Tannic acid-functionalized micromotor with enhanced bacterial capture and antibacterial effects, preparation method, and application thereof

Abstract

The application discloses a tannic acid functionalized micro motor with enhanced bacterial capture and bacteriostatic effect and a preparation method and application thereof; the micro motor takes a Cu7S4 / vSiO2 particle with a virus-like structure as a carrier, and tannic acid is loaded on the surface of the carrier through a self-assembly method. On one hand, TA has an antibacterial effect and has a high affinity effect with abundant peptidoglycan on the cell wall of gram-positive bacteria; on the other hand, the virus-like structure with a spike structure has a high adhesion to bacteria, and the micro motor Cu7S4 / vSiO2 / TA has an enhanced capture effect on gram-positive bacteria; in addition, the photothermal effect of Cu7S4 under infrared light irradiation and self-driving can cooperate with TA to realize high-efficiency sterilization.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a tannic acid functionalized micromotor with enhanced bacteria capture and antibacterial effects, and a preparation method and application thereof. Background Art

[0002] Gram-positive bacteria impose a significant burden on human health, healthcare, and the socioeconomic landscape. Currently developed antimicrobial agents, such as metal or metal oxide nanoparticles, can be used to kill pathogenic microorganisms in water, but suffer from low targeting efficiency and the need for macroscopic agitation to effectively engage the pathogens. For example, antimicrobial materials based on advanced oxidation technology (AOP) have insufficient ability to capture pathogenic microorganisms in water, making it difficult to actively capture, enrich, and kill them in situ, resulting in inefficient sterilization.

[0003] Antibacterial micro / nanomotors have attracted widespread attention from researchers due to their high antibacterial properties, integrated multifunctionality (such as photothermal and photodynamic properties), and precise delivery and enrichment. Currently, researchers primarily functionalize nanomaterials through electrostatic interactions and substances that can bind to bacterial surfaces (such as antibodies, aptamers, and antibiotics) to rapidly capture pathogenic microorganisms in water. However, these methods suffer from unsatisfactory issues such as high cost. The natural product tannic acid (TA) has antioxidant, antibacterial, and anti-inflammatory properties, and has a high affinity for the abundant peptidoglycan on the cell walls of Gram-positive bacteria, enabling selective capture of Gram-positive bacteria. Furthermore, inspired by the localized "capture and kill" effect caused by the spike structures on the surface of natural bacteriophages, a tannic acid-functionalized, photothermally active, virus-like micromotor was constructed. This not only allows for efficient in situ capture of pathogens, but also achieves efficient sterilization through photothermal action, achieving "active capture and in situ inactivation" of Gram-positive bacteria.

[0004] So far, there has been no research or report on tannic acid-functionalized photothermal antibacterial micromotors with virus-like structures. Summary of the Invention

[0005] In view of this, the present invention provides a tannic acid-functionalized micromotor with enhanced bacteria capture and antibacterial effects, which is used to solve the technical problem of low performance of traditional fungicides in killing pathogenic microorganisms in the prior art.

[0006] A first aspect of the present invention provides a method for preparing a tannic acid-functionalized micromotor with enhanced bacteria capture and antibacterial effects.

[0007] To this end, the first technical solution provided by the present invention is as follows:

[0008] A tannic acid-functionalized micromotor with enhanced bacterial capture and antibacterial effects comprises Cu7S4 / vSiO2 particles with a virus-like structure and an antibacterial natural product, wherein the natural product is loaded on the surface of the Cu7S4 / vSiO2 particles with a virus-like structure.

[0009] Furthermore, in the above-mentioned tannic acid functionalized micromotor with enhanced bacterial capture and antibacterial effects, SiO2 coats Cu7S4 in the virus-like structured Cu7S4 / vSiO2 particles.

[0010] Furthermore, in the above-mentioned tannic acid-functionalized micromotor with enhanced bacteria capture and antibacterial effects, the natural product is tannic acid.

[0011] Furthermore, in the aforementioned tannic acid-functionalized micromotor with enhanced bacterial capture and antibacterial properties, the molar ratio of Cu7S4 particles, TA, and vSiO2 is Cu7S4 particles: tannic acid: vSiO2 = (20-80) mg: (10-60) mg: (3-6) mmol. More preferably, the molar ratio of Cu7S4 particles, TA, and vSiO2 is Cu7S4 particles: tannic acid: vSiO2 = 50 mg: 34 mg: 4.5 mmol.

[0012] Furthermore, in the above-mentioned tannic acid-functionalized micromotor with enhanced bacteria capture and antibacterial effects, the particle size of the virus-like structured Cu7S4 / vSiO2 particles is 450 nm.

[0013] The second technical solution provided by the present invention is a method for preparing the above-mentioned tannic acid-functionalized micromotor with enhanced bacteria capture and antibacterial effects, comprising the steps of:

[0014] Step S1: dissolving copper salt in deionized water, adding hydrazine hydrate for reduction, and then adding sodium sulfide to obtain Cu7S4 particles;

[0015] Step S2: Dispersing Cu7S4 particles, hexadecyltrimethylammonium bromide, and sodium hydroxide in water, adding tetraethoxysilane prepared with cyclohexane, and carrying out hydrothermal reaction at 30-90°C for 24-72h to obtain virus-like structure Cu7S4 / vSiO2 particles;

[0016] Step S3: Mix the virus-like structure Cu7S4 / vSiO2 particle dispersion and tannic acid solution, and load tannic acid on the surface of the virus-like structure Cu7S4 / vSiO2 particles through electrostatic self-assembly to obtain an antibacterial micromotor with bacteria capture ability.

[0017] In step S3, the virus-like structure Cu7S4 / vSiO2 particle dispersion is obtained by mixing Cu7S4 / vSiO2 particles with deionized water in a mass volume ratio of (50-200) mg: (10-30) mL and ultrasonically dispersing for 10-30 min;

[0018] The tannic acid solution is obtained by mixing tannic acid TA and deionized water in a mass volume ratio of (20-100) mg: (10-30) mL using a vortex mixer;

[0019] The electrostatic self-assembly is performed by placing the mixed solution in a constant temperature shaker, with the reaction temperature being 20-60° C. and the reaction time being 1-4 hours.

[0020] Furthermore, in the above-mentioned method for preparing a tannic acid-functionalized micromotor with enhanced bacteria capture and antibacterial effects, in step S1, the copper salt is copper nitrate hexahydrate;

[0021] The last technical solution provided by the present invention is the use of the above-mentioned tannic acid-functionalized micromotor with enhanced bacteria capture and antibacterial effects in capturing and killing pathogenic microorganisms.

[0022] The pathogenic microorganisms are Escherichia coli and methicillin-resistant Staphylococcus aureus.

[0023] A third aspect of the present invention provides a tannic acid-functionalized micromotor with enhanced bacterial capture and antibacterial properties for use in killing pathogenic microorganisms. Preferably, the pathogenic microorganisms include Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA).

[0024] The novel tannic acid-functionalized micromotor with enhanced bacterial capture and antibacterial properties prepared by the present invention has the following advantages and beneficial effects:

[0025] (1) Tannic acid TA has a high affinity for the abundant peptidoglycan on the cell wall of Gram-positive bacteria; on the other hand, the virus-like structure with a spike structure has a high adhesion to bacteria. Therefore, the micromotor Cu7S4 / vSiO2 / TA has an enhanced capture effect on Gram-positive bacteria (methicillin-resistant Staphylococcus aureus).

[0026] (2) The photothermal effect and self-driving of Cu7S4 under infrared light irradiation can cooperate with TA to achieve efficient sterilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 TEM image of the micromotor Cu7S4 / vSiO2 / TA;

[0028] Figure 2FT-IR and Zeta potential diagrams of the samples;

[0029] Figure 3 Temperature-raising cycle image (a), motion trajectory (b), motion rate at different powers (c), and motion time-lapse image (d) of the micromotor Cu7S4 / vSiO2 / TA under near-infrared light of 1064 nm;

[0030] Figure 4 Figure 2 shows the growth status of MRSA: (a) blank control group; (b) blank control group + 1064nm infrared light; (c) micromotor Cu7S4 / vSiO2 / TA + 1064nm infrared light;

[0031] Figure 5 Comparison of the capture rate of MRSA by different samples (a) and TEM image of MRSA capture by micromotor Cu7S4 / vSiO2 / TA (b). DETAILED DESCRIPTION

[0032] The technical solution of the present application will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments.

[0033] Example 1

[0034] This Example 1 provides a tannic acid-functionalized micromotor with enhanced bacterial capture and antibacterial effects. Cu7S4 particles are prepared by a reduction method, Cu7S4 / vSiO2 particles are prepared by a hydrothermal reaction method, and a micromotor Cu7S4 / vSiO2 / TA is prepared by an electrostatic self-assembly method, wherein the mass molar ratio of the Cu7S4 particles, TA, and vSiO2 is Cu7S4 particles: tannic acid: vSiO2 = 50 mg:34 mg:4.5 mmol.

[0035] It is prepared by the following method:

[0036] 1) The steps for preparing Cu7S4 particles by the reduction method are as follows: dissolve copper salt copper nitrate hexahydrate (0.28 g) in deionized water, add 2.5 mL of PVP (50 mg / mL) as a dispersant, and stir magnetically until fully dissolved. Then, add 35 mL of hydrazine hydrate for reduction, stir magnetically for 25 minutes, and then add 1.5 mL of sodium sulfide solution (7.8 mg / mL) and stir magnetically for 10 minutes. After the reaction is completed, the product is collected, washed with ethanol and deionized water, and dried to obtain Cu7S4 particles.

[0037] 2) The hydrothermal reaction method for preparing Cu7S4 / vSiO2 particles is as follows: 50 mg of Cu7S4 particles, 250 mg of hexadecyltrimethylammonium bromide, and 0.15 mL of sodium hydroxide (0.1 mol / mL) were dispersed in a round-bottom flask filled with deionized water (10 mL). The mixture was then stirred in an oil bath at 60°C for 2 h. A mixture of 1 mL of tetraethoxysilane and 9 mL of cyclohexane was then added, and the mixture was reacted in an oil bath at 60°C for 48 h. After the reaction, the mixture was cooled to room temperature. The product was collected, washed with anhydrous ethanol and deionized water, and dried to obtain Cu7S4 / vSiO2 particles.

[0038] 3) The steps for preparing Cu7S4 / vSiO2 / TA micromotors by electrostatic self-assembly are as follows: ultrasonically disperse 100 mg of virus-like structured Cu7S4 / vSiO2 particles in 20 mL of deionized water, add 20 mL of TA (2.5 mg / mL) and mix, place it in a constant temperature shaker, shake it at 200 r / min and 40°C for 2 h, collect the product, wash it with anhydrous ethanol and deionized water, and dry it to obtain the virus-like structured micromotors Cu7S4 / vSiO2 / TA.

[0039] Among them, the TEM images of Cu7S4 particles, Cu7S4 / vSiO2 particles and micromotor Cu7S4 / vSiO2 / TA are as follows: Figure 1 As shown in Figures a to c, it can be seen from the figures that the Cu7S4 prepared in Example 1 of the present application is irregular in shape and has a particle size of about 100 nm; Cu7S4 / vSiO2 is a virus-like structure, with a spike structure on the surface and is relatively rough; the morphology of the micromotor Cu7S4 / vSiO2 / TA is similar to that of Cu7S4 / vSiO2, showing a virus-like structure with a spike structure, indicating that the loading of TA does not change its morphology.

[0040] The FT-IR and Zeta potential diagrams of the micromotor Cu7S4 / vSiO2 / TA are shown in the figure below. Figure 2 As shown, from Figure 2 It can be concluded from a that the FT-IR spectrum of Cu7S4 / vSiO2 / TA has typical characteristic peaks of Cu7S4 / vSiO2 and TA, proving that TA has been successfully loaded on the surface of Cu7S4 / vSiO2. Figure 2 b It can be concluded that Cu7S4 / vSiO2 and TA have opposite potentials. The micromotor Cu7S4 / vSiO2 / TA can be prepared by electrostatic self-assembly, and the surface of the final product is negatively charged.

[0041] Example 2

[0042] The present embodiment 2 provides a tannic acid functionalized micro-motor with enhanced bacterial capture and bacteriostatic effect, Cu7S4 particles are prepared by reduction method, Cu7S4 / vSiO2 particles are prepared by hydrothermal reaction method, and micro-motor Cu7S4 / vSiO2 / TA is prepared by electrostatic self-assembly method, wherein the mass molar ratio of the Cu7S4 particles, TA and vSiO2 is Cu7S4 particles: tannic acid: vSiO2 = 20 mg: 10 mg: 3 mmol.

[0043] It is prepared by the following method:

[0044] 1) The steps of preparing Cu7S4 particles by reduction method are as follows: copper salt copper nitrate hexahydrate (0.28 g) is dissolved in deionized water, 2.5 mL of dispersant PVP (50 mg / mL) is added, and magnetic stirring is performed until complete dissolution, 35 mL of hydrazine hydrate is added for reduction, magnetic stirring is performed for 25 min, and then 1.5 mL of sodium sulfide solution (7.8 mg / mL) is added, and magnetic stirring is performed for 10 min; after the reaction is completed, the product is washed with ethanol and deionized water respectively, and dried to obtain Cu7S4 particles;

[0045] 2) The steps of preparing Cu7S4 / vSiO2 particles by hydrothermal reaction method are as follows: Cu7S4 particles (20 mg), cetyltrimethylammonium bromide (166.7 mg) and 0.15 mL of sodium hydroxide (0.07 mol / mL) are dispersed in a round-bottom flask containing deionized water (10 mL), and placed in an oil bath at 60°C for magnetic stirring reaction for 2 h. Then a mixture of 0.7 mL of tetraethoxysilane and 6 mL of cyclohexane is added, and the reaction is carried out in an oil bath at 60°C for 48 h. After the reaction is completed, it is cooled to room temperature, and the product is collected and washed with anhydrous ethanol and deionized water, and dried to obtain Cu7S4 / vSiO2 particles.

[0046] 3) The steps of preparing Cu7S4 / vSiO2 / TA micro-motor by electrostatic self-assembly method are as follows: 100 mg of virus-like structure Cu7S4 / vSiO2 particles are ultrasonically dispersed in 20 mL of deionized water, 20 mL of TA (0.75 mg / mL) is added and mixed, and then placed in a constant temperature shaker at 200 r / min and 40°C for 2 h. After the product is collected, it is washed with anhydrous ethanol and deionized water, and dried to obtain virus-like structure micro-motor Cu7S4 / vSiO2 / TA.

[0047] Embodiment 3

[0048] The present embodiment 3 provides a tannic acid functionalized micro-motor with enhanced bacterial capture and bacteriostatic effect, Cu7S4 particles are prepared by reduction method, Cu7S4 / vSiO2 particles are prepared by hydrothermal reaction method, and micro-motor Cu7S4 / vSiO2 / TA is prepared by electrostatic self-assembly method, wherein the mass molar ratio of the Cu7S4 particles, TA and vSiO2 is Cu7S4 particles: tannic acid: vSiO2 = 80 mg: 60 mg: 6 mmol.

[0049] It is prepared by the following method:

[0050] 1) The steps of preparing Cu7S4 particles by reduction method are as follows: copper salt copper nitrate hexahydrate (0.28 g) is dissolved in deionized water, 2.5 mL of dispersant PVP (50 mg / mL) is added, and magnetic stirring is performed until complete dissolution, 35 mL of hydrazine hydrate is added for reduction, magnetic stirring is performed for 25 min, and then 1.5 mL of sodium sulfide solution (7.8 mg / mL) is added, and magnetic stirring is performed for 10 min; after the reaction is completed, the product is washed with ethanol and deionized water respectively, and dried to obtain Cu7S4 particles;

[0051] 2) The steps of preparing Cu7S4 / vSiO2 particles by hydrothermal reaction method are as follows: Cu7S4 particles (80 mg), cetyltrimethylammonium bromide (333.3 mg) and 0.15 mL of sodium hydroxide (0.13 mol / mL) are dispersed in a round-bottom flask containing deionized water (10 mL), and placed in an oil bath at 60°C for magnetic stirring reaction for 2 h. Then a mixture of 1.3 mL of tetraethoxysilane and 12 mL of cyclohexane is added, and the reaction is carried out in an oil bath at 60°C for 48 h. After the reaction is completed, it is cooled to room temperature, and the product is collected and washed with anhydrous ethanol and deionized water, and dried to obtain Cu7S4 / vSiO2 particles.

[0052] 3) The steps of preparing Cu7S4 / vSiO2 / TA micro-motor by electrostatic self-assembly method are as follows: 100 mg of virus-like structure Cu7S4 / vSiO2 particles are ultrasonically dispersed in 20 mL of deionized water, 20 mL of TA (4.4 mg / mL) is added and mixed, and then placed in a constant temperature shaker at 200 r / min and 40°C for 2 h. After the product is collected, it is washed with anhydrous ethanol and deionized water, and dried to obtain virus-like structure micro-motor Cu7S4 / vSiO2 / TA.

[0053] Embodiment 4

[0054] The present embodiment tests the motion of the micro-motor Cu7S4 / vSiO2 / TA prepared in embodiment 1.

[0055] The 0.1 mg Cu7S4 / vSiO2 / TA micromotor provided in Example 1 was placed in a 96-well plate containing 100 μL of water, and its motion characteristics in water were observed with and without the application of 1064 nm infrared light.

[0056] The results are as follows Figure 3 As shown, from Figure 3 a It can be seen that the micromotor Cu7S4 / vSiO2 / TA heats up from 25°C to 75°C within 8 minutes and can be recycled 3 times, indicating that Cu7S4 / vSiO2 / TA has good photothermal effect and recyclability. Figure 3 b is its motion trajectory under near-infrared light irradiation. The motion speed of the micromotor Cu7S4 / vSiO2 / TA under near-infrared light is positively correlated with the light power, as shown in Figure 3 As shown in c, when the optical power increases from 1 to 2 mW / cm 2 When the speed of the movement increases from 2 to 7 μm / s. Figure 3 d is the time-lapse image of the micromotor Cu7S4 / vSiO2 / TA moving in water.

[0057] Example 5

[0058] This example tests the antibacterial performance of the micromotor Cu7S4 / vSiO2 / TA prepared in Example 1 against MRSA.

[0059] The bactericidal performance test of the micromotor Cu7S4 / vSiO2 / TA was conducted as follows: 0.1 mg of the micromotor Cu7S4 / vSiO2 / TA provided in Example 1 was added to the MRSA bacterial solution of the experimental group. The concentration of the micromotor in the bacterial solution was 0.5 mg / mL. A 1064 nm infrared laser was used as the photothermal light source at an optical power of 1.5 mW / cm 2 After irradiation for 8 minutes, 100 μL of the bacterial suspension was removed, diluted 10×10-fold, and 100 μL was evenly spread on LB solid medium. Three replicates were performed on each sterilization plate, and the cells were then incubated in a 37°C incubator for 24 hours. A control group of MRSA bacteria was tested without the addition of antimicrobial micromotors. The same testing procedures were followed, and the antimicrobial activity of the Cu7S4 / vSiO2 / TA micromotors was evaluated by observing the number of MRSA colonies in both experimental and control groups.

[0060] The antibacterial results of the micromotor Cu7S4 / vSiO2 / TA are as follows Figure 4 As shown in Figure 3, Figure c shows an excellent antibacterial effect compared to Figure a and Figure b. At the same time, Figure b also proves that at 1.5mW / cm 2 Irradiation with 1064nm infrared laser for 8min did not affect the normal growth of MRSA.

[0061] Example 6

[0062] This example tests the MRSA capture performance of the sample prepared in Example 1.

[0063] The micromotor's MRSA capture performance test process is as follows: 1 mg of the micromotor Cu7S4 / vSiO2 / TA provided in Example 1 was added to the MRSA bacterial solution of the experimental group. The concentration of the antibacterial micromotor in the bacterial solution after addition was 0.5 mg / mL. A 1064 nm infrared laser was used as the photothermal light source at an optical power of 1.5 mW / cm 2 Irradiate and react for 8 minutes. After the reaction is complete, let it stand for 5 minutes, collect the supernatant from the bacterial solution, measure the OD600 absorbance, and compare it with the initial bacterial solution. Simultaneously, take 1 mL of the post-reaction bacterial solution, fix it with 4% paraformaldehyde for 6 hours, then centrifuge it. The bacteria are then graded and dehydrated using 30%, 50%, 70%, and 90% methanol, respectively, and finally stored in 100% methanol.

[0064] The test results of the sample's MRSA capture performance are as follows: Figure 5 As shown, from Figure 5 a It can be seen that compared with Cu7S4 and Cu7S4 / vSiO2, the capture rate of MRSA by the micromotor Cu7S4 / vSiO2 / TA is significantly improved. Figure 5 b is the TEM image of the micromotor Cu7S4 / vSiO2 / TA capturing MRSA. It can be seen that Cu7S4 / vSiO2 / TA is tightly aggregated on MRSA, which intuitively demonstrates its capture ability.

[0065] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A tannic acid-functionalized micromotor with enhanced bacterial capture and antibacterial properties, characterized in that: It includes Cu7S4 / vSiO2 particles with a virus-like structure and an antibacterial natural product, wherein the antibacterial natural product is loaded on the surface of the Cu7S4 / vSiO2 particles with a virus-like structure; The antibacterial natural product is tannic acid; It is prepared by the following steps in sequence: Step S1: dissolving copper salt in deionized water, adding hydrazine hydrate for reduction, and then adding sodium sulfide to obtain Cu7S4 particles; Step S2: Disperse Cu7S4 particles, hexadecyltrimethylammonium bromide, and sodium hydroxide in water, add tetraethoxysilane prepared with cyclohexane, and perform hydrothermal reaction at 30-90°C for 24-72 hours to obtain virus-like structure Cu7S4 / vSiO2 particles; Step S3: The virus-like structure Cu7S4 / vSiO2 particle dispersion is mixed with the tannic acid solution to obtain a mixed solution, and the tannic acid is loaded on the surface of the virus-like structure Cu7S4 / vSiO2 particles through electrostatic self-assembly to obtain a tannic acid-functionalized micromotor with enhanced bacterial capture and antibacterial effects.

2. The tannic acid-functionalized micromotor with enhanced bacteria capture and antibacterial effect according to claim 1, characterized in that: In the virus-like structured Cu7S4 / vSiO2 particles, SiO2 coats Cu7S4.

3. The tannic acid-functionalized micromotor with enhanced bacteria capture and antibacterial effects according to claim 2, characterized in that: The mass molar ratio of the Cu7S4 particles, TA and vSiO2 is Cu7S4 particles: tannic acid: vSiO2 = (20-80) mg: (10-60) mg: (3-6) mmol.

4. The tannic acid-functionalized micromotor with enhanced bacteria capture and antibacterial effects according to claim 3, characterized in that: The particle size of the virus-like structure Cu7S4 / vSiO2 particles is 450 nm.

5. The tannic acid-functionalized micromotor with enhanced bacteria capture and antibacterial effects according to claim 1, characterized in that: In step S1, the copper salt is copper nitrate hexahydrate.

6. The tannic acid-functionalized micromotor with enhanced bacteria capture and antibacterial effects according to claim 1, characterized in that: In step S3, the virus-like structure Cu7S4 / vSiO2 particle dispersion is obtained by mixing Cu7S4 / vSiO2 particles with deionized water at a mass volume ratio of (50-200) mg: (10-30) mL, and ultrasonically dispersing for 10-30 min; The tannic acid solution is obtained by mixing tannic acid TA and deionized water in a mass volume ratio of (20-100) mg: (10-30) mL using a vortex mixer; The electrostatic self-assembly is performed by placing the mixed solution in a constant temperature shaker, with a reaction temperature of 20-60°C and a reaction time of 1-4 hours.

7. Use of the tannic acid-functionalized micromotor with enhanced bacteria capture and antibacterial effects according to any one of claims 1 to 6 in capturing and killing pathogenic microorganisms.

8. The use according to claim 7, characterized in that The pathogenic microorganisms are Escherichia coli and methicillin-resistant Staphylococcus aureus.

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