Manganese calcium doped mesoporous silica particle antibacterial agent, method for preparing the same, and use thereof

Abstract

This invention discloses a manganese-calcium-doped mesoporous silica particle antibacterial agent, its preparation method, and its application. The antibacterial agent uses mesoporous silica as a carrier, with manganese and calcium ions loaded within the pores of the mesoporous silica. This antibacterial agent can be applied to various filter materials. Through biological inhibition zone and ultraviolet absorption tests, the antibacterial effect of the filter material incorporating the antibacterial agent on common airborne bacteria was tested. The results show that the filter material incorporating the antibacterial agent has a strong inhibitory ability against bacteria.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial agent preparation technology, specifically relating to a manganese-calcium doped mesoporous silica particle antibacterial agent, its preparation method, and its application. Background Technology

[0002] In recent years, with the development of the national economy and the improvement of people's living standards, air pollution has received increasing public attention, especially in public places such as hospitals, canteens, and offices. Previously, disinfection of airborne pathogens was mainly carried out using ultraviolet light, ozone, and peracetic acid spraying, but these methods are highly harmful to human health and have low sterilization efficiency.

[0003] Common antibacterial agents include TiO2 and CuO particles. TiO2 is non-toxic, biocompatible, and highly stable, making it commonly used in textiles; however, its antibacterial properties only manifest under ultraviolet light. CuO particles are easily mixed with polymers, have relatively stable physical and chemical properties, and exhibit excellent photocatalytic performance under visible light, making them suitable for treating pollutants in water and as antibacterial agents in textiles. However, a weakness of using CuO nanoparticles as antibacterial agents on cotton fabrics is that they exhibit only weak binding forces on the textile fiber surface. Silver, as the most common antibacterial metal-based ion, is also widely used. However, the high specific surface area and surface energy of nanosilver, along with the size instability of silver nanoparticles, cause them to agglomerate into large nanoparticles during loading. This makes the nanosilver easily washed away from the fiber surface of the filter material, resulting in the loss of its antibacterial 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 possess relatively high specific surface areas and large pore sizes, allowing for effective loading and sustained release of antibacterial materials, achieving highly efficient antibacterial effects. Mesoporous silica is a porous material with high surface area and tunable pore structure. Due to its unique channel structure, mesoporous silica has attracted widespread attention in drug delivery, biomedicine, and antibacterial fields. Its large-pore structure can be used for drug loading, while the pore surface can be used to adsorb antibacterial drugs, thereby achieving controlled drug release and enhanced antibacterial performance. Developing an antibacterial agent with excellent antibacterial activity, antibacterial durability, and stability is a pressing technical problem that needs to be solved. Summary of the Invention

[0005] To address the aforementioned technical problems, this 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 within the pores of the mesoporous silica.

[0006] According to an embodiment of the present invention, the mesoporous silica has a particle size of 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 manganese ions on the support is 12-20 wt%, preferably 14-18 wt%, and the loading amount of calcium ions on the support is 1-6 wt%, preferably 2-4 wt%.

[0009] This invention provides a method for preparing the above-mentioned manganese-calcium-doped mesoporous silica particle 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 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 combat Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, hemolytic cocci, and Corynebacterium diphtheriae.

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

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

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

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

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

[0021] This invention also provides the application of the above-mentioned manganese-calcium doped mesoporous silica particle antibacterial agent in filter materials, such as in medical filter materials.

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

[0023] The beneficial effects of this invention are:

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

[0025] 2. The antibacterial agent of this invention can be applied to various filter materials. Through biological inhibition zone and ultraviolet absorption tests, the antibacterial effect of filter materials incorporating the antibacterial agent on common airborne bacteria was tested. The results show that the filter materials incorporating the antibacterial agent have a strong inhibitory effect on bacteria.

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

[0027] 4. The antibacterial agent of this invention uses mesoporous silica (SiO2) as a template, avoiding the problems of agglomeration and poor bonding of common antibacterial materials on filter materials. Nano-sized SiO2 particles can form covalent bonds with the surface of filter materials (e.g., cotton fabrics), improving the softness and flexibility of the filter material. Manganese can generate ROS through a Fenton-like reaction, achieving antibacterial properties even under visible light, with excellent antibacterial effects. Calcium can cause bacterial calcium death and has a hemostatic effect. Manganese and calcium can achieve antibacterial effects under visible light, achieving effective and long-lasting antibacterial effects. The uniformly distributed mesoporous silica particles can be evenly dispersed on the surface of the filter material. The actual antibacterial effect was tested through inhibition zone experiments and colony counting methods. After adding the antibacterial agent of this invention to the filter material, the resulting filter material showed a strong and long-lasting antibacterial effect against common airborne bacteria, including but not limited to Escherichia coli and Staphylococcus aureus.

[0028] 5. This invention uses antibacterial agents in filter materials. By combining the antibacterial agents with the filter materials, the antibacterial activity, antibacterial durability, and stability of the filter materials are further improved. Attached Figure Description

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

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

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

[0032] Figure 4 This is the result of the plate counting method in the test case.

[0033] Figure 5 This is the result of the antibacterial zone experiment of the filter material in the test example.

[0034] Figure 6 These are SEM images of the filter material and bacteria after incubation in the test case.

[0035] Figure 7 This is the antibacterial effect of the filter material in the test case against Escherichia coli (left) and Staphylococcus aureus (right) (inhibition zone experiment). Detailed Implementation

[0036] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory 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 covered within the scope of protection intended by the present invention.

[0037] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products 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 were added to a round-bottom flask. After 30 min, MnCl2 and CaCl2 were added, followed by hexamethylenetetramine (HXMT) after another 30 min. The mixture was reacted overnight at 90 °C and then freeze-dried to obtain the antibacterial agent. The mass ratio of MnCl2, CaCl2, hexamethylenetetramine (HXMT), and silica nanospheres was 0.8:0.2:1:2.

[0041] Test case

[0042] 1. The composition and morphology of the antibacterial agent in Example 1 were characterized by EDS mapping, SEM and TEM.

[0043] 2. Antibacterial test

[0044] 2.1 Solid culture medium preparation shall be carried out according to the following method:

[0045] Mix ultrapure water, sodium chloride, yeast powder, peptone and agar powder evenly, cover with a breathable membrane and aluminum foil, put in an autoclave for 2 hours, and take it out for use when the temperature drops to 50℃.

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

[0047] 2.2 Ultraviolet absorbance experiment:

[0048] Antibacterial agent solutions of different mass concentrations as in Example 1 (antibacterial agent content of 1.0% wt, 2.0% wt, 3.0% wt, 4.0% wt, and 5.0% wt, respectively) and a blank control group were prepared. Filter material (cotton fabric (polyethylene terephthalate (PET))) was cut into small round pieces with a diameter of 6 mm. 5 mL of activated bacterial solution (0.5 McFarland turbidity standard: concentration 1.5 × 10⁻⁶) was placed in EP tubes. 8 The bacterial cell density was determined by mixing the filter material with antibacterial agent solutions of different concentrations (CFU / mL) until the bacterial solution and the filter material containing the antibacterial agent were completely dried. The bacterial solution and the filter material containing the antibacterial agent were then shaken in a shaker for 2 hours to mix them evenly. The ultraviolet absorption of the solution was recorded at certain time intervals under visible light at a wavelength of 600 nm to determine the bacterial cell density.

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

[0050] Filter media containing a 5% wt antibacterial agent solution was placed on a solid culture medium coated with two types of bacteria. The solid culture medium was then incubated at 37°C. The antibacterial effect was observed and photographed after 24 hours. A blank control group (PBS) was placed in the center of the solid culture medium.

[0051] 2.4 Plate counting experiment

[0052] The dilution-coating plate count method was used to evaluate the antibacterial qualitative effect and survival rate of the material against Escherichia coli and Staphylococcus aureus. The specific procedure was as follows:

[0053] Dilute the logarithmic-phase Escherichia coli and Staphylococcus aureus bacterial suspensions to a final concentration of 10. 8 CFU / mL, and different concentrations of the antibacterial agent in Example 1 (the concentrations of the antibacterial agent were 0, 2.0, 4.0, 6.0, and 8.0%wt, respectively, corresponding to...) Figure 4 Filter media with concentrations of 0, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, and 250 μg / mL were used. The bacterial solution was then mixed with the filter media containing the antibacterial agent in EP tubes and incubated on a shaker for 2 hours. After incubation, 300 μL of the solution was taken and added dropwise to solid culture medium, spread evenly with a spreader. The solid culture medium was then incubated at 37°C. The antibacterial effect was observed and photographed after 24 hours. PBS was used as the blank control group.

[0054] 2.5 After processing, the morphological characteristics of the bacteria were observed using transmission electron microscopy and scanning electron microscopy. The specific steps are as follows: After incubation with the filter material for 12 hours, the bacterial culture was centrifuged at 6000 rpm for 5 minutes with PBS buffer, and the supernatant was removed. The mixture was washed three times and fixed with 2.5% glutaraldehyde for 2 hours. The sample was then washed with sterile deionized water by centrifugation, followed by gradient elution with a series of ethanol concentrations (30%, 40%, 50%, 70%, 90%), each concentration for 10 minutes. This was followed by two more dehydration cycles with 100% ethanol, and finally, the bacterial cells were dissolved in 100% ethanol. The samples were then dropped onto a silicon wafer for scanning electron microscopy observation, and simultaneously dropped onto a copper grid for transmission electron microscopy observation.

[0055] Experimental conclusions and analysis

[0056] Figure 1 These are transmission electron microscope (TEM) and scanning electron microscope (SEM) images of the antibacterial agent in Example 1. Figure 2 This is an EDS (Energy Dispersive X-ray Diode) image of the antibacterial agent in Example 1. From... Figure 1 The antibacterial agent clearly exhibits a mesoporous structure; after being loaded with manganese and calcium, the size of the antibacterial agent is approximately 100 nm. Figure 2 EDS analysis showed that the manganese doping content was 16.44 wt% and the calcium doping content was 2.11 wt%.

[0057] Figure 3 This is a UV absorbance graph of bacteria at different time points (0, 4h, 8h, 12h and 24h) when the antibacterial agent concentration is 200μg / mL (1 is Escherichia coli, 2 is Staphylococcus aureus). Figure 3 In the experiment, after the bacterial solution and filter material were incubated for different times, the absorbance of the bacterial solution at a wavelength of 600 nm was measured. Figure 3As can be seen, 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 absorbance value remained stable thereafter, indicating that the antibacterial performance of the material is stable. The growth of bacteria was greatly inhibited after 4 hours, indicating that the antibacterial agent can be well penetrated from the filter material to achieve the antibacterial effect.

[0058] Figure 4 This is the result of the plate counting method. For example... Figure 4 As shown, the blank group plates had a higher number of colonies and better growth. The colony counts indicated that the survival rate of bacteria in both groups decreased with increasing material concentration. This is attributed to the synergistic effect of oxidative stress caused by the Fenton-like reaction of manganese and calcium ions. When the material concentration exceeded 100 μg / mL, the colony count significantly decreased. At a concentration of 250 μg / mL, the bactericidal effect against *Escherichia coli* and *Staphylococcus aureus* was significantly enhanced.

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

[0060] Figure 6 This is a SEM image of the filter material after bacterial incubation. (Example:) Figure 6 As shown, the control group of bacteria without filter material treatment (leftmost) exhibits intact bacterial morphology, smooth and rounded surfaces, and complete cell structure. However, in the experimental group exposed to the antibacterial material, the morphology of both bacteria is clearly disrupted, with no intact cell membranes remaining (leftmost, leftmost, rightmost image). The cells rupture and lose structural integrity, accompanied by the leakage of cell contents. This further confirms that the material causes severe structural damage to Escherichia coli and Staphylococcus aureus, leading to bacterial death.

[0061] Figure 7 This shows the antibacterial effect of the filter material against *Escherichia coli* (left) and *Staphylococcus aureus* (right) (inhibition zone experiment). The antibacterial effect of the filter material on a solid plate was observed after 7 days. Figure 7As can be seen, besides the area around the filter material, the solid plate was covered with bacteria. Even after 7 days, a clear inhibition zone remained around the filter material, with no bacterial colonies growing there. This indicates that the manganese-calcium mesoporous nano-silica particles in the filter material achieved continuous release, resulting in excellent antibacterial effects and a long-lasting antibacterial duration. The above provides 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 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 within the scope of protection of the present invention.

Claims

1. A method for preparing a manganese-calcium-doped mesoporous silica particle antibacterial agent, characterized in that, The antibacterial agent uses mesoporous silica as a carrier, with manganese and calcium ions loaded within the pores of the mesoporous silica. The method for preparing the antibacterial agent includes: The antibacterial agent is prepared by mixing a precursor containing manganese ions, a precursor containing calcium ions, a reducing agent, and mesoporous silica; the reducing agent is selected from hexamethylenetetramine. The mixing temperature is 60-120℃; The mesoporous silica has a particle size of 80-150 nm; The loading amount of manganese ions on the carrier is 12-20 wt%, and the loading amount of calcium ions on the carrier is 1-6 wt%.

2. The method according to claim 1, characterized in that, The particle size of the antibacterial agent is 100-230 nm.

3. The method according to claim 1, characterized in that, The loading amount of manganese ions on the carrier is 14-18 wt%, and the loading amount of calcium ions on the carrier is 2-4 wt%.

4. The method according to claim 1, characterized in that, The precursor containing manganese ions is MnCl2; The precursor containing calcium ions is CaCl2.

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

6. The method according to claim 1, characterized in that, The antibacterial agent is used to combat at least one of Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, hemolytic cocci, and Corynebacterium diphtheriae.

7. The method according to claim 4, characterized in that, The mixing time is 30 min to 24 h.

8. The antibacterial agent prepared by the method according to any one of claims 1-7.

9. The use of the antibacterial agent according to claim 8 in filter materials.

10. The application according to claim 9, characterized in that, Used in medical filter materials.

11. The application according to claim 10, characterized in that, The filter material is selected from cotton fabrics.

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