Nano material with photodynamic and sonodynamic combined antibacterial effect as well as preparation method and application of nano material

Abstract

The invention discloses a nano material with photodynamic and sonodynamic combined antibacterial effects and a preparation method and application thereof, and the nano material is prepared by loading eugenol and chlorin e6 on a metal organic framework material with a zeolite topological structure. The ternary nano composite material prepared by the invention can synergistically enhance photodynamic and sonodynamic effects, and active oxygen ROS is fully utilized. The material prepared by the invention has a remarkable antibacterial effect on staphylococcus aureus and escherichia coli, and has a remarkable inhibition effect on bacterial biofilms. The prepared material has multiple antibacterial mechanisms such as the bacteriostatic effect of natural antibacterial components, the bacteriostatic effect of ROS generated by photodynamic force and the penetrating effect improved by ultrasound, the antibacterial spectrum is widened, the overall antibacterial efficacy is enhanced, the used material is in the food grade, the overall preparation method is simple and easy to implement, and a new thought is provided for food bacteriostasis and preservation.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of food industry and nanotechnology, and particularly relates to a nano material with a combined photodynamic and sonodynamic antibacterial effect and a preparation method and application thereof. BACKGROUND

[0002] Fresh food is prone to microbial contamination and spoilage during storage and transportation. Traditional chemical preservatives have safety concerns and limited effectiveness, and physical sterilization methods often face the problems of poor penetration and impact on food quality. Photodynamic antibacterial technology (PDT) as a new non-thermal sterilization method uses photosensitizers to produce reactive oxygen species (ROS) to kill microorganisms under specific light excitation, has the advantages of high efficiency, low residue and difficulty in developing drug resistance, and shows great potential in food preservation. However, the effect of single PDT is often limited by the depth of light penetration and the complex matrix of food. Sonodynamic antibacterial technology (SDT) uses sonosensitizers to generate ROS under the action of ultrasound, which can make up for the deficiency of PDT in penetration. Dihydrophenanthrene e6 (Ce6) is derived from chlorophyll and is a highly efficient natural photosensitizer, but its water solubility is poor, it is easy to aggregate and inactivate, and its targeting performance in food matrix is poor, which limits its practical application. Metal-organic framework material ZIF-8 has high drug loading capacity, pH-responsive degradation, good biocompatibility and "nano reactor" effect on ROS, but there is no report on loading Ce6 and plant-derived antibacterial agent eugenol (EU) in ZIF-8 at the same time. There is also no report on using the obtained composite material as a photosensitizer and sonosensitizer to realize the synergistic antibacterial preservation of PDT-SDT in food storage. SUMMARY

[0003] The application provides a nano material with a combined photodynamic and sonodynamic antibacterial effect and a preparation method thereof, which can synergistically enhance the photodynamic and sonodynamic effects, fully utilize reactive oxygen species ROS, and maintain high and stable antibacterial activity in the storage environment of fresh food, thereby effectively prolonging the shelf life of food.

[0004] The application also provides the application of the nano material with a combined photodynamic and sonodynamic antibacterial effect.

[0005] Technical scheme: In order to achieve the above-mentioned purpose, the nano material with a combined photodynamic and sonodynamic antibacterial effect is prepared by loading eugenol (EU) and dihydrophenanthrene e6 (Ce6) on a metal-organic framework material with a zeolite topological structure.

[0006] The metal-organic framework material with a zeolite topological structure is ZIF-8, which is formed by the reaction of zinc acetate dihydrate and 2-methyl imidazole.

[0007] The preparation method of the nanomaterial with the antibacterial effect of the combination of the photodynamic effect and the sonodynamic effect comprises the following steps:

[0008] (1) The preparation method of ZIF-8 is as follows: zinc acetate dihydrate and 2-methylimidazole are respectively dissolved in deionized water, the zinc acetate dihydrate solution is added to the 2-methylimidazole solution, the stirred suspension is centrifuged, and the precipitate is washed and dried for 12-24 hours to obtain the required sample;

[0009] (2) The synthesis method of EU / Ce6@ZIF-8 is as follows:

[0010] Zinc acetate dihydrate and 2-methylimidazole are respectively dissolved in deionized water, then eugenol solution and chlorin e6 solution are respectively prepared, the eugenol solution and the chlorin e6 solution are mixed with the 2-methylimidazole solution and stirred uniformly, the prepared zinc acetate dihydrate solution is added dropwise, stirring is continued, after the reaction is completed, the precipitate is collected by centrifugation, washed and dried to obtain EU / Ce6@ZIF-8 powder.

[0011] The molar ratio of the zinc acetate dihydrate to the 2-methylimidazole is 1:20-50.

[0012] The mass ratio of the eugenol solution to the chlorin e6 is 10-20:1.

[0013] The volume ratio of the eugenol solution, the chlorin solution, the zinc acetate dihydrate solution and the 2-methylimidazole solution is 1:1:1:2-5.

[0014] After the reaction is completed, the nanoparticles are collected by centrifugation, washed with methanol three times after centrifugation, and dried in a 30 DEG C vacuum drying oven for 12-24 hours to obtain EU / Ce6@ZIF-8 powder.

[0015] The application of the nanomaterial with the antibacterial effect of the combination of the photodynamic effect and the sonodynamic effect to the preparation of an antibacterial agent.

[0016] The antibacterial agent includes Staphylococcus aureus and Escherichia coli.

[0017] The antibacterial product contains the nanomaterial with the antibacterial effect of the combination of the photodynamic effect and the sonodynamic effect.

[0018] The EU / Ce6@ZIF-8 composite nanomaterial prepared by the method is characterized by SEM, TEM, XRD, FT-IR and TGA, and the influence of the nanomaterial on the generation of singlet oxygen is evaluated.

[0019] The application is through mixing ZIF-8, EU, EU / Ce6@ZIF-8 three bacteriostatic materials with staphylococcus aureus and escherichia coli liquid, and then treating under the condition of light treatment: exposure for 5 min under 650 nm laser, and the light intensity is 15 mW / cm 2 , and the condition of light combined with ultrasonic treatment is: the light is 15 mW / cm 2 of 650 nm red light, and the ultrasonic power is 100 W.

[0020] Further, the bacteriostatic activity of the composite nanomaterial on staphylococcus aureus and escherichia coli is evaluated by adopting colony counting method, and the bacteriostatic effect of the photodynamic combined with the sonodynamic is evaluated.

[0021] Further, the inhibition effect of the bacteriostatic agent on the bacterial biofilm is explored by measuring the change of the bacterial biofilm biomass after the bacteriostatic material is added into the bacterial liquid.

[0022] The application is through loading Ce6 and the plant source antibacterial agent eugenol (EU) in ZIF-8 at the same time, and the obtained composite material is used as a photosensitizer and a sonosensitizer at the same time, so that the application of PDT-SDT synergistic bacteriostatic preservation in food storage and transportation is realized. The application develops a ternary nanosystem of EU / Ce6 / ZIF-8, which can effectively load and protect the natural photosensitizer, and can construct a stable and efficient light-sonodynamic synergistic antibacterial platform, so that ROS is efficiently generated under the synergistic action of a low-dose light source and ultrasonic, and the film targeting destruction of the natural antibacterial agent is also possessed. The application has significant technical and economic value for breaking through the pollution of drug-resistant bacteria in the process of fresh food storage and transportation, prolonging the shelf life and reducing the dependence on chemical preservatives.

[0023] The core innovation of the application is not the improvement of the antibacterial performance of a single component, but the construction of a “chemical auxiliary-light dynamic-sonodynamic” three-in-one composite antibacterial mechanism, which breaks through the defects of traditional single light dynamic antibacterial “weak penetration, limited ROS diffusion range”, and single chemical antibacterial “easy to produce drug resistance, short time limit”. The application verifies the light dynamic-sonodynamic combined antibacterial effect of the ternary composite material of EU / Ce6@ZIF-8.

[0024] Advantages: compared with the prior art, the application has the following advantages:

[0025] The ternary nanocomposite material of EU / Ce6@ZIF-8 prepared by the application can generate an excess of reactive oxygen species (ROS) under the action of light, and the ultrasonic action is helpful to improve the penetration of cells, which is beneficial to the ROS generated by the light dynamic to enter the bacterial cells, so that the light-sonodynamic synergistic bacteriostatic effect is remarkable, and the antibacterial efficiency is greatly improved.

[0026] The ZIF-8 skeleton effectively coats and protects the photosensitizer Ce6 and the natural bacteriostatic agent eugenol, significantly improves the stability of the two in a complex food environment (such as salt, different pH values, light), reduces inactivation and degradation. Meanwhile, the material has good sustained-release properties, can continuously release active ingredients for a long time, and prolongs the antibacterial preservation time.

[0027] The application innovatively co-loads the efficient photosensitizer Ce6 with good sonosensitization activity and the natural component eugenol with inherent bacteriostatic activity in ZIF-8. Ce6 dominates the high-efficiency photodynamic effect and can effectively enhance the photoacoustic dynamic response, and the volatile bacteriostatic component of eugenol itself has the function of destroying the bacterial cell membrane, can synergize with ROS, forms a multiple antibacterial barrier, widens the antibacterial spectrum and enhances the overall bacteriostatic efficiency.

[0028] The material used in the application includes the natural photosensitizer Ce6 and the natural bacteriostatic component eugenol recognized as safe, and the biocompatible ZIF-8 carrier, and the material is high in safety, meets the requirements of food-grade application. The preparation process is simple, the conditions are mild, the raw materials are easy to obtain, has good repeatability and large-scale production prospect. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Fig. 1 is a scanning electron microscope image of EU / Ce6@ZIF-8, a transmission electron microscope image and particle size and zeta potential of ZIF-8 and EU / Ce6@ZIF-8.

[0030] Figure 2 Fig. 3 is a characterization diagram of different materials, (a) Fourier infrared spectrum, (b) XRD spectrum, (c) thermal gravimetric analysis diagram.

[0031] Figure 3 Fig. 4 is the ROS production of different materials.

[0032] Figure 4 Fig. 5 is the survival rate of Staphylococcus aureus in the bacterial solution and the survival rate of Escherichia coli in the bacterial solution.

[0033] Figure 5 Fig. 6 is the antibacterial effect of EU / Ce6@ZIF-8 on Escherichia coli.

[0034] Figure 6 Fig. 7 is the antibacterial effect of EU / Ce6@ZIF-8 on Staphylococcus aureus.

[0035] Figure 7 Fig. 8 is a scanning electron microscope analysis diagram of Staphylococcus aureus.

[0036] Figure 8 Fig. 9 is a scanning electron microscope analysis diagram of Escherichia coli.

[0037] Figure 9 (a)(c) are the inhibitory effects of EU / Ce6@ZIF-8 on Staphylococcus aureus biofilm, and (b)(d) are the inhibitory effects of EU / Ce6@ZIF-8 on Escherichia coli biofilm. DETAILED DESCRIPTION

[0038] The application will be further described below in conjunction with examples.

[0039] The experimental methods described in the examples are all conventional methods unless otherwise specified; and the reagents and materials described are all commercially available unless otherwise specified.

[0040] Example 1

[0041] The ternary composite nanomaterial with the combined photodynamic and sonodynamic antibacterial effect of the present example is prepared according to the following steps:

[0042] The preparation method of ZIF-8 is as follows: 0.2 mmol of zinc acetate dihydrate is dissolved in 1 mL of deionized water, and 6 mmol of 2-methylimidazole is dissolved in 3 mL of deionized water. After complete dissolution, the zinc acetate dihydrate solution is added to the 2-methylimidazole solution under stirring for 15 min. During stirring, the solution changes from transparent to milky suspension. The stirred suspension is centrifuged at a speed of 10000 rpm for 10 min. After centrifugation, the precipitate is washed with methanol three times to obtain a white substance. The white substance is placed in a 30 ℃ vacuum drying oven for drying for 12 h to obtain the required sample ZIF-8.

[0043] The synthesis method of EU / Ce6@ZIF-8 is as follows: 0.2 mmol of zinc acetate dihydrate is dissolved in 1 mL of deionized water, and 6 mmol of 2-methylimidazole is dissolved in 3 mL of deionized water. Then, 15 mg / mL of eugenol methanol solution (EU solution) and 1 mg / mL of chlorin e6 methanol solution (Ce6 solution) are prepared respectively. 1 mL of eugenol solution (EU) and 1 mL of Ce6 solution are mixed with the 2-methylimidazole solution and stirred uniformly. After 5 min, the prepared zinc acetate dihydrate solution is added dropwise, and stirring is continued for 30 min. After the reaction is completed, the nanoparticles are collected by centrifugation (10000 rpm, 10 min). After centrifugation, the precipitate is washed with methanol three times and dried in a 30 ℃ vacuum drying oven for 12 h to obtain EU / Ce6@ZIF-8 powder.

[0044] The characterization method of the antibacterial material is as follows:

[0045] The particle size and zeta potential of 0.2 mg / L of ZIF-8 and EU / Ce6@ZIF-8 water suspensions are determined by a Malvern laser particle size analyzer.

[0046] SEM (Scanning Electron Microscopy) and TEM (Transmission Electron Microscopy): Morphological characteristics of EU / Ce6@ZIF-8 samples were analyzed. An appropriate amount of EU / Ce6@ZIF-8 was dissolved in methanol solution and sonicated for 30 min to ensure complete dispersion. Five μL of each of the two completely dispersed samples was dropped onto appropriately sized silicon wafers, which were then attached to the sample stage using conductive adhesive. The sample stage was sputter-coated with gold for 60 s, and the samples were observed using an electron microscope.

[0047] Fourier transform infrared spectroscopy (FTIR): The chemical structures of eugenol EU, Ce6, and EU / Ce6@ZIF-8 samples were analyzed using the ATR-FTIR method.

[0048] Thermogravimetric analysis (TGA): This analyzes the thermal stability of the samples. Samples need to be dried for a period of time to remove moisture before thermogravimetric analysis. The test conditions are: under nitrogen atmosphere, the temperature is increased from room temperature to 800 °C at a rate of 10 °C / min. The thermal stability of eugenol, Ce6, ZIF-8, and EU / Ce6@ZIF-8 samples is analyzed.

[0049] X-ray diffraction (XRD): The effects of EU and Ce6 embedding on the ZIF-8 crystal structure were analyzed using XRD.

[0050] from Figure 1 The particle size results show that the ZIF-8 particles prepared by this method have a particle size of 66.5 nm. The EU / Ce6@ZIF-8 particles are larger than the standard ZIF-8 particles, with a particle size of 109.7 nm, indicating that the particle size of ZIF-8 increases after encapsulating EU and Ce6. Scanning electron microscopy and transmission electron microscopy show that EU / Ce6@ZIF-8 exhibits a similar rhombic dodecahedral morphology. Figure 2 FTIR results show that EU and Ce6 are effectively embedded within the internal structure of EU / Ce6@ZIF-8. XRD results indicate that the crystal form of EU / Ce6@ZIF-8 did not change significantly after embedding. Thermogravimetric analysis results show that EU / Ce6@ZIF-8 improves thermal stability compared to EU and Ce6 alone.

[0051] Example 2

[0052] The influence of EU / Ce6@ZIF-8 nanomaterials on ROS generation.

[0053] The singlet oxygen content of the EU / Ce6@ZIF-8 nanocomposite was evaluated using the chemical probe 1,3-diphenylisobenzofuran (DPBF). 1 O2) is generated, and the probe is... 1O2 causes an irreversible reaction, leading to DPBF degradation. For this assay, 1 mL of DPBF solution (20 μg / mL DMSO solution) was added to dispersions of Ce6, ZIF-8, and EU / Ce6@ZIF-8 (20 μg / mL for each dispersion, 1 mL). The samples were then exposed to a power density of 15 mW / cm². 2 The absorption peak intensities were recorded at different times using a 650 nm laser. The absorption peak of DPBF at 410 nm was continuously monitored, with DPBF used alone as a negative control. All experimental procedures were performed under light-confined conditions to prevent light interference.

[0054] The results are as follows Figure 3 As shown, the rate of decrease in absorbance is related to 1 The O2 production is directly proportional. Under 650 nm red light irradiation, the absorbance of free Ce6 decreases rapidly, while the absorbance of EU / Ce6@ZIF-8 gradually decreases with increasing irradiation time, indicating that EU / Ce6@ZIF-8 contains... 1 The O2 generation efficiency is higher, and the encapsulated ternary complex synergistically enhances the photodynamic generation of reactive oxygen species.

[0055] Example 3

[0056] The antibacterial properties of EU / Ce6@ZIF-8 nanomaterials were analyzed. Antibacterial experiments were conducted on Gram-negative bacteria *Escherichia coli* and Gram-positive bacteria *Staphylococcus aureus* to assess the antibacterial effect of the EU / Ce6@ZIF-8 nanomaterials prepared in Example 1. The specific steps are as follows:

[0057] To analyze the inhibitory effect on bacterial growth, wild-type Escherichia coli and Staphylococcus aureus in the logarithmic growth phase were analyzed at concentrations of OD600. nm = 0.1, inoculated into LB liquid medium containing EU / Ce6@ZIF-8 (concentration of 0.5 mg / mL) (with sterile deionized water as the control group). The next treatment was divided into three groups, one of which exposed the culture to a power density of 15 mW / cm². 2 One group was treated with 650 nm red light for 5 minutes, and the other group was exposed to 15 mW / cm² light. 2 The cells were treated with 650 nm red light and 100 W sonication for 5 minutes, with one control group incubated in the dark without light or sonication. They were then incubated at 37 °C for 4 hours. The cells were inoculated onto LB agar using the dilution plating method, and colony counts were performed after single colonies formed.

[0058] Antibacterial results as follows Figure 4As shown, EU / Ce6@ZIF-8 with a concentration of 0.5 mg / mL had a certain inhibitory effect on S. aureus and E. coli under dark conditions, with inhibition rates of 38% and 25%, respectively. The inhibition rate was greatly improved under simple light treatment, and the inhibitory effect was best under the combined treatment of light and ultrasound: the inhibition rates of 0.5 mg / mL EU / Ce6@ZIF-8 on S. aureus and E. coli were 80% and 76%, respectively. From Figure 5 and Figure 6 It can be seen that under the combined treatment of light and ultrasound, only a few colonies grew on the EU / Ce6@ZIF-8 culture plate, which was significantly reduced compared with the control group. Figure 7 and Figure 8 It can be seen that compared with the control group, the bacterial morphology of the EU / Ce6@ZIF-8 treatment group changed significantly, the bacterial surface roughness increased, and the cell membrane appeared wrinkled and even ruptured. The above results show that EU / Ce6@ZIF-8 has a significant synergistic antibacterial effect under the combined action of light and ultrasound.

[0059] Example 4

[0060] Inhibition of EU / Ce6@ZIF-8 nanomaterials on bacterial biofilm.

[0061] The analysis of biofilm biomass was carried out by ultraviolet-visible light measurement. Wild-type E. coli and S. aureus were cultured to an optical density (OD600 nm) of 0.1. 100 μL of the cultured bacteria were added to each well of a 96-well plate, followed by the addition of 100 μL of the prepared EU / Ce6@ZIF-8 nanomaterial suspension with a concentration of 2 mg / mL. After 20 hours of culture, the culture medium was discarded and the wells were gently washed with PBS three times to eliminate planktonic bacteria. The bacterial biofilm was fixed with methanol for 15 minutes, then stained with 0.1% crystal violet solution for 20 minutes. After staining, the wells were carefully washed twice with PBS to remove excess dye, and then air-dried for 1 hour. After the drying process was completed, 33% acetic acid solution was added to the wells, and the absorbance of the remaining crystal violet at 600 nm was measured using an enzyme marker to analyze the biomass of the biofilm.

[0062] Biofilm is the excretion produced during bacterial growth, mainly including polysaccharides, proteins and other extracellular matrix, which has a protective barrier effect on bacteria and helps bacteria to develop drug resistance. From Figure 9As can be seen from the results, under the synergistic effect of light and ultrasound, the biofilm of E. coli in the EU / Ce6@ZIF-8 treatment group decreased by 84.21%, and the biofilm of S. aureus decreased by 86.11% compared with the control group. This shows that the combination of photodynamic and sonodynamic treatment and the release of EU have a significant inhibitory effect on bacterial biofilm, further demonstrating the excellent antibacterial effect of EU / Ce6@ZIF-8.

Claims

1. A nanomaterial having a combined photodynamic and sonodynamic antibacterial effect, characterized in that, The nanomaterial takes a metal organic framework material with a zeolite topology as a carrier, and loads eugenol (EU) and chlorin e6 (Ce6).

2. The nano-material with the combined photo-dynamic and sonodynamic antibacterial effect according to claim 1, characterized in that, The metal organic framework material with a zeolite topology is ZIF-8, which is formed by reacting zinc acetate dihydrate and 2-methylimidazole.

3. The method for preparing the nano-material with the combined antibacterial effect of the photodynamic and sonodynamic effects according to claim 2, characterized in that, The method comprises the following steps: (1) The ZIF-8 is prepared as follows: zinc acetate dihydrate and 2-methylimidazole are respectively dissolved in deionized water, the zinc acetate dihydrate solution is added to the 2-methylimidazole solution, the stirred suspension is centrifuged, the precipitate is washed and dried for 12-24 hours, and the desired sample is obtained; (2) The synthesis method of EU / Ce6@ZIF-8 is as follows: Zinc acetate dihydrate and 2-methylimidazole are respectively dissolved in deionized water, then eugenol solution and chlorin e6 solution are respectively prepared, the eugenol solution and the chlorin e6 solution are mixed with the 2-methylimidazole solution and stirred uniformly, the prepared zinc acetate dihydrate solution is added dropwise, stirring is continued, after the reaction is completed, the precipitate is collected by centrifugation, washed and dried, and EU / Ce6@ZIF-8 powder is obtained.

4. The method for preparing nanomaterials with combined photodynamic and acoustic antibacterial effects according to claim 3, characterized in that, The molar ratio of the zinc acetate dihydrate to the 2-methylimidazole is 1:20-50.

5. The method for preparing nanomaterials with combined photodynamic and acoustic antibacterial effects according to claim 3, characterized in that, The mass ratio of the eugenol solution to the chlorin e6 is 10-20:

1.

6. The method for preparing nanomaterials with combined photodynamic and acoustic antibacterial effects according to claim 3, characterized in that, The volume ratio of the eugenol solution, the chlorin solution, the zinc acetate dihydrate solution and the 2-methylimidazole solution is 1:1:1:2-5.

7. The method for preparing nanomaterials with combined photodynamic and acoustic antibacterial effects according to claim 3, characterized in that, After the reaction is completed, the nanoparticles are collected by centrifugation, washed and dried, and EU / Ce6@ZIF-8 powder is obtained.

8. The use of the nanomaterial with the photodynamic combined sonodynamic antibacterial effect of claim 1 in combination with light and ultrasound in the preparation of an antibacterial agent.

9. Use according to claim 8, characterized in that, The antibacterial agent includes Staphylococcus aureus and Escherichia coli.

10. An antibacterial product containing the nanomaterial with the photodynamic combined sonodynamic antibacterial effect of claim 1.

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