Preparation method of ultrasonic response type copper ion-loaded magnetic microbubble and generator

Abstract

The invention provides an ultrasonic response type copper ion-loaded magnetic microbubble generator. According to the preparation method and generator of the ultrasonic response type copper ion-loaded magnetic microbubbles, an operation table is included, supporting legs are arranged on the lower surface of the operation table, a second placement groove is formed in the upper surface of the operation table, a magnetic stirrer is fixedly connected to the inner bottom wall of the second placement groove, and a heating device is arranged on the upper surface of the magnetic stirrer; the upper surface of the heating device is provided with a water bath heating barrel, the interior of the water bath heating barrel is provided with a reaction barrel, and the upper surface of the operation table is provided with a high-speed homogenizer and a centrifugal machine. The microjet generated by the cavitation effect can physically destroy the structure of the bacterial biofilm and responsively release the drug at the same time, so that the drug has the ability to permeate into the deep part of the bacterial biofilm.

Description

Technical Field

[0001] The present invention relates to the technical field of antibacterial biofilms of nanomaterials, and specifically to a preparation method and a generator of ultrasound-responsive copper ion-loaded magnetic microbubbles. Background Art

[0002] The main reason why the bacterial biofilm infection on the surface of titanium implants is difficult to treat is that the biofilm provides a protective barrier for bacteria, making it difficult for antibiotics to penetrate and reach an effective concentration to kill or inhibit the growth of bacteria. The bacteria in the biofilm can reduce their metabolic activities and proliferation rates, making them more resistant to antibiotics. In addition, the biofilm can hinder the defense mechanisms of the host immune system, inhibit the phagocytosis and bactericidal efficacy of immune cells, further exacerbating the complexity and challenge of treatment. The combined effect of these factors makes the biofilm infection on the surface of titanium implants a clinically difficult problem to solve.

[0003] While enhancing drug penetration with the help of ultrasonic energy and having the ability to efficiently kill bacteria in different metabolic states inside the biofilm are the keys to completely removing the biofilm and resolving the dilemma of frequent recurrence of biofilm infections. Metal ions are becoming effective drugs in the fields of immunomodulation and antibacterial strategies. Their unique ability to interact with biological systems provides a promising method for enhancing immune responses and combating stubborn microbial infections. It has been reported that Cu-doped titanium implants enhance their bactericidal effect by promoting the M1 polarization of macrophages. Due to the biofilm matrix, it is difficult for copper ions to be transported deep into the bacterial biofilm and reach an effective inhibitory concentration. Ultrasound-responsive microbubbles are micron-sized spheres with a gas core and a stable shell. Due to their unique acoustic properties, they have been regarded as an alternative platform for drug delivery. MB can oscillate and contract, and generate local microflows through inertial cavitation, thereby destroying the structure of the biofilm and enhancing cell uptake. Therefore, using ultrasound-responsive microbubbles to deliver copper ions may prove to be a promising method for treating bacterial infections in the immune microenvironment. Summary of the Invention

[0004] (1) Technical Problems to be Solved

[0005] In view of the deficiencies of the prior art, the present invention provides a preparation method and a generator of ultrasound-responsive copper ion-loaded magnetic microbubbles, which solve the problem that copper ions, with their unique mechanism of action, exhibit excellent bactericidal efficacy, can quickly destroy the cell membranes of bacteria, interfere with the enzyme systems and metabolic processes of bacteria, and thus efficiently kill bacteria, and facilitate the acquisition of temperature by realizing the stability of the probe through a positioning device.

[0006] (2) Technical Solutions

[0007] To achieve the above object, the present invention is realized by the following technical solutions: An ultrasound-responsive copper ion-loaded magnetic microbubble generator, comprising: an operating table, the lower surface of the operating table is provided with legs, the upper surface of the operating table is provided with a second placement groove, the bottom inner wall of the second placement groove is fixedly connected with a magnetic stirrer, the upper surface of the magnetic stirrer is provided with a heating device, the rear end of the magnetic stirrer is fixedly connected with a connecting wire, the front end of the connecting wire is fixedly connected with a connecting rod, the lower end of the connecting rod is provided with a temperature probe, a water bath heating bucket is placed on the upper surface of the heating device, a reaction bucket is arranged inside the water bath heating bucket, a high-speed homogenizer is placed on the upper surface of the operating table, the lower surface of the high-speed homogenizer is fixedly connected with a support column, the upper surface of the operating table is provided with a mounting hole, the upper surface of the operating table is provided with a first placement groove, and a centrifuge is placed inside the first placement groove;

[0008] A positioning device, which is located on the upper surface of the operating table, and the positioning device includes a support frame, a sliding rod, a first fixing member, a threaded plug, a slider, a sliding groove, a second fixing member and a through hole.

[0009] Preferably: an operation hole is provided on the front surface of the operating table, and the magnetic stirrer is controlled through the operation hole.

[0010] Preferably: a third fixing member is fixedly connected to the upper surface of the reaction bucket, and the lower surface of the third fixing member contacts the water bath heating bucket.

[0011] Preferably: the inner wall of the mounting hole contacts the support column, the number of the mounting holes is four and they are evenly distributed, and the number of the support columns is four and they are evenly distributed.

[0012] Preferably: the lower surface of the support frame is fixedly connected to the operating table, and the number of the support frames is two and they are symmetrically distributed.

[0013] Preferably: both ends of the sliding rod are fixedly connected to the support frame, the outer surface of the sliding rod is slidably connected to the slider through the sliding groove, and the upper surface of the slider is fixedly connected to the first fixing member.

[0014] Preferably: the front surface of the slider is fixedly connected to the second fixing member, the inner wall of the first fixing member is threadedly connected to the threaded plug, the inner wall of the threaded plug contacts the connecting wire, and the inside of the second fixing member contacts the connecting rod through the through hole.

[0015] A method for preparing ultrasonic responsive copper ion-carrying magnetic microbubbles, based on any one of the ultrasonic responsive copper ion-carrying magnetic microbubble generators, comprises the following steps: S1, dissolving Fe3O4 nanoparticles, tannic acid TA, and CuCl2·2H2O in ultrapure water, adjusting the pH to neutral, bathing at 45°C for 4 hours, and magnetically stirring at a speed of 500 rpm to encapsulate a layer of metal-polyphenol chelate Fe-CuTA on the surface of the Fe3O4 nanoparticles, wherein the molar ratio of TA to CuCl2·2H2O is about 0.004:1, and the mass ratio of Fe3O4 nanoparticles to the formed tannic acid copper CuTA is about 10:1, and adjusting the pH to 7.4 with 1M NaOH under vortexing;

[0016] S2, washing the product in S1 to remove unchelated tannic acid and Cu2+, and obtaining Fe-CuTA nanoparticles with an average size of 150 nm;

[0017] S3, adding Fe-CuTA nanoparticles to sodium dodecyl sulfate SDS solution, forming drug-loaded microbubbles by high-speed homogenization, the concentration of Fe-CuTA nanoparticles is 10 mg / mL, based on Fe3O4 nanoparticles, the concentration of SDS is 10 mM, the high-speed homogenization speed is 20000 rpm, and the process is carried out for 3 minutes in an ice water bath;

[0018] S4. Magnetic separation of drug-loaded microbubbles and washing with ultrapure water. Magnetic separation of drug-loaded microbubbles needs to be performed after the microbubbles are tightly self-assembled overnight.

[0019] (III) Beneficial effects

[0020] The present invention provides a preparation method and a generator of ultrasonic responsive copper ion-carrying magnetic microbubbles, which have the following beneficial effects:

[0021] A preparation method and generator of ultrasound-responsive copper ion-loaded magnetic microbubbles. Under the action of ultrasound, the drug-loaded microbubbles can produce cavitation. The microjets produced by the cavitation can physically destroy the structure of the bacterial biofilm and simultaneously release drugs responsively, so that the drugs are able to penetrate deep into the bacterial biofilm. In the acidic microenvironment of bacterial infection, the metal-polyphenol chelate CuTA encapsulated on the surface of Fe3O4 nanoparticles will gradually dissociate and continuously release high concentrations of copper ions. Copper ions, by virtue of their unique mechanism of action, exhibit excellent bactericidal efficacy, can quickly destroy bacterial cell membranes, interfere with bacterial enzyme systems and metabolic processes, and thus efficiently kill bacteria. More importantly, unlike traditional antibiotics, when copper ions exert their antibacterial effects, it is difficult for bacteria to develop drug resistance through conventional gene mutations or drug resistance mechanisms, which greatly ensures the durability and stability of the antibacterial effect and reduces the risk of recurrence of infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a schematic diagram of the overall structure of the present invention;

[0023] Figure 2 is a schematic diagram of the installation of the magnetic stirrer of the present invention;

[0024] Figure 3 is a schematic diagram of the position of the positioning device of the present invention;

[0025] Figure 4 is a schematic diagram of the structure of the positioning device of the present invention;

[0026] Figure 5 is a schematic diagram of the position of the high-speed homogenizer of the present invention;

[0027] Figure 6 is a schematic diagram of the position of the centrifuge of the present invention.

[0028] Among them, 1, operating platform; 2, positioning device; 3, sliding rod; 4, magnetic stirrer; 5, heating device; 6, connecting wire; 7, connecting rod; 8, temperature probe; 9, first fixing member; 10, threaded plug; 11, slider; 12, chute; 13, second fixing member; 14, through hole; 15, high-speed homogenizer; 16, mounting hole; 17, support column; 18, first placement groove; 19, centrifuge; 20, operation hole; 21, water bath heating barrel; 22, reaction barrel; 23, third fixing member; 24, leg; 25, second placement groove; 26, support frame. Detailed implementation manners

[0029] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0030] Such as Figure 1-6As shown in the figure, an embodiment of the present invention provides an ultrasound-responsive copper ion-loaded magnetic microbubble generator, including an operating table 1. Legs 24 are provided on the lower surface of the operating table 1. A second placement groove 25 is provided on the upper surface of the operating table 1. A magnetic stirrer 4 is fixedly connected to the bottom inner wall of the second placement groove 25. A heating device 5 is provided on the upper surface of the magnetic stirrer 4. A connecting line 6 is fixedly connected to the rear end of the magnetic stirrer 4. A connecting rod 7 is fixedly connected to the front end of the connecting line 6. A temperature probe 8 is provided at the lower end of the connecting rod 7. A water bath heating bucket 21 is placed on the upper surface of the heating device 5. A reaction bucket 22 is provided inside the water bath heating bucket 21. A high-speed homogenizer 15 is placed on the upper surface of the operating table 1. A support column 17 is fixedly connected to the lower surface of the high-speed homogenizer 15. An installation hole 16 is provided on the upper surface of the operating table 1. A first placement groove 18 is provided on the upper surface of the operating table 1. A centrifuge 19 is placed inside the first placement groove 18. An operation hole 20 is provided on the front surface of the operating table 1. The magnetic stirrer 4 is controlled through the operation hole 20. A third fixing member 23 is fixedly connected to the upper surface of the reaction bucket 22. The lower surface of the third fixing member 23 contacts the water bath heating bucket 21. The inner wall of the installation hole 16 contacts the support column 17. The number of the installation holes 16 is four and they are evenly distributed. The number of the support columns 17 is four and they are evenly distributed.

[0031] A positioning device 2, which is located on the upper surface of the operating table 1. The positioning device 2 includes a support frame 26, a sliding rod 3, a first fixing member 9, a threaded plug 10, a slider 11, a sliding groove 12, a second fixing member 13 and a through hole 14. The lower surface of the support frame 26 is fixedly connected to the operating table 1. The number of the support frames 26 is two and they are symmetrically distributed. Both ends of the sliding rod 3 are fixedly connected to the support frame 26. The outer surface of the sliding rod 3 is slidably connected to the slider 11 through the sliding groove 12. The upper surface of the slider 11 is fixedly connected to the first fixing member 9. The front surface of the slider 11 is fixedly connected to the second fixing member 13. The inner wall of the first fixing member 9 is threadedly connected to the threaded plug 10. The inner wall of the threaded plug 10 contacts the connecting line 6. The inside of the second fixing member 13 contacts the connecting rod 7 through the through hole 14.

[0032] A preparation method of ultrasound-responsive copper ion-loaded magnetic microbubbles, based on an ultrasound-responsive copper ion-loaded magnetic microbubble generator according to any one of the above, includes the following steps:

[0033] S1. Dissolve Fe3O4 nanoparticles, tannic acid TA, and CuCl2·2H2O in ultrapure water, adjust the pH to neutral, perform a water bath at 45 °C for 4 hours, and magnetically stir at a speed of 500 rpm to coat a metal-polyphenol chelate Fe-CuTA on the surface of the Fe3O4 nanoparticles. The molar ratio of TA to CuCl2·2H2O is about 0.004:1, and the mass ratio of Fe3O4 nanoparticles to the formed copper tannate CuTA is about 10:1. Adjust the pH to 7.4 with 1M NaOH under vortexing.

[0034] S2. Wash the product in S1 to remove unchelated tannic acid and Cu2+, and the obtained Fe-CuTA nanoparticles have an average size of 150 nm.

[0035] S3. Add the Fe-CuTA nanoparticles into a sodium dodecyl sulfate (SDS) solution, and form drug-loaded microbubbles through high-speed homogenization. The concentration of Fe-CuTA nanoparticles is 10 mg / mL (calculated as Fe3O4 nanoparticles), the concentration of SDS is 10 mM, the rotation speed of high-speed homogenization is 20,000 rpm, and perform for 3 minutes, and keep it in an ice-water bath throughout the process.

[0036] S4. Magnetically separate the drug-loaded microbubbles and wash them with ultrapure water. The magnetic separation of the drug-loaded microbubbles needs to be carried out after the microbubbles are tightly self-assembled overnight.

[0037] Working principle: Install the supporting leg 24 on the lower surface of the operating table 1, fix the magnetic stirrer 4 on the bottom inner wall of the second placement groove 25, connect the connecting wire 6, the connecting rod 7 and the temperature probe 8, install the heating device 5, the water bath heating barrel 21 and the reaction barrel 22, install the high-speed homogenizer 15 on the mounting hole 16 through the support column 17, place the centrifuge 19 in the first placement groove 18, install the positioning device 2, adjust the position of the slider 11 on the slide rod 3, fix the connecting wire 6 and the connecting rod 7 through the threaded plug 10 and the second fixing member 13, turn on the power supply, and debug equipment such as the magnetic stirrer 4 and the high-speed homogenizer 15 to ensure their normal operation.

[0038] Proceed as follows: Accurately weigh a certain amount of Fe3O4 nanoparticles, tannic acid (TA), and CuCl2·2H2O, add them into ultrapure water, use a pH regulator to adjust the pH to neutral, place the reaction vessel into the reaction barrel 22 in the water bath heating barrel 21, turn on the magnetic stirrer 4, set the rotation speed to 500 rpm, set the temperature to 45 °C, and react for 4 hours. During the reaction process, monitor the temperature in real time through the temperature probe 8 to ensure the temperature is stable. When the reaction is approaching the end, adjust the pH to 7.4 with 1 M NaOH under vortexing.

[0039] After the reaction is completed, transfer the reaction product to a centrifuge tube, place it in centrifuge 19, set appropriate centrifugation speed and time, centrifuge the product to separate it, remove the supernatant, wash the precipitate with ultrapure water multiple times until no unchelated tannic acid and Cu2+ can be detected in the washing solution, to obtain Fe-CuTA nanoparticles. Add the obtained Fe-CuTA nanoparticles to a solution containing sodium dodecyl sulfate (SDS), place the mixed solution in the working container of high-speed homogenizer 15, set the high-speed homogenization rotation speed to 20,000 rpm and the time to 3 minutes, place the working container in an ice-water bath, turn on high-speed homogenizer 15 for homogenization treatment, let the homogenized product stand overnight to make the microbubbles self-assemble tightly, and then use a magnetic separation device to magnetically separate the drug-loaded microbubbles. Wash the separated drug-loaded microbubbles with ultrapure water multiple times to obtain pure ultrasound-responsive copper ion-loaded magnetic microbubbles.

[0040] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An ultrasonically responsive copper ion-carrying magnetic microbubble generator, characterized in that: include: An operating table (1), wherein the lower surface of the operating table (1) is provided with a support leg (24), the upper surface of the operating table (1) is provided with a second placement groove (25), the bottom inner wall of the second placement groove (25) is fixedly connected to a magnetic stirrer (4), the upper surface of the magnetic stirrer (4) is provided with a heating device (5), the rear end of the magnetic stirrer (4) is fixedly connected to a connecting wire (6), the front end of the connecting wire (6) is fixedly connected to a connecting rod (7), and the lower end of the connecting rod (7) is provided with a temperature probe (8) , a water bath heating barrel (21) is placed on the upper surface of the heating device (5), a reaction barrel (22) is arranged inside the water bath heating barrel (21), a high-speed homogenizer (15) is placed on the upper surface of the operating table (1), a support column (17) is fixedly connected to the lower surface of the high-speed homogenizer (15), a mounting hole (16) is arranged on the upper surface of the operating table (1), a first placement groove (18) is arranged on the upper surface of the operating table (1), and a centrifuge (19) is placed inside the first placement groove (18); A positioning device (2) is located on the upper surface of the operating table (1), and the positioning device (2) comprises a support frame (26), a sliding rod (3), a first fixing member (9), a threaded plug (10), a sliding block (11), a sliding groove (12), a second fixing member (13) and a through hole (14).

2. The ultrasonic responsive copper ion-carrying magnetic microbubble generator according to claim 1, characterized in that: The front surface of the operating table (1) is provided with an operating hole (20), and the magnetic stirrer (4) is controlled through the operating hole (20).

3. The ultrasonic responsive copper ion-carrying magnetic microbubble generator according to claim 1, characterized in that: The upper surface of the reaction barrel (22) is fixedly connected to a third fixing member (23), and the lower surface of the third fixing member (23) is in contact with the water bath heating barrel (21).

4. The ultrasonic responsive copper ion-carrying magnetic microbubble generator according to claim 1, characterized in that: The inner wall of the mounting hole (16) is in contact with the support column (17); the number of the mounting holes (16) is four and they are evenly distributed; the number of the support columns (17) is four and they are evenly distributed.

5. The ultrasonic responsive copper ion-carrying magnetic microbubble generator according to claim 1, characterized in that: The lower surface of the support frame (26) is fixedly connected to the operating table (1), and the number of the support frames (26) is two and they are symmetrically distributed.

6. The ultrasonic responsive copper ion-carrying magnetic microbubble generator according to claim 1, characterized in that: The two ends of the sliding rod (3) are fixedly connected to the support frame (26), the outer surface of the sliding rod (3) is slidably connected to the slider (11) through a sliding groove (12), and the upper surface of the slider (11) is fixedly connected to the first fixing member (9).

7. The ultrasonic responsive copper ion-carrying magnetic microbubble generator according to claim 1, characterized in that: The front surface of the slider (11) is fixedly connected to the second fixing member (13), the inner wall of the first fixing member (9) is threadedly connected to the threaded plug (10), the inner wall of the threaded plug (10) is in contact with the connecting line (6), and the interior of the second fixing member (13) is in contact with the connecting rod (7) through the through hole (14).

8. A method for preparing ultrasonically responsive copper-loaded magnetic microbubbles, based on an ultrasonically responsive copper-loaded magnetic microbubble generator according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1. Dissolve Fe3O4 nanoparticles, tannic acid TA, and CuCl2·2H2O in ultrapure water, adjust the pH to neutral, place in a water bath at 45°C for 4 hours, and stir magnetically at 500 rpm to encapsulate a layer of metal-polyphenol chelate Fe-CuTA on the surface of the Fe3O4 nanoparticles, the molar ratio of TA to CuCl2·2H2O is about 0.004:1, and the mass ratio of Fe3O4 nanoparticles to the formed tannic acid copper CuTA is about 10:

1. Adjust the pH to 7.4 with 1 M NaOH under vortexing; S2, washing the product in S1 to remove unchelated tannic acid and Cu2+, and obtaining Fe-CuTA nanoparticles with an average size of 150 nm; S3, adding Fe-CuTA nanoparticles to sodium dodecyl sulfate SDS solution, forming drug-loaded microbubbles by high-speed homogenization, the concentration of Fe-CuTA nanoparticles is 10 mg / mL, based on Fe3O4 nanoparticles, the concentration of SDS is 10 mM, the high-speed homogenization speed is 20000 rpm, and the process is carried out for 3 minutes in an ice water bath; S4. Magnetic separation of drug-loaded microbubbles and washing with ultrapure water. Magnetic separation of drug-loaded microbubbles needs to be performed after the microbubbles are tightly self-assembled overnight.

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