Preparation method of polyphenol nanodot sound-sensitive agent and application of polyphenol nanodot sound-sensitive agent in sonodynamic antibiosis

Abstract

The invention is suitable for the technical field of sonodynamic antibacterial materials, and provides a preparation method of a polyphenol nanodot sonosensitizer and application of the polyphenol nanodot sonosensitizer in sonodynamic antibiosis. The method comprises the following steps: dissolving tannic acid (TA) in deionized water, stirring at room temperature until the tannic acid is completely dissolved, carrying out oxidative polymerization reaction on the dissolved solution, assembling the tannic acid through oxidative polymerization to obtain the polyphenol nanodot sound-sensitive agent (TANDs), cooling after the reaction is finished, collecting the product, filtering, transferring the filtrate into a dialysis bag for dialysis, changing water once every 6 hours, and drying to obtain the polyphenol nanodot sound-sensitive agent (TANDs). Freeze-drying the dialyzed solution into solid powder, and dispersing the freeze-dried solid powder into deionized water for later use; tA is used as an element, the TANDs with the sound-sensitive property are prepared through simple oxidative polymerization reaction self-assembly, the TANDs show excellent singlet oxygen (1O2) generation capacity under the ultrasonic (US) condition, and the TANDs also show excellent antibacterial performance in the actual antibacterial application process.

Description

Technical Field

[0001] The invention belongs to the technical field of sonodynamic antibacterial materials, and in particular relates to a preparation method of a polyphenol nanodot sonosensitizer and application thereof in sonodynamic antibacterial. Background Art

[0002] In daily life, there is a complex symbiotic relationship between the human body and bacteria. When the bacterial population is out of balance, it will cause a series of bacterial infectious diseases, such as pneumonia, wound infection and periodontitis. Some local inflammatory diseases caused by bacterial infection may evolve into systemic diseases such as sepsis if they are not treated in time or improperly handled, posing a serious threat to human health and becoming a major public health issue worldwide. Therefore, it is particularly important to propose efficient and convenient antibacterial strategies and treatment methods, but it also faces huge challenges.

[0003] At present, antibiotic treatment or antibiotic-assisted administration is considered to be one of the most effective treatments for bacterial infectious diseases. However, long-term use of antibiotics can lead to bacterial resistance, affecting the treatment effect. In order to solve this problem, sonodynamic antibacterial strategy has emerged as a new type of non-invasive antibacterial method. This strategy uses low-intensity ultrasound (US) to activate sonosensitizers to produce reactive oxygen species (ROS), causing serious damage to bacterial membranes, DNA, enzymes and proteins, and combined with the cavitation effect produced during the ultrasound process, synergistically induces bacterial death. The penetration depth of US can reach more than 10 cm. Compared with traditional photodynamic therapy, sonodynamic therapy has higher tissue penetration, which makes up for the defect of insufficient penetration ability of photodynamic therapy light source. In addition, the cavitation effect generated by ultrasound can effectively reduce the density of biofilms, which is conducive to the removal of biofilms and improves the bioavailability of materials.

[0004] However, there are still some problems with the sonosensitizers used in current sonodynamic antibacterial strategies. Inorganic sonosensitizers are often derived from traditional inorganic photosensitizers such as TiO 2 Or carbon nitride and some derivatives, have a long retention time in the body, are difficult to degrade, and have certain biological toxicity. Traditional organic sonosensitizers face the problems of strong hydrophobicity, poor biocompatibility and low ROS yield in practical applications. Therefore, it is necessary to improve the biocompatibility of sonosensitizers and explore new sonosensitizers to give full play to their performance. To this end, the present invention proposes a preparation method of a polyphenol nanodot sonosensitizer and its application in sonodynamic antibacterial. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a polyphenol nanodot sonosensitizer and its application in sonodynamic antibacterial, aiming to solve the problems raised in the above background technology.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] Preparation method of polyphenol nanodot sonosensitizer, comprising the following steps:

[0008] Dissolve tannic acid in deionized water, stir at room temperature until completely dissolved, perform oxidative polymerization reaction on the dissolved solution, and obtain polyphenol nanodot sonosensitizer through oxidative polymerization assembly of tannic acid. After the reaction is completed, cool, collect the product and filter it, transfer the filtrate to a dialysis bag for dialysis, change the water every 6 h, lyophilize the dialyzed solution into a solid powder, and finally disperse the lyophilized solid powder in deionized water for standby.

[0009] Further, the addition ratio of tannic acid to deionized water is 1 g:50 mL.

[0010] Further, the dissolved solution reacts at 75-90 °C for 12 h.

[0011] Further, the product is filtered using a 0.22 μm aqueous filter head.

[0012] Further, transfer the filtrate to a dialysis bag with a molecular weight of 3000 for dialysis for 48 h.

[0013] Polyphenol nanodot sonosensitizer prepared according to the preparation method of the polyphenol nanodot sonosensitizer described above.

[0014] Application of the polyphenol nanodot sonosensitizer described above in the preparation of sonodynamic antibacterial materials.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] The present invention uses natural polyphenol monomer tannic acid (TA) as a building block, and a novel polyphenol nanodot sonosensitizer (TANDs) with sonosensitive properties is prepared through simple oxidative polymerization reaction and self-assembly. Under ultrasonic (US) conditions, it exhibits excellent singlet oxygen ( 1 O 2 ) generation ability, and TANDs also exhibit excellent antibacterial performance during actual antibacterial applications. Description of the drawings

[0017] Figure 1 It is a morphological and size characteristic diagram of TA NDs; wherein (a) is a transmission electron microscope photograph of TANDs, and (b) is a statistical diagram of the particle size distribution of TANDs.

[0018] Figure 2UV absorption spectra of TA monomer and TA NDs, detection results of TA monomer during the oxidative polymerization self-assembly process, and detection results of TA NDs; among them, (a) is the UV absorption spectra of TA monomer and TA NDs, (b) is the detection results of TA monomer at different time intervals under room temperature (RT) conditions, and (c) is the detection results of TA NDs at different time intervals under 80 °C conditions.

[0019] Figure 3 It is the fluorescence spectrum of TA NDs.

[0020] Figure 4 It is the infrared spectrum of TA NDs.

[0021] Figure 5 It is the Zeta potential map of TA NDs.

[0022] Figure 6 It is the detection of the phenolic hydroxyl content in TA NDs.

[0023] Figure 7 It is the femtosecond transient absorption spectra of TA monomer and TANDs; among them, (a) is the femtosecond transient absorption spectrum of TA monomer, and (b) is the femtosecond transient absorption spectrum of TA NDs.

[0024] Figure 8 It is the total ROS production test of the Ctrl group, TA monomer group, and TA NDs group under ultrasonic conditions; among them, (a) is the total ROS production test of the Ctrl group under ultrasonic conditions, (b) is the total ROS production test of the TA monomer group under ultrasonic conditions, and (c) is the total ROS production test of the TA NDs group under ultrasonic conditions.

[0025] Figure 9 It is the electron paramagnetic resonance test for the activation of TA NDs to generate singlet oxygen ( 1 O 2 ) under ultrasonic conditions; among them, (a) is the electron paramagnetic resonance spectrum of TA NDs after 5 min of ultrasonic treatment, (b) is the electron paramagnetic resonance spectrum of TA NDs at different time intervals of ultrasonic treatment, and (c) is the fitting curve of the yield change of singlet oxygen ( 1 O 2 ) of TA NDs during 0 - 5 min of ultrasonic treatment.

[0026] Figure 10 It is to explore the adhesion of TA NDs to bacteria using a quartz crystal microbalance analyzer (QCM); among them, (a) is the adhesion result of TA to bacteria, and (b) is the adhesion result of TA NDs to bacteria.

[0027] Figure 11Performance test of TA NDs for sonodynamic antibacterial (S. mutans, E. faecalis); among which (a) is the agar plate photos of S. mutans and E. faecalis after being treated with each group of materials, (b) is the bacterial survival rate of S. mutans after being treated with each group of materials, and (c) is the bacterial survival rate of E. faecalis after being treated with each group of materials.

[0028] Figure 12 Morphological changes of S. mutans and E. faecalis after being treated with each group of materials.

[0029] Figure 13 Detection of ROS production in bacteria using the fluorescent probe DCFH-DA.

[0030] Figure 14 Determination of the biofilm removal effect of the materials; among which (a) is the fluorescence photos of the biofilm after being treated with each group of materials, and (b) is the SEM photos of the biofilm removal effect after being treated with each group of materials.

[0031] Figure 15 Cytotoxicity evaluation of TA NDs on mouse fibroblasts (L929); among which (a) is the Live / Dead staining results of PBS, TA, and TANDs, and (b) is the Live / Dead staining results of 6 groups of experiments. Detailed implementation manners

[0032] For a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solutions of the present invention are described in detail below, but it should not be construed as a limitation on the implementable scope of the present invention.

[0033] The present invention provides a preparation method of a polyphenol nanodot photosensitizer, comprising the following steps:

[0034] Natural polyphenol monomer tannic acid (TA) is dissolved in deionized water (the addition ratio of TA to deionized water is 1 g:50 mL), stirred at room temperature until completely dissolved, and the dissolved solution is reacted at 75-90 °C for 12 h. The tannic acid is assembled by oxidative polymerization to obtain the polyphenol nanodot photosensitizer (TANDs). After the reaction, it is cooled, the product is collected and filtered using a 0.22 μm aqueous filter head, the filtrate is transferred to a dialysis bag with a molecular weight of 3000 and dialyzed for 48 h, and the water is changed every 6 h. The dialyzed solution is freeze-dried into a solid powder, and finally the freeze-dried solid powder is dispersed in deionized water for standby.

[0035] The following describes the specific implementation of the present invention in detail with specific embodiments.

[0036] Example 1: 1 g of TA was dissolved in 50 mL of deionized water and stirred at room temperature until completely dissolved. The resulting solution was reacted in a water bath at 75 °C for 12 h. The tannic acid was assembled into polyphenol nanodot sonosensitizer (TANDs) through oxidative polymerization. After the reaction, it was cooled, the product was collected and filtered using a 0.22 μm aqueous filter head. The filtrate was transferred to a dialysis bag with a molecular weight cut-off of 3000 and dialyzed for 48 h, changing the water every 6 h. The dialyzed solution was lyophilized into a solid powder, and finally the lyophilized solid powder was dispersed in deionized water for standby.

[0037] Example 2: 1 g of TA was dissolved in 50 mL of deionized water and stirred at room temperature until completely dissolved. The resulting solution was reacted in a water bath at 80 °C for 12 h. The tannic acid was assembled into polyphenol nanodot sonosensitizer (TANDs) through oxidative polymerization. After the reaction, it was cooled, the product was collected and filtered using a 0.22 μm aqueous filter head. The filtrate was transferred to a dialysis bag with a molecular weight cut-off of 3000 and dialyzed for 48 h, changing the water every 6 h. The dialyzed solution was lyophilized into a solid powder, and finally the lyophilized solid powder was dispersed in deionized water for standby.

[0038] Example 3: 1 g of TA was dissolved in 50 mL of deionized water and stirred at room temperature until completely dissolved. The resulting solution was reacted in a water bath at 85 °C for 12 h. The tannic acid was assembled into polyphenol nanodot sonosensitizer (TANDs) through oxidative polymerization. After the reaction, it was cooled, the product was collected and filtered using a 0.22 μm aqueous filter head. The filtrate was transferred to a dialysis bag with a molecular weight cut-off of 3000 and dialyzed for 48 h, changing the water every 6 h. The dialyzed solution was lyophilized into a solid powder, and finally the lyophilized solid powder was dispersed in deionized water for standby.

[0039] Example 4: 1 g of TA was dissolved in 50 mL of deionized water and stirred at room temperature until completely dissolved. The resulting solution was reacted in a water bath at 90 °C for 12 h. The tannic acid was assembled into polyphenol nanodot sonosensitizer (TANDs) through oxidative polymerization. After the reaction, it was cooled, the product was collected and filtered using a 0.22 μm aqueous filter head. The filtrate was transferred to a dialysis bag with a molecular weight cut-off of 3000 and dialyzed for 48 h, changing the water every 6 h. The dialyzed solution was lyophilized into a solid powder, and finally the lyophilized solid powder was dispersed in deionized water for standby.

[0040] Example 5: 500 mg of TA was dissolved in 25 mL of deionized water and stirred at room temperature until completely dissolved. The resulting solution was placed in a reaction kettle and reacted at 80 °C for 12 h. The tannic acid was assembled into polyphenol nanodot sonosensitizer (TANDs) through oxidative polymerization. After the reaction, it was cooled, the product was collected and filtered using a 0.22 μm aqueous filter head. The filtrate was transferred to a dialysis bag with a molecular weight cut-off of 3000 and dialyzed for 48 h, changing the water every 6 h. The dialyzed solution was lyophilized into a solid powder, and finally the lyophilized solid powder was dispersed in deionized water for standby.

[0041] Next, the products obtained in Example 2 were used for the following investigations:

[0042] (1) Relevant data on the morphology of TA NDs.

[0043] Figure 1 In (a) is the transmission electron microscope image of TA NDs, taken using a JEM-2100F electron microscope (200 kV). From Figure 1 In (b), it was observed that TA NDs are nanodots with a diameter in the range of 1 - 3 nm, and the average particle size is 2.03 nm.

[0044] (2) Relevant data on the structure of TA NDs.

[0045] Figure 2 In (a) is the ultraviolet absorption spectrum of TA monomer and TA NDs, tested using a Shimadzu 2600 ultraviolet-visible spectrometer. It was observed that compared with the TA monomer, the absorption of TANDs obtained by oxidative polymerization assembly at 80 °C is significantly enhanced near 350 nm. This is because during the oxidative polymerization process, the phenolic hydroxyl groups are gradually oxidized to form o-benzoquinone structures. After the polymerization of TA monomers, rich conjugated structures are formed, the band gap becomes narrower, and the absorption of TANDs undergoes a red shift. As Figure 2 shown in (b), the TA monomer at room temperature was detected at different time intervals. Its ultraviolet absorption spectrum did not change significantly within 0 - 12 h, indicating that the TA monomer does not undergo oxidative polymerization at room temperature. As Figure 2 shown in (c), the TANDs products at 80 °C were detected at different time intervals to investigate the kinetics of the oxidative polymerization self-assembly process. It was found that as the reaction time continuously extended, the ultraviolet absorption of the product near 350 nm gradually increased, showing a positive correlation with the reaction time.

[0046] Figure 3 This is the fluorescence spectrum of TA NDs, tested using a Shimadzu RF-6000 fluorescence spectrometer. The excitation wavelength is 365 nm. The TA monomer has an obvious emission near 455 nm. When excited at the same concentration with the same excitation wavelength of 365 nm, the emission of TANDs is red-shifted to 470 nm compared with the TA monomer, and the fluorescence intensity weakens.

[0047] Figure 4 This is the infrared spectrum of TA NDs, tested using a Bruker VERTEX 80V infrared spectrometer. The infrared spectrum of TA NDs is in the range of 1620 - 750 cm -1The relative intensity of multiple peaks within the range decreases, which is due to the changes in C-H, phenolic hydroxyl groups, and benzene rings on the benzene ring in the product TANDs after the oxidative polymerization of TA; 3500 - 3200 cm -1 The range is for the stretching vibration of intermolecular hydrogen bond O-H. The vibration peak in this range of TA NDs is significantly narrower than that of TA, indicating a reduction in intermolecular hydrogen bonds after oxidative polymerization.

[0048] Figure 5 This is the Zeta potential diagram of TA NDs, which was tested using a Malvern Zetasizer Pro. It was observed that the surface potential of TANDs is negative, with a potential of -32.1 ± 0.4 mV.

[0049] Figure 6 This is the detection of the phenolic hydroxyl group content in TA NDs - the Folin-Ciocalteu method. The absorbance at 750 nm after the reaction of Folin-Ciocalteu with TA NDs was measured using an InfiniteM Nano full-wavelength light absorption microplate reader. Compared with TA monomers, the phenolic hydroxyl group content in TANDs did not change significantly.

[0050] Figure 7 This is the femtosecond transient absorption spectrum of TA monomers and TA NDs, which was tested using an Ultrafast Systems, HeliosFire femtosecond transient absorption spectrometer with an excitation wavelength of 365 nm. From Figure 7 It can be seen from (a) and (b) that TA NDs show obvious excited state absorption (ESA) compared with TA monomers, proving that TA NDs have rich excited state energy levels. The TA NDs prepared by self-assembly through oxidative polymerization effectively improve the non-radiative transition efficiency of the excited state energy of the original TA monomers, which is conducive to promoting the generation of ROS.

[0051] (3) Relevant data on the activation of TA NDs to generate ROS under ultrasonic conditions.

[0052] Figure 8Total ROS production tests of the Ctrl group, TA monomer group, and TA NDs group under ultrasonic conditions were performed using a Shimadzu RF-6000 fluorescence spectrometer. Using DCFH-DA as a probe, with a TA NDs concentration of 200 μg / mL and a DCFH-DA working solution concentration of 10 mM, 1 μL of DCFH-DA was added to 2 mL of the TA NDs sample. After ultrasonic treatment for a certain time, the test was carried out at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Observe the reaction of ROS generated by TA NDs under ultrasonic conditions with DCFH-DA and the change in fluorescence intensity at 525 nm. Among them, the Ctrl group and the TA monomer group were the same as the TA NDs group in all aspects except that the above-mentioned 2 mL of TA NDs sample was changed to 2 mL of deionized water and 2 mL of a TA monomer solution with a concentration of 200 μg / mL, respectively. From Figure 8 As can be seen from (a)-(c) in 2 , after ultrasonic treatment, compared with the Ctrl group, the fluorescence of the TA monomer group at 525 nm did not change significantly, while the fluorescence of the TA NDs group at 525 nm increased significantly, proving that under ultrasonic conditions, TA NDs can be activated to generate ROS. Ultrasonic parameters: 1.5 W / cm

[0053] Figure 9 For the electron paramagnetic resonance test of TA NDs activated to generate singlet oxygen ( 1 O 2 ) under ultrasonic conditions, a Bruker EMXnano electron paramagnetic resonance spectrometer was used for the test. Using 2,2,6,6-tetramethyl-4-piperidone hydrochloride (TEMP) as a scavenger to capture 1 O 2 generated by TA NDs under ultrasonic conditions. TEMP is converted into a stable nitroxide radical to prove the generation of 1 O 2 . The final concentration of TEMP used in the test process was 200 mM, the TA NDs concentration was 1 mg / mL, and the ultrasonic parameters were: 1.5 W / cm 2 , 1 MHz, duty cycle 50%, 5 min. As can be seen from (a)-(c) in Figure 9 , as the ultrasonic time increased, the amount of 1 O 2 captured also gradually increased, that is, the generated 1 O 2 was positively correlated with the ultrasonic time.

[0054] (4) Relevant data on the sonodynamic antibacterial properties of TA NDs.

[0055] Figure 10 To investigate the adhesion of TA NDs to bacteria using a quartz crystal microbalance analyzer (QCM), a Q-Sense Analyzer quartz crystal microbalance analyzer was used for testing. During the test, first, PBS was used. After the frequency stabilized, it was switched to S.mutans (PBS) at a concentration of 10 8 CFU / mL -1 . After sufficient adhesion and frequency stabilization, it was switched back to PBS to rinse off the S.mutans that was not firmly adhered. After stabilization again, it was switched to TA / TA NDs to observe the adhesion ability of the bacterial surface to TA / TA NDs. The results are shown in Figure 10 (a) and (b). In the QCM data graph, the frequency change value in the red part decreased significantly, indicating that the bacterial surface has good adhesion to TA / TANDs.

[0056] Figure 11 For the performance test of sonodynamic antibacterial of TA NDs (S.mutans, E.faecalis). S.mutans was cultured in brain heart infusion medium (BHI) at 37 °C under anaerobic conditions for 24 h, and E.faecalis was cultured under normal conditions for 24 h. It was diluted with BHI medium, and 6 groups of experiments were set up, namely the Ctrl US(-) group without ultrasonic treatment (US), the TA US(-) group, the TA NDs US(-) group, the Ctrl US(+) group with ultrasonic treatment (US), the TA US(+) group, and the TA NDs US(+) group. The final concentration of TA / TA NDs was 200 μg / mL, and an equal volume of PBS was added to the other control groups; ultrasonic parameters: 1.5 W / cm 2 , 1 MHz, duty cycle 50%, 5 min. It can be seen from Figure 11 (a)-(c) that compared with the control group, the TA NDs group produced 1 O 2 under ultrasonic conditions, showing good antibacterial effects. The sonodynamic antibacterial rates against the two bacteria S.mutans and E.faecalis were 99.9% and 97.1% respectively.

[0057] Figure 12Morphological changes of S. mutans and E. faecalis after treatment with each group of materials. Six groups of experiments were set up, namely the Ctrl US(-) group, TA US(-) group, TA NDs US(-) group without ultrasonic treatment (US), and the Ctrl US(+) group, TA US(+) group, TA NDs US(+) group with ultrasonic treatment (US). The final concentration of TA / TA NDs was 200 μg / mL, and an equal volume of PBS was added to the other control groups; ultrasonic parameters: 1.5 W / cm 2 , 1 MHz, duty cycle 50%, 5 min. Then, it was fixed with 2.5% glutaraldehyde for 24 h, gradually dehydrated with 20%, 50%, 80%, 90%, and 100% ethanol, and observed by SEM. The bacterial morphology of S. m in the Ctrl US(-) group, TA US(-) group, TANDs US(-) group, Ctrl US(+) group, and TA US(+) group was intact and the surface was smooth, while in the TANDs US(+) group, due to the high toxicity generated under ultrasonic conditions 1 O 2 it could damage the bacterial membrane and DNA. Therefore, the bacterial membranes of the bacteria in this group showed varying degrees of shrinkage and depression, and the bacterial membranes of some bacteria were directly broken with obvious perforations, indicating the good sonodynamic antibacterial performance of TA NDs.

[0058] Figure 13 To detect the production of ROS in bacteria using the fluorescent probe DCFH-DA. Six groups of experiments were set up, namely the Ctrl US(-) group, TA US(-) group, TA NDs US(-) group without ultrasonic treatment (US), and the Ctrl US(+) group, TA US(+) group, TA NDs US(+) group with ultrasonic treatment (US). The final concentration of TA / TA NDs was 200 μg / mL, and an equal volume of PBS was added to the other control groups; ultrasonic parameters: 1.5 W / cm 2 , 1 MHz, duty cycle 50%, 5 min. After treatment with DCFH-DA, it was observed using a confocal microscope with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The TANDs US(+) group after ultrasonic treatment showed obvious green fluorescence, indicating that ROS was generated during the sonodynamic antibacterial process. Referring to the previous experimental part, it can be determined that the type of this ROS is 1 O 2 .

[0059] Figure 14Determination of the biofilm removal effect of the material. During the growth of S. mutans bacterial biofilms, Alexa Fluor 647-dextran conjugate was used to label EPS. Six groups of experiments were set up, namely the Ctrl US(-) group without ultrasonic treatment (US), the TA US(-) group, the TA NDs US(-) group, the Ctrl US(+) group with ultrasonic treatment (US), the TA US(+) group, and the TA NDs US(+) group. The final concentration of TA / TA NDs was 200 μg / mL, and an equal volume of PBS was added to the other control groups. Ultrasonic parameters: 1.5 W / cm 2 , 1 MHz, duty cycle 50%, 3 min, and observed with a confocal microscope. It can be observed from the taken fluorescence photos that the biofilm of the Ctrl US(-) group is very complete, and there are no obvious changes in the thickness and integrity of the biofilms in the TA US(-) group and the TA NDs US(-) group. Compared with the control group, the thickness of the biofilms in the Ctrl US(+) group and the TA US(+) group becomes thinner ( Figure 14 as shown in (a)), indicating that only through ultrasonic treatment, a certain biofilm removal effect can be achieved. In the TA NDs US(+) group, due to the generation of 1 O 2 during the ultrasonic treatment process, further damage is caused to the biofilm, and only a small part of the biofilm remains, achieving effective biofilm removal. The SEM was used to observe the biofilm removal effect in the six groups of experiments, showing consistent results. The removal effect of the TA NDs US(+) group is the most obvious, and obvious damage also appears in the morphology of bacteria in the broken part of the biofilm, proving that antibacterial effects can be exerted while removing the biofilm ( Figure 14 as shown in (b)).

[0060] Figure 15 Cytotoxicity evaluation of TA NDs on mouse fibroblasts (L929). The cells were cultured in a confocal dish and treated with PBS, TA (200 μg / mL), and TA NDs (200 μg / mL) respectively, and co-incubated for 24 h. The Calcein AM / PI cell viability and cytotoxicity detection kit was used for Live / Dead staining, and observed with a confocal microscope. TA NDs have no obvious cytotoxicity. Figure 15(a)). Six groups of experiments were set up, namely the Ctrl US(-) group without ultrasonic treatment (US), the TAUS(-) group, the TA NDs US(-) group, the Ctrl US(+) group with ultrasonic treatment (US), the TA US(+) group, and the TA NDs US(+) group. The Live / Dead staining was performed using the Calcein AM / PI cell viability and cytotoxicity detection kit, and it was observed through a confocal microscope that short-term ultrasonic treatment did not cause obvious cytotoxicity. Figure 15 (b)).

[0061] The above are only the preferred embodiments of the present invention. It should be noted that for those skilled in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicability of the patent.

Claims

1. A method for preparing a polyphenol nanodot sonosensitizer, characterized in that: The following steps are involved: Tannic acid was dissolved in deionized water and stirred at room temperature until completely dissolved. The dissolved solution was subjected to oxidative polymerization reaction. Tannic acid was assembled through oxidative polymerization to obtain polyphenol nanodot sonosensitizer. After the reaction was completed, the solution was cooled and the product was collected and filtered. The filtrate was transferred to a dialysis bag for dialysis. The water was changed every 6 hours. The dialyzed solution was freeze-dried into a solid powder. Finally, the freeze-dried solid powder was dispersed in deionized water for later use.

2. The method for preparing the polyphenol nanodot sonosensitizer according to claim 1, characterized in that: The addition ratio of the tannic acid to deionized water is 1 g:50 mL.

3. The method for preparing the polyphenol nanodot sonosensitizer according to claim 1, characterized in that: The dissolved solution is reacted at 75-90° C. for 12 hours.

4. The method for preparing the polyphenol nanodot sonosensitizer according to claim 1, characterized in that: The product was filtered using a 0.22 μm aqueous filter.

5. The method for preparing the polyphenol nanodot sonosensitizer according to claim 1, characterized in that: The filtrate was transferred to a dialysis bag with a molecular weight of 3000 and dialyzed for 48 hours.

6. The polyphenol nanodot sonosensitizer prepared according to the preparation method of the polyphenol nanodot sonosensitizer according to any one of claims 1 to 5.

7. Use of the polyphenol nanodot sonosensitizer according to claim 6 in the preparation of sonodynamic antibacterial materials.

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