solution and the filter material combined with the antibacterial agent in a shaker for 2 hours to mix them evenly, and record the ultraviolet absorption of the solution at a certain interval under visible light with a wavelength of 600nm to determine the cell density of bacteria.

[0049] 2.3 Inhibition zone method: Select Escherichia coli and Staphylococcus aureus, prepare 300 μL of bacterial solution, place it on solid culture medium, and spread it evenly with a spreading stick.

[0050] The filter material with 5%wt antibacterial solution was placed on the solid culture medium coated with two bacteria, and then the solid culture medium was placed in a 37°C constant temperature incubator for culture, and its antibacterial effect was observed after 24 hours, and photographed and recorded. A blank control group PBS was placed in the center of the solid culture medium.

[0051] 2.4 Plate count experiment

[0052] The dilution plate counting method was used to evaluate the qualitative antibacterial effect of the material on Escherichia coli and Staphylococcus aureus and its influence on the survival rate. The specific process is as follows:

[0053] Take the logarithmic phase Escherichia coli and Staphylococcus aureus and dilute them to a final concentration of 10 8 CFU / mL, and different concentrations of the antimicrobial agent in Example 1 (the concentrations of the antimicrobial agent were 0, 2.0, 4.0, 6.0, 8.0% wt, which corresponded to Figure 4 0, 50μg / mL, 100μg / mL, 150μg / mL, 200μg / mL, 250μg / mL) of filter material, then mix the bacterial solution and the filter material combined with the antibacterial agent in an EP tube and place it in a shaker for incubation for 2h. Take out 300 microliters of the solution after full incubation, add it dropwise to the solid culture medium, spread it evenly with a coating rod, and then the solid culture medium will be placed in a 37℃ constant temperature incubator for incubation. After 24h, observe its antibacterial effect and take pictures for record. The blank control group is PBS.

[0054] 2.5 After treatment, the morphological characteristics of the bacteria were observed using a transmission electron microscope and a scanning electron microscope. The specific steps are as follows: after incubation with the filter material for 12 hours, the bacterial solution was centrifuged at 6000r / min for 5 minutes with PBS buffer, and the supernatant was removed. Repeat the washing three times and add 2.5% glutaraldehyde for fixation for 2 hours. Centrifuge and wash with sterile deionized water, and then gradient elute the sample with a series of ethanol concentrations (30%, 40%, 50%, 70%, 90%), dehydrate for 10 minutes at each concentration, and then dehydrate with 100% ethanol twice, and finally dissolve the bacteria in 100% ethanol. The sample was dripped on a silicon wafer for scanning electron microscopy observation, and the sample was dripped on a copper mesh for transmission electron microscopy observation.

[0055] Experimental conclusion and analysis

[0056] Figure 1 The transmission electron microscope (TEM) and scanning electron microscope (SEM) images of the antibacterial agent in Example 1 are shown. Figure 2 It is the EDS spectrum image of the antibacterial agent in Example 1. Figure 1 It can be clearly seen that the antibacterial agent has a mesoporous structure. The size of the antibacterial agent after loading manganese and calcium is about 100nm. Figure 2 EDS analysis shows that the doping amount of manganese is 16.44wt% and that of calcium is 2.11wt%.

[0057] Figure 3 This is the UV absorbance graph of bacteria at different times (0, 4h, 8h, 12h and 24h) when the antibacterial agent concentration is 200μg / mL (1 is Escherichia coli, 2 is Coccus aureus). Figure 3 After the bacterial solution and the filter material were incubated for different time periods, the absorbance of the bacterial solution at a wavelength of 600 nm was tested. Figure 3It can be seen that the absorbance of the two groups of bacteria decreased by about 50% after 4 hours, indicating that the material has excellent antibacterial effect. With the extension of incubation time, the absorbance of the two groups of bacteria decreased by about 60%, and the subsequent absorbance values ​​were stable, indicating that the antibacterial performance of the material was stable. After 4 hours, the growth of bacteria had been greatly inhibited, indicating that the antibacterial agent can penetrate from the filter material very well to achieve the antibacterial effect.

[0058] Figure 4 is the plate count result. Figure 4 As shown in the figure, the number of colonies on the blank plate is large and the growth state is good. From the perspective of the number of colonies, the survival rate of the two groups of bacteria decreases with the increase of the material concentration. This is the synergistic effect of oxidative stress caused by the "Fenton-like" reaction of manganese and calcium ions. When the material concentration is greater than 100μg / mL, the number of colonies decreases significantly. When the material concentration reaches 250μg / mL, it has a significantly enhanced bactericidal effect on Escherichia coli and Staphylococcus aureus.

[0059] Figure 5 The results of the antibacterial zone experiment of the filter material (Escherichia coli on the left and Staphylococcus aureus on the right). The blank control group PBS was placed in the center of the solid culture medium. After culturing in a 37°C constant temperature incubator for 24 hours, the diameter of the antibacterial zone was measured. The diameter of the antibacterial zone for Escherichia coli was 13.9 mm, and the diameter of the antibacterial zone for Staphylococcus aureus was 11.8 mm, indicating that the antibacterial agent of the present invention has excellent antibacterial effect.

[0060] Figure 6 This is the SEM picture of the filter material after incubation with bacteria. Figure 6 As shown, in the control group of bacteria that were not treated with the filter material (i.e., the first on the left), the bacteria were intact in morphology, with a round and smooth surface and intact cell structure. However, in the experimental group exposed to the antibacterial material, it was clearly observed that the morphology of the two bacteria was destroyed, and there was no intact cell membrane (i.e., the second and third on the left, and the right picture). The cells ruptured and lost their structural integrity, accompanied by the outflow of cell contents, which further confirmed that the material caused serious structural damage to Escherichia coli and Staphylococcus aureus, leading to the death of the bacteria.

[0061] Figure 7 The antibacterial effect of the filter material on Escherichia coli (left) and Staphylococcus aureus (right) (inhibition zone test). The antibacterial effect of the filter material on the solid plate was observed after 7 days. Figure 7It can be seen that, except for the area around the filter material, the solid plate is covered with bacteria. Even after 7 days, there is still an obvious antibacterial zone around the filter material, and no colonies grow around it, indicating that the manganese-calcium mesoporous nano-silica particles in the filter material achieve sustained release, excellent antibacterial effect, and long antibacterial time. The above is an exemplary description of the embodiments of the present invention. However, the scope of protection of the present invention is not limited to the above-mentioned embodiments. Any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A manganese-calcium doped mesoporous silica particle antibacterial agent, characterized in that: The antibacterial agent uses mesoporous silica as a carrier, and manganese ions and calcium ions are loaded in the pores of the mesoporous silica.

2. The antibacterial agent according to claim 1, characterized in that The particle size of the mesoporous silica is 80-150 nm. Preferably, the particle size of the antibacterial agent is 100-230 nm, preferably 100-180 nm.

3. The antibacterial agent according to claim 1 or 2, characterized in that The loading amount of the manganese ions on the carrier is 12-20 wt %, preferably 14-18 wt %, and the loading amount of the calcium ions on the carrier is 1-6 wt %, preferably 2-4 wt %.

4. The method for preparing the antibacterial agent according to any one of claims 1 to 3, characterized in that: The method comprises: The antibacterial agent is prepared by mixing a precursor containing manganese ions, a precursor containing calcium ions, a reducing agent and mesoporous silica.

5. The method according to claim 4, characterized in that The precursor containing manganese ions is MnCl2; The precursor containing calcium ions is CaCl2; The reducing agent is selected from hexamethylenetetramine.

6. The method according to claim 4, characterized in that The mass ratio of the precursor containing manganese ions, the precursor containing calcium ions, the reducing agent and the mesoporous silica is 0.1-3:0.05-2:1:1-5.

7. The method according to claim 4, characterized in that The antibacterial agent is used for antibacterial of at least one of Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, hemolytic cocci and Corynebacterium diphtheriae.

8. The method according to claim 4, characterized in that The mixing temperature is 60-120°C. Preferably, the mixing time is 30 min-24 h.

9. Use of the antibacterial agent according to any one of claims 1 to 3 in filter materials, for example in medical filter materials.

10. The use according to claim 9, characterized in that: The filter material is selected from cotton fabric.

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