Method, device and application of in-situ discharge preparation of plasma-activated microbubbles in microbubbles

Abstract

The application belongs to the field of plasma medicine, and relates to a method, device and application of in-situ discharge preparation of plasma-activated microbubbles. The application provides a method for in-situ discharge preparation of plasma-activated microbubbles, which utilizes an external electric field to activate the formed microbubbles in-situ, so that the microbubbles load and deliver plasma active substances. That is, under the action of the external electric field, the gas core, gas-liquid interface and / or surrounding liquid phase of the microbubbles are activated in-situ to obtain plasma-activated microbubbles. The application also provides a device for in-situ discharge preparation of plasma-activated microbubbles, which comprises an excitation power supply, a high-voltage electrode, a grounding electrode, a dielectric container, a liquid pipeline and a driving pump system; and the dielectric container is located in an electric field region formed by the high-voltage electrode and the grounding electrode. The application separates the microbubble preparation and plasma activation processes, reduces the decay of the active substances in the subsequent process, and can improve the activation uniformity and preparation efficiency through circulation flow.

Description

Technical Field

[0001] This invention relates to the field of plasma medicine technology, and in particular to a method, apparatus and application for preparing plasma-activated microbubbles by in-situ discharge within microbubbles. Background Technology

[0002] In 1968, Gramiak discovered and recognized that core-shell microbubbles (MBs) could be used as ultrasound contrast agents to enhance ultrasound imaging signals. Later, researchers found that under ultrasound-guided stimulation, MBs could induce cavitation at a targeted location, stimulating surrounding cells to undergo endocytosis, improving cell membrane permeability, and thus enabling drug delivery into the cell. Other researchers have also achieved drug delivery by mounting anticancer drugs on or inside the surface of MBs. Therefore, microbubbles have been widely used in ultrasound imaging and drug delivery.

[0003] Cold Atmospheric Plasma (CAP) can generate a variety of reactive oxygen and nitrogen species (RONS) under low-temperature conditions, including hydrogen peroxide, nitrite, nitrate, ozone, hydroxyl radicals, nitric oxide, and peroxynitrite. This makes CAP potentially valuable for a wide range of applications in antibacterial, wound healing, and cancer treatment. Currently, the applications of cold plasma in biomedicine mainly fall into two categories: direct and indirect treatment. Direct treatment involves applying the plasma discharge region directly to biological tissue. This method can rapidly generate a strong active effect in a localized area, but its treatment range, depth of action, and equipment layout are easily limited by discharge spacing, target morphology, tissue wettability, and safety parameters. Indirect treatment utilizes plasma to pre-activate water, saline, culture medium, or other liquid media to form a plasma-activated solution, which is then delivered via rinsing, injection, or perfusion. This method improves the storage and transfer capabilities of plasma active components, but ordinary plasma activation solutions lack targeted release and local enrichment mechanisms, and are easily diluted and diffused after entering complex biological systems, resulting in reduced efficiency.

[0004] To improve the delivery efficiency of plasma RONS, existing technologies attempt to combine plasma RONS with microbubbles to form plasma-activated microbubbles. Theoretically, this type of system can simultaneously utilize the biological effects of plasma RONS and the delivery-enhancing effect of microbubbles. However, existing plasma-activated microbubbles mostly introduce plasma RONS simultaneously during microbubble preparation. The microbubble generation process and the plasma activation process are coupled, resulting in limitations on discharge voltage, gas flow rate, processing time, and activation intensity due to the microbubble preparation process, making it difficult to fully increase the RONS loading.

[0005] Furthermore, after microbubble preparation, in order to obtain a microbubble suspension with relatively uniform particle size, post-processing steps such as centrifugation, filtration, washing, sieving, or concentration are usually required to remove free shell material, large particulate impurities, and broken microbubbles. However, plasma RONS has a short lifespan. If plasma RONS is introduced during the microbubble generation stage, subsequent processing can easily cause RONS attenuation, diffusion, or loss, thereby reducing the effective activity and operational stability of the final plasma-activated microbubbles.

[0006] Therefore, current technologies still lack a method for the immediate, efficient, and controllable activation of microbubbles after they have been formed, purified, sieved, or concentrated. If the microbubble preparation process can be separated from the plasma activation process, and the formed microbubbles can be activated in situ using a discharge electric field, generating and enriching RONS in the microbubble core, gas-liquid interface, and / or surrounding liquid phase, it is expected to improve the preparation efficiency, RONS loading, and local delivery effect of plasma-activated microbubbles. Summary of the Invention

[0007] To address the shortcomings of existing plasma-activated microbubbles in terms of effective activity and stability, this invention provides a method for preparing plasma-activated microbubbles through in-situ discharge within microbubbles. This method utilizes an external electric field to in-situ activate the formed microbubbles, loading and delivering plasma RONS (Reactive Oxygen Spectrostomy System). Specifically, under the influence of the external electric field, in-situ activation reactions occur in the microbubble core, gas-liquid interface, and / or surrounding liquid phase, generating and enriching RONS to obtain plasma-activated microbubbles. This invention also provides an apparatus for preparing plasma-activated microbubbles through in-situ discharge within microbubbles. The apparatus includes an excitation power supply, a high-voltage electrode, a grounding electrode, a dielectric container, liquid pipelines, and a drive pump system. The dielectric container is located within the electric field region formed by the high-voltage electrode and the grounding electrode and carries the microbubble suspension. This invention separates the microbubble preparation from the plasma activation process, reducing RONS attenuation during post-processing and storage, and improving activation uniformity and preparation efficiency through circulating flow.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing plasma-activated microbubbles by in-situ discharge within microbubbles, comprising the following steps: S10. Obtain a microbubble suspension with uniform particle size distribution and a core and shell structure; S11. Place the microbubble suspension in a dielectric container within the electric field region; S12. An electric field is applied to the microbubble suspension in the medium container, causing the microbubble core, the microbubble gas-liquid interface and / or the liquid around the microbubble to undergo an in-situ activation reaction under preset conditions, generating a plasma-activated microbubble suspension containing active oxygen and nitrogen substances. S13. Collect plasma-activated microbubble suspensions.

[0009] As one possible implementation, the microbubble suspension preparation method is any one of ultrasonic emulsification, mechanical stirring, membrane emulsification, microfluidics, or other preparation methods capable of forming micron-sized bubbles; The microbubble suspension is subjected to at least one of the following post-treatments: centrifugation, static stratification, filtration, washing, sieving, concentration, or resuspension, to obtain a microbubble suspension with uniform particle size distribution. The gas in the microbubble core includes a rare gas or at least one of air, oxygen, and nitrogen. The microbubble shell material includes at least one of phospholipids, surfactants, proteins, and polymers. The liquid medium of the microbubble suspension includes at least one of water, physiological saline, phosphate buffer, culture medium, or liquid medium suitable for the delivery of reactive oxygen and nitrogen substances. The microbubble suspension contains microbubbles with an average particle size of 1~10 μm and a microbubble concentration of 10. 6 ~10 10 cells / mL; The volume of the microbubble suspension is 0.5~100mL.

[0010] As one possible implementation, the rare gas in the gas includes helium, argon, or other rare gases, and the volume fraction of the rare gas is 50% to 100%. When the gas contains air, oxygen, and nitrogen, the total volume fraction of the air, oxygen, and nitrogen incorporated is 0.1% to 50%.

[0011] As one possible implementation, the voltage of the applied electric field is 1~20kV, the discharge frequency is 1~500kHz, and the discharge time is 10s~30min.

[0012] As one possible implementation, the preset conditions are that the liquid flow rate of the microbubble suspension is 0.1 to 100 mL / min, and the number of cycles of the microbubble suspension flowing in the electric field region is 2 to 100.

[0013] In a second aspect, the present invention provides an apparatus for preparing plasma-activated microbubbles by in-situ discharge within microbubbles, used to prepare plasma-activated microbubble suspensions by implementing the method described in the first aspect, specifically including an excitation power supply, a high-voltage electrode, a grounding electrode, a dielectric container, a liquid pipeline, and a drive pump system. The excitation power source is at least one of AC power, pulse power, and radio frequency power; The high-voltage electrode and the ground electrode are respectively electrically connected to the excitation power supply. The excitation power supply applies an electric field to the plasma discharge structure formed by the high-voltage electrode and the ground electrode, generating an electric field region between the high-voltage electrode and the ground electrode, so that the microbubble core, microbubble gas-liquid interface and / or liquid phase around the microbubble in the microbubble suspension undergo in-situ activation reaction, generating and enriching active oxygen and nitrogen substances, thereby forming a plasma-activated microbubble suspension. The high-voltage electrode and the grounding electrode are arranged parallel to each other, with a spacing of 1~20mm; The high-voltage electrode can be suspended above or in contact with the microbubble suspension. The dielectric container is disposed in the electric field region formed by the high-voltage electrode and the grounding electrode. The dielectric container is placed on the high-voltage electrode or the grounding electrode. The dielectric container is used to carry the microbubble suspension and also serves as the dielectric layer of the plasma discharge structure. The liquid pipeline is connected to the drive pump system and the medium container for the circulation or collection of the microbubble suspension; The drive pump system includes at least one of a liquid storage container, a circulation pump, a diaphragm pump, a peristaltic pump, a syringe pump, and a flow control device, for causing the microbubble suspension to flow continuously or in a circulating manner through the electric field region.

[0014] As one possible implementation, the high-voltage electrode is at least one of a plate electrode, a needle electrode, a ring electrode, a mesh electrode, a rod electrode, a coaxial electrode, and a thin film electrode; the grounding electrode is at least one of a plate electrode, a needle electrode, a ring electrode, a mesh electrode, a rod electrode, a coaxial electrode, and a thin film electrode.

[0015] As one possible implementation, the medium container is made of insulating material and has a wall thickness of 0.1~5mm.

[0016] As one possible implementation, the insulating material is at least one of glass, quartz, ceramic, polytetrafluoroethylene, polycarbonate, and polydimethylsiloxane.

[0017] Thirdly, the present invention provides an application of in-situ discharge within microbubbles to prepare plasma-activated microbubbles, wherein the plasma-activated microbubbles are used to prepare adjuvant preparations for in vitro antibacterial disinfection, biofilm disruption, wound care, local delivery of reactive oxygen and nitrogen substances, or local tumor treatment. When used, the adjuvant preparations are subjected to ultrasonic treatment, causing the plasma-activated microbubbles to oscillate, cavitate, or rupture, thereby promoting the local release or transmembrane delivery of reactive oxygen and nitrogen substances.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The method for preparing plasma-activated microbubbles by in-situ discharge within microbubbles provided by the present invention separates the microbubble preparation process from the plasma activation process. The microbubbles can first be purified, sieved, or concentrated to obtain higher purity and more uniform particle size, and then be activated in situ immediately before use to reduce the attenuation of RONS during post-processing and storage.

[0019] 2. The method for preparing plasma-activated microbubbles by in-situ discharge within microbubbles provided by the present invention is not limited by the microbubble preparation device and preparation process. The discharge voltage, frequency, waveform, processing time, gas composition, liquid volume and liquid layer thickness can be independently adjusted, thereby increasing the amount of RONS generated and the degree of freedom of control.

[0020] 3. The method for preparing plasma-activated microbubbles by in-situ discharge within microbubbles provided by the present invention utilizes the microbubble core and gas-liquid interface as the interface for RONS generation and enrichment, so that the prepared plasma-activated microbubbles have the functions of RONS storage, local release and delivery enhancement. Attached Figure Description

[0021] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a schematic flowchart of a method for preparing plasma-activated microbubbles through in-situ discharge within microbubbles, provided in an embodiment of the present invention. Figure 2 A diagram illustrating the apparatus for preparing plasma-activated microbubbles via in-situ discharge within microbubbles, as provided in an embodiment of the present invention. Figure 3 This is a partial schematic diagram of the in-situ activation reaction of microbubbles provided in an embodiment of the present invention; Figure 4 A physical image of the microbubble in-situ activation experimental device provided in an embodiment of the present invention; Figure 5 These are microscopic morphology images of microbubbles before and after treatment with an electric field or plasma discharge in Embodiment 1 of the present invention. Figure 6 This is a graph showing the change in average particle size of microbubbles before and after plasma discharge activation in Example 1 of the present invention; Figure 7 This is a graph showing the changes in microbubble concentration before and after plasma activation treatment in Example 1 of the present invention; Figure 8 The graph shows the detection results of hydrogen peroxide and nitrite concentrations in ordinary microbubbles and plasma-activated microbubbles of this application in Examples 1, 2 and Comparative Example 2 of this invention; Figure 9The graph shows the detection results of CCK-8 cell activity of HN-6 cancer cells by plasma-activated microbubbles in Examples 1 and 2 of the present invention, as well as activated water in Comparative Example 1 and ordinary microbubbles in Comparative Example 2.

[0022] Figure Labels 1-Excitation power supply, 2-High voltage electrode, 3-Grounding electrode, 4-Dielectric container, 5-Microbubble suspension, 6-Microbubble, 7-Liquid pipeline, 8-Drive pump system, 9-Storage container, 10-Raw material container, 11-Active oxygen and nitrogen substances, 12-Excitation electric field. Detailed Implementation

[0023] To facilitate a clear description of the technical solutions in the embodiments of the present invention, the terms "first" and "second" are used to distinguish identical or similar items with essentially the same function and effect. For example, the first threshold and the second threshold are merely used to distinguish different thresholds and do not limit their order. Those skilled in the art will understand that the terms "first" and "second" do not limit the quantity or execution order, and that the terms "first" and "second" are not necessarily different.

[0024] It should be noted that in this invention, the terms "exemplary" or "for example" are used to indicate examples, illustrations, or descriptions. Any embodiment or design described as "exemplary" or "for example" in this invention should not be construed as being more preferred or advantageous than other embodiments or designs. Specifically, the use of terms such as "exemplary" or "for example" is intended to present the relevant concepts in a concrete manner.

[0025] In this invention, "at least one" refers to one or more, and "more than one" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone, where A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects are in an "or" relationship. "At least one" or similar expressions refer to any combination of these items, including any combination of singular or plural items. For example, "at least one of a, b, or c" can represent: a, b, c, a combination of a and b, a combination of a and c, a combination of b and c, or a, b, and c, where a, b, and c can be single or multiple.

[0026] To address the issues of insufficient effective activity and stability in the use of plasma-activated microbubbles manufactured using existing technologies, this invention provides a method for preparing plasma-activated microbubbles through in-situ discharge within microbubbles. This method utilizes an external electric field to in-situ activate the formed microbubbles, loading and delivering plasma RONS (Reactive Oxygen Spectrostomy) within the microbubbles. Specifically, under the influence of the external electric field, in-situ activation reactions occur in the microbubble core, gas-liquid interface, and / or surrounding liquid phase, generating and enriching RONS to obtain plasma-activated microbubbles. This invention also provides an apparatus for preparing plasma-activated microbubbles through in-situ discharge within microbubbles. The apparatus includes an excitation power supply, a high-voltage electrode, a grounding electrode, a dielectric container, liquid pipelines, and a drive pump system. The dielectric container is located within the electric field region formed by the high-voltage electrode and the grounding electrode and carries the microbubble suspension. This invention separates the microbubble preparation from the plasma activation process, reducing the attenuation of RONS during post-processing and storage, and improving activation uniformity and preparation efficiency through circulating flow.

[0027] In a first aspect, the present invention provides a method for preparing plasma-activated microbubbles by in-situ discharge within microbubbles, comprising the following steps: S10. Obtain a microbubble suspension with uniform particle size distribution and a core and shell structure; S11. Place the microbubble suspension in a dielectric container within the electric field region; S12. An electric field is applied to the microbubble suspension in the medium container, causing the microbubble core, the microbubble gas-liquid interface and / or the liquid around the microbubble to undergo an in-situ activation reaction under preset conditions, generating a plasma-activated microbubble suspension containing active oxygen and nitrogen substances. S13. Collect plasma-activated microbubble suspensions.

[0028] The gas core is the core component of the microbubble structure, typically composed of gas and surrounded by a liquid film or shell. In this embodiment of plasma-activated microbubbles, the gas core is not only the site of the discharge reaction but also a key region for in-situ plasma generation.

[0029] When an electric field is applied, the gas molecules within the gas core are ionized and excited under the influence of the high-energy electric field, generating a large amount of high-energy electrons, ions, and free radical plasma. These active particles further interact with the gas-liquid interface and the surrounding liquid, driving the production of reactive oxygen species (such as...). 1 O2, O2 − ) and reactive nitrogen (such as NO, NO 2、 NO2 − NO3 − ONOO - The formation of matter.

[0030] The shell is the outer part of the microbubble structure that encloses the gas core. It is usually composed of a liquid film or functional materials and plays a key role in stabilizing the microbubble structure and regulating the discharge process in plasma-activated microbubble technology.

[0031] In-situ activation reaction refers to the direct application of an electric field within the microbubble and its interface region, triggering a localized plasma discharge process in the gas core, gas-liquid interface, and surrounding liquid, thereby efficiently generating reactive oxygen species (such as...). 1 O2, O2 − ) and reactive nitrogen (such as NO, NO 2、 NO2 − NO3 − ONOO - For chemical reactions of substances, see [link to relevant documentation]. Figure 3 As shown, microbubble 6 undergoes in-situ activation in the excitation electric field 12 formed by high-voltage electrode 2 and grounding electrode 3. The excitation electric field 12 induces the gas in the core of microbubble 2 to generate active oxygen and nitrogen substances 11. The core characteristics of this reaction include: Spatial concentration: The reaction is concentrated in the core and interface region of the microbubble, forming a high-energy closed microenvironment that can significantly improve the free radical yield.

[0032] Highly adjustable: By adjusting parameters such as electric field strength, pulse frequency, and gas type (e.g., O2, N2, air), the type and concentration of RONS can be controlled.

[0033] The implementation steps of this invention are as follows: Figure 1 The key step lies in separating the microbubble preparation process from the plasma activation process. Specifically, microbubbles are first prepared, and after processing to obtain uniform microbubbles, they are then activated in situ using a discharge electric field. Unlike existing technologies that simultaneously introduce plasma RONS during microbubble generation, this invention uses pre-formed microbubbles as a gas-liquid interface carrier. Under the action of an external electric field, in-situ plasma activation reactions occur in the gas inside the microbubble, the gas-liquid interface of the microbubble, and / or the liquid phase surrounding the microbubble, thereby generating and enriching RONS.

[0034] This technical solution avoids the limitations of microbubble preparation processes on discharge voltage, gas flow rate, and activation time, thus improving the freedom of discharge parameter control and microbubble activation efficiency. Furthermore, this method allows for the pre-processing of microbubble purification and particle size sieving, enabling microbubbles to achieve high purity and uniform particle size before immediate plasma activation, thereby reducing RONS attenuation during centrifugation, filtration, storage, and transfer.

[0035] The plasma-activated microbubbles prepared by this technical solution can not only provide RONS in the liquid phase, but also store and deliver RONS using the microbubble core and gas-liquid interface, thereby improving the stability and utilization efficiency of plasma RONS.

[0036] As one possible implementation, the microbubble suspension preparation method is any one of ultrasonic emulsification, mechanical stirring, membrane emulsification, microfluidics, or other preparation methods capable of forming micron-sized bubbles; The microbubble suspension is subjected to at least one of the following post-treatments: centrifugation, static stratification, filtration, washing, sieving, concentration, or resuspension, to obtain a microbubble suspension with uniform particle size distribution. The gas in the microbubble core includes a rare gas or at least one of air, oxygen, and nitrogen. The microbubble shell material includes at least one of phospholipids, surfactants, proteins, and polymers. The liquid medium of the microbubble suspension includes at least one of water, physiological saline, phosphate buffer, culture medium, or a liquid medium suitable for RONS delivery. The microbubble suspension contains microbubbles with an average particle size of 1~10 μm and a microbubble concentration of 10. 6 ~10 10 cells / mL; The volume of the microbubble suspension is 0.5~100mL.

[0037] The microbubble suspension can be prepared using existing mature technologies, and the appropriate method can be selected based on the actual application requirements.

[0038] As an example, when efficiency and cost control are required, mechanical stirring or ultrasonic emulsification can be selected; when particle size uniformity and stability are required, membrane emulsification or microfluidics are recommended.

[0039] The post-processing step aims to obtain a microbubble suspension with a uniform particle size distribution. Uniform particle size distribution improves discharge uniformity and reaction consistency, which is beneficial for subsequent large-scale applications. As an example, the post-processing procedure for the microbubble suspension is as follows: The obtained microbubble suspension is centrifuged and filtered to remove large particles of unencapsulated gas, free shell material, broken microbubbles, and other impurities, resulting in a microbubble suspension with relatively uniform particle size distribution and stable concentration. The centrifugation speed can be 500-3000 rpm, the centrifugation time can be 3-10 min, and the filter pore size can be 10-20 μm. The treated microbubble suspension can be directly used for subsequent plasma activation.

[0040] Among them, the gas in the gas core directly affects the plasma discharge characteristics and the generation efficiency of active species. One or more gases can be mixed according to the requirements.

[0041] As an example, this embodiment provides specific functions of gas in a microbubble core for reference and selection: Helium (He): It has the characteristics of strong chemical inertness, good thermal conductivity and high discharge uniformity, which is conducive to the formation of mild and stable low-temperature plasma, and is suitable for biomedical applications that are sensitive to temperature rise.

[0042] Argon (Ar): It has a low discharge threshold, is easy to break down and maintain stable discharge, and can improve the ionization efficiency at the microbubble core and gas-liquid interface. It is suitable as the preferred base gas in the preparation of plasma-activated microbubbles.

[0043] Oxygen (O2): Directly provides an oxygen source, which is beneficial for the formation of ·OH. 1 O2 and other reactive oxygen species enhance oxidative capacity, stimulate oxidative stress responses in cells, and promote apoptosis.

[0044] Nitrogen (N2): can promote the generation of reactive nitrogen such as NO and ONOO⁻, and is suitable for antibacterial, anti-inflammatory and other biological applications.

[0045] Air: Low cost and readily available, produces a wide variety of RONS, but its discharge stability is slightly lower than that of pure gas.

[0046] The microbubble shell material is a water-soluble organic compound. These materials can form a relatively uniform membrane structure, improving the stability of microbubbles in the microbubble suspension and extending the stable storage time of the microbubble suspension. Furthermore, these materials can be used alone or in combination to construct composite shells, balancing stability, responsiveness, and functionality to meet the needs of different applications (such as ultrasound imaging and RONS delivery). When necessary, the microbubble shell or surface can be functionalized to introduce targeted ligands or functional molecules, thereby enhancing the enrichment and delivery capabilities of plasma-activated microbubbles in specific tissues or lesion areas.

[0047] As an example, this embodiment provides the functionality of commonly used materials for reference selection: Phospholipids (such as DSPC and DPPC) can form monolayer membrane structures with good elasticity and biocompatibility, which can effectively slow down gas diffusion and improve the acoustic responsiveness and biocompatibility of microbubbles. Surfactants, such as polyethylene glycol (PEG) modified lipid / surfactant combinations, can enhance shell stability, prevent microbubble aggregation, and prolong blood circulation time. Proteins: Commonly used proteins include albumin, which self-assembles to form a robust shell. Albumin microvesicle shells retain near-natural protein structures and possess good acoustic responsiveness and drug loading capacity. Polymer materials, such as polylactic acid (PLA) and polycaprolactone (PCL), can provide stronger mechanical strength and longer retention time, making them suitable for sustained-release or targeted delivery scenarios.

[0048] The selection of the liquid medium in the microbubble suspension mainly considers its impact on the stability of the microbubble structure, biocompatibility, retention of active substances, and subsequent delivery efficiency. A suitable liquid medium can reduce the risk of microbubble aggregation, rupture, or rapid dissipation, and is beneficial for maintaining the stability of the core-shell structure of microbubbles during in-situ plasma activation, thereby improving the generation, preservation, and delivery of RONS.

[0049] In this embodiment, water, physiological saline, and phosphate-bubble buffer (PBS) are all commonly used liquid media in the biomedical field and can be selected according to the actual application scenario. Furthermore, the liquid media suitable for RONS delivery can be optimized by adjusting pH value, ionic strength, osmotic pressure, or viscosity, and appropriate amounts of stabilizers, protectants, or biocompatible additives can be added to improve the dispersion stability, local retention capacity, and active substance retention capacity of the microbubble suspension.

[0050] Furthermore, this technical solution optimizes the parameters (particle size, concentration) of microbubbles in the microbubble suspension. These optimized parameters make the microbubbles prepared in this embodiment suitable for medical ultrasound imaging and drug delivery.

[0051] The optimal volume of the microbubble suspension is chosen to obtain microbubbles with corresponding functions. As an example, this embodiment provides the influence of microbubble suspensions with different volume ranges on the final performance of the microbubbles for reference: When the liquid volume is 0.5–5 mL, it is used to prepare highly active, low-dose plasma-activated microbubble solutions. When the liquid volume is 5-20 mL, it is used to balance RONS generation efficiency and microbubble stability. When the liquid volume is 20-100 mL, it is used in conjunction with a circulating flow system for batch preparation of plasma-activated microbubble solutions.

[0052] As one possible implementation, the rare gas in the gas includes helium, argon, or other rare gases, and the volume fraction of the rare gas is 50% to 100%. When the gas contains air, oxygen, and nitrogen, the total volume fraction of the air, oxygen, and nitrogen incorporated is 0.1% to 50%.

[0053] During the microbubble preparation process, the internal gas composition of the microbubble can be controlled according to the requirements of subsequent plasma discharge and RONS generation. Specifically, the air or filling gas in the microbubble preparation system can be replaced with easily ionized rare gases such as He and Ar to reduce the conditions required for local ionization within the microbubble core and improve the stability of plasma activation reactions at the gas and gas-liquid interface within the microbubble.

[0054] The volume fraction of rare gases in the microbubble core can be 50%-100%, meaning the core can use only rare gases or a mixture of rare gases with air, oxygen, nitrogen, etc.

[0055] Furthermore, air, O2, N2, or a mixture thereof are incorporated into the rare gas, with the volume fraction of the incorporated gas controlled within the range of 0.1%-50%, in order to regulate the type and concentration of RONS during the subsequent discharge process.

[0056] Accordingly, air was chosen as the gas core for the microbubble, which simplifies the preparation process and reduces the preparation cost.

[0057] As one possible implementation, the voltage of the applied electric field is 1~20kV, the discharge frequency is 1~500kHz, and the discharge time is 10s~30min.

[0058] This embodiment provides external reaction conditions for microbubble plasma activation reaction. An electric field is applied as an external reaction condition. Multiple parameters jointly determine the generation efficiency and reaction intensity of active species in plasma-activated microbubbles. These parameters are applicable to the device provided in the second aspect of this embodiment, which takes into account both reaction efficiency and system stability, and can be flexibly adjusted according to the target application scenario.

[0059] As one possible implementation, the preset conditions are that the liquid flow rate of the microbubble suspension is 0.1 to 100 mL / min, and the number of cycles of the microbubble suspension flowing in the electric field region is 2 to 100.

[0060] This embodiment provides the internal conditions for the microbubble plasma activation reaction. By adjusting external conditions such as discharge voltage, discharge frequency, and processing time, and internal conditions such as liquid volume, liquid layer thickness, gas composition, liquid flow rate, number of cycles, or cooling conditions, the type, concentration, release behavior, and storage stability of RONS in the plasma-activated microbubble solution can be controlled.

[0061] As an example, the effect of adjusting certain parameters on the final microbubble performance in this embodiment is as follows: The energy input per unit volume and residence time of microbubbles within the discharge region are controlled by adjusting the liquid flow rate and the number of cycles. The liquid flow rate is 0.1–50 mL / min. When the liquid flow rate is 0.1–10 mL / min, it is used to improve the uniformity of microbubble activation and maintain microbubble stability. When the liquid flow rate is 10–50 mL / min, it is used to improve the continuous preparation throughput of plasma-activated microbubble solutions; Among them, the number of cycles is 2 to 100. When the number of cycles is 2 to 5, the electric field effect on the microbubbles is relatively weak, which is suitable for preparing low-dose plasma-activated microbubbles. When the number of cycles is 5 to 30, the microbubbles can pass through the discharge region multiple times, the RONS concentration gradually accumulates, and the activation uniformity is significantly improved. When the number of cycles is 30 to 100, the reaction depth can be further enhanced, but the energy consumption and the tolerance of the microbubble structure need to be weighed.

[0062] In a second aspect, the present invention provides an apparatus for preparing plasma-activated microbubbles by in-situ discharge within microbubbles, used to prepare plasma-activated microbubble suspensions by implementing the method described in the first aspect, specifically including an excitation power supply, a high-voltage electrode, a grounding electrode, a dielectric container, a liquid pipeline, and a drive pump system. The excitation power source is at least one of AC power, pulse power, and radio frequency power; The high-voltage electrode and the ground electrode are respectively electrically connected to the excitation power supply. The excitation power supply applies an electric field to the plasma discharge structure formed by the high-voltage electrode and the ground electrode, generating an electric field region between the high-voltage electrode and the ground electrode, so that the microbubble core, microbubble gas-liquid interface and / or liquid phase around the microbubble in the microbubble suspension undergo in-situ activation reaction, generating and enriching active oxygen and nitrogen substances, thereby forming a plasma-activated microbubble suspension. The high-voltage electrode and the grounding electrode are arranged parallel to each other, with a spacing of 1~20mm; The high-voltage electrode can be suspended above or in contact with the microbubble suspension. The dielectric container is disposed in the electric field region formed by the high-voltage electrode and the grounding electrode. The dielectric container is placed on the high-voltage electrode or the grounding electrode. The dielectric container is used to carry the microbubble suspension and also serves as the dielectric layer of the plasma discharge structure. The liquid pipeline is connected to the drive pump system and the medium container for the circulation or collection of the microbubble suspension; The drive pump system includes at least one of a liquid storage container, a circulation pump, a diaphragm pump, a peristaltic pump, a syringe pump, and a flow control device, for causing the microbubble suspension to flow continuously or in a circulating manner through the electric field region.

[0063] See Figure 2The apparatus for preparing plasma-activated microbubbles by in-situ discharge within microbubbles provided in this embodiment includes an excitation power supply 1, a high-voltage electrode 2, a grounding electrode 3, a dielectric container 4, a liquid pipeline 7, and a drive pump system 8.

[0064] The excitation power supply 1, high voltage electrode 2, grounding electrode 3 and dielectric container 4 form a plasma discharge structure. The excitation power supply 1 is connected to the high voltage electrode 2 and the grounding electrode 3. The high voltage electrode 2 and the grounding electrode 3 are set on opposite sides of the dielectric container 4. The dielectric container 4 can be placed on any electrode.

[0065] The dielectric container 4 refers to the insulating liquid container used to hold the microbubble suspension 5, which can be made of glass, quartz, ceramic, polytetrafluoroethylene, polycarbonate, polydimethylsiloxane, or other insulating materials. The dielectric container 4 serves both as the container for the microbubble suspension 5 and as the dielectric layer of the plasma discharge structure.

[0066] The dielectric container 4 is located in the electric field region formed between the high-voltage electrode 2 and the ground electrode 3; the microbubble suspension 5 is placed in the dielectric container 4, and the microbubbles 6 in the microbubble suspension 5 are located in the electric field region formed between the high-voltage electrode 2 and the ground electrode 3, and undergo in-situ plasma activation reaction under the action of AC voltage, pulse voltage or radio frequency voltage applied by the excitation power supply 1.

[0067] The drive pump system 8 is connected to the medium container 4 via a liquid pipeline 7. The microbubble suspension can be circulated or collected through the liquid pipeline 7 under the action of the drive pump system 8. The drive pump system includes, but is not limited to, at least one of a circulating pump, diaphragm pump, peristaltic pump, syringe pump, and flow control device. The drive pump system can continuously process a large number of microbubbles and ensure that the flow of microbubbles in the medium container allows each microbubble to receive uniform treatment.

[0068] Figure 4 This is a photograph of the microbubble in-situ activation experimental apparatus in Embodiment 1 of this application. As can be seen from the figure, the microbubble suspension is placed within the electric field region formed by the high-voltage electrode and the grounding electrode. During the discharge process, a distinct plasma luminescence region is generated between the electrodes. This apparatus allows for the immediate in-situ activation of the formed microbubble suspension after it has been shaped and purified or sieved. This allows the microbubble core, gas-liquid interface, and / or surrounding liquid phase to be subjected to an electric field or plasma discharge, thereby obtaining plasma-activated microbubbles.

[0069] Specifically, the device for preparing plasma-activated microbubbles by in-situ discharge within microbubbles provided in this embodiment can perform plasma activation on the formed microbubbles after microbubble preparation, purification, or sieving. The dielectric container is used to carry the microbubble suspension and serve as the dielectric layer for plasma discharge. The high-voltage electrode and the grounding electrode are used to form an electric field region. The excitation power supply is used to provide the discharge voltage, so that the microbubble core, gas-liquid interface, and / or surrounding liquid phase are generated and enriched with RONS. The drive pump system and liquid pipeline are used to drive the microbubbles to repeatedly pass through the activation region, thereby improving the activation efficiency and RONS loading.

[0070] Meanwhile, the device can achieve different activation methods. As an example, when the static activation method is used, the microbubble suspension 5 is placed directly in the medium container 4 and located in the discharge area formed between the high voltage electrode 2 and the ground electrode 3. When the continuous flow activation method is adopted, the microbubble suspension 5 enters the medium container 4 from the storage container 9 through the liquid pipeline 7, is activated in the discharge area, and flows out from the liquid pipeline 7 and is collected. When the circulating flow activation method is adopted, the microbubble suspension 5 flows out of the medium container 4 and is circulated into the discharge area by the driving pump system for repeated processing.

[0071] Specifically, depending on the preparation method and volume requirements of plasma-activated microbubbles, different settings can be made for the treatment of microbubble suspension 5.

[0072] When small-volume, on-demand preparation is required, the microbubble suspension 5, after centrifugation, filtration, washing, or resuspension, can be directly added to the medium container 4, placing it within the discharge area formed between the high-voltage electrode 2 and the grounding electrode 3.

[0073] As an example, 2-20 mL of purified microbubble suspension can be added to medium container 4, the liquid layer thickness in medium container 4 is 1-10 mm, the electrode spacing is 1-20 mm, and an AC high voltage signal is applied to the high voltage electrode to cause in-situ plasma activation reaction in the microbubble core, microbubble gas-liquid interface and / or liquid phase around the microbubble.

[0074] When continuous preparation of plasma-activated microbubbles is required, the microbubble suspension enters the dielectric container 4 from the storage container via the inlet pipe. After being activated in the discharge region, it flows out from the liquid pipeline 7 and is collected, forming a continuously prepared plasma-activated microbubble solution. This method allows the microbubble suspension in the container to be in a continuous flow state, thereby realizing the batch preparation of plasma-activated microbubbles.

[0075] As an example, when it is necessary to prepare more than 100 mL of plasma-activated microbubble suspension, or when it is necessary to continuously obtain plasma-activated microbubble suspension, a continuous flow activation method can be used. The microbubble suspension 5 is placed in the raw material container 10 and transported to the medium container 4 through the liquid pipeline 7 and the drive pump system 8. The flow rate of the microbubble suspension 5 is 0.1–100 mL / min. The microbubble suspension 5 is activated in situ as it flows through the electric field region formed by the high-voltage electrode 2 and the grounding electrode 3, and then flows out through the outlet pipe and is collected in the storage container 9, thereby realizing the continuous preparation of plasma-activated microbubble suspension.

[0076] When it is necessary to improve the processing uniformity of microbubble suspension and the RONS generation efficiency, a circulating flow method can be adopted. The microbubble suspension is repeatedly introduced into the medium container 4 under the drive of the pump system 8 and passes through the discharge area multiple times, thereby increasing the contact probability between the microbubble and the discharge area and enhancing the in-situ activation reaction of the microbubble core and the gas-liquid interface.

[0077] As an example, when the concentration of RONS loaded in the microbubbles needs to be above 100 μM, a circulating flow method can be used. 20-100 mL of purified microbubble suspension 5 is added to the storage container 9, connecting the storage container 9, liquid pipeline 7, drive pump system 8, and medium container 4 to form a circulation loop. The drive pump system 8 is a peristaltic pump, controlling the microbubble suspension 5 to circulate through the medium container 4 at a flow rate of 10-50 mL / min, with 2-100 cycles.

[0078] Through the above-described technical methods, the embodiments of the present invention rationally configure the connection relationships between the excitation power supply 1, high-voltage electrode 2, grounding electrode 3, dielectric container 4, microbubble suspension 5, microbubbles 6, liquid pipeline 7, and drive pump system 8, enabling the microbubble suspension 5 to be activated in situ within the discharge region. Simultaneously, through a circulating flow, the microbubbles 6 repeatedly pass through the discharge activation region, thereby improving the uniformity of microbubble activation and the RONS generation efficiency. With the increase in the number of cycles or processing time, the RONS concentration inside the microbubbles, at the microbubble gas-liquid interface, and in the liquid phase gradually increases, thus obtaining a plasma-activated microbubble solution with a higher RONS content.

[0079] This invention can be combined with a continuous flow or circulating flow system to allow the microbubble suspension to repeatedly pass through the discharge activation region, thereby improving the uniformity of the electric field applied to different microbubbles and avoiding the problems of over-activation of some microbubbles or insufficient activation of others. This structure is suitable for both small-volume experimental preparation and continuous, batch preparation of larger-volume plasma-activated microbubble solutions, exhibiting good process scale-up potential and ease of application.

[0080] Compared to traditional plasma activation solutions, the plasma-activated microbubbles prepared in this invention can not only carry and release RONS, but also oscillate, cavitate, or rupture under ultrasound, achieving local release and enhanced transmembrane delivery in the lesion area. Therefore, this invention retains the advantages of plasma RONS, such as antibacterial activity, biomembrane disruption, and redox environment regulation, while utilizing the targeted triggering characteristics of microbubbles to improve local delivery efficiency. This avoids the need for large amounts of plasma activation solution to enhance therapeutic effects, thereby reducing potential oxidative stimulation and toxic side effects on normal tissues.

[0081] Through the above technical solutions, the embodiments of the present invention achieve an organic combination of plasma RONS generation, storage, delivery and local release functions, providing a more efficient, safe and easy-to-control plasma microbubble preparation method and device for applications such as antibacterial disinfection, biofilm destruction, wound repair and local tumor treatment.

[0082] As one possible implementation, the high-voltage electrode is at least one of a plate electrode, a needle electrode, a ring electrode, a mesh electrode, a rod electrode, a coaxial electrode, and a thin film electrode; the grounding electrode is at least one of a plate electrode, a needle electrode, a ring electrode, a mesh electrode, a rod electrode, a coaxial electrode, and a thin film electrode.

[0083] The high-voltage electrode and the grounding electrode can be made of stainless steel, copper, aluminum, tungsten, titanium, platinum, conductive glass, conductive film, conductive mesh or other conductive materials; and different forms of electrode pairing can be selected according to requirements. As an example, the needle-plate electrode combination can be used to study discharge priority and triggering mechanism; the coaxial structure is used to optimize electric field utilization and discharge stability.

[0084] In practical applications, the electrode type and pairing method should be selected comprehensively based on factors such as the characteristics of the discharge medium, the size of the reaction space, and the energy input requirements.

[0085] As one possible implementation, the medium container is made of insulating material and has a wall thickness of 0.1~5mm.

[0086] The dielectric container is made of insulating material with a wall thickness of 0.1~5mm. This design is intended to enable the dielectric container to play a key role in electric field isolation and structural support in the plasma-activated microbubble system.

[0087] If the wall thickness is too thin, such as less than 0.1 mm, the mechanical and physical strength of the medium container will be insufficient, making it easy to break under the pressure generated by the liquid flow or unable to withstand the electric field and break down. If the wall thickness is too thick, such as greater than 5 mm, it may weaken the effect of the electric field.

[0088] These structural parameters ensure the safety and repeatability of the discharge process, while allowing the electric field to be effectively coupled to the microbubble suspension, promoting core ionization and the generation of active species.

[0089] As one possible implementation, the insulating material is at least one of glass, quartz, ceramic, polytetrafluoroethylene, polycarbonate, and polydimethylsiloxane.

[0090] The material provided in this embodiment is widely used in dielectric containers, electrode supports, or isolation structures of plasma activation systems due to its excellent electrical insulation properties and chemical stability.

[0091] Thirdly, the present invention provides an application of in-situ discharge within microbubbles to prepare plasma-activated microbubbles, wherein the plasma-activated microbubbles are used to prepare adjuvant preparations for in vitro antibacterial disinfection, biofilm disruption, wound care, local delivery of reactive oxygen and nitrogen substances, or local tumor treatment. When used, the adjuvant preparations are subjected to ultrasonic treatment, causing the plasma-activated microbubbles to oscillate, cavitate, or rupture, thereby promoting the local release or transmembrane delivery of reactive oxygen and nitrogen substances.

[0092] Plasma-activated microbubbles can be used to prepare adjuvant agents for in vitro antibacterial disinfection, biofilm disruption, wound care, local RONS delivery, and local tumor treatment. Their core advantage lies in the in-situ generation of a microenvironment rich in reactive oxygen species (ROS) and reactive nitrogen species (RNS) through physical means, achieving efficient, precise, and drug-resistant bioregulation. When using adjuvant agents, ultrasound is applied, causing the plasma-activated microbubbles to "explode" near the cells, propelling active substances into the cells and enhancing therapeutic efficacy.

[0093] As an example, specific applications are as follows: In vitro antibacterial disinfection: reactive oxygen species (such as OH-), 1 O2 can disrupt bacterial cell membranes and DNA structures, and has a broad-spectrum killing effect on common pathogenic bacteria such as Staphylococcus aureus and Escherichia coli, making it suitable for skin disinfection, medical device surface treatment, and other applications.

[0094] Biofilm disruption: Biofilms formed by microorganisms are highly resistant to antibiotics. Plasma-activated microbubbles can combine RONS oxidation and ultrasonic cavitation effects to weaken the extracellular polymeric substance (EPS) structure and enhance the efficiency of active substances entering the biofilm, thereby improving the biofilm removal effect.

[0095] Wound care: During the wound healing process, activated microbubbles can not only continuously release bactericidal components, but also promote capillary regeneration and tissue regeneration, improve local microcirculation, and accelerate the repair of difficult-to-heal wounds such as chronic ulcers and burns.

[0096] Local RONS delivery: Microbubbles can be used as carriers to deliver RONS to specific tissues and release them triggered by an external electric field, achieving precise treatment with spatiotemporal control.

[0097] Adjunctive local tumor treatment: By inducing oxidative stress in cancer cells, activating apoptosis pathways, and potentially stimulating distal immune effects, it enhances the sensitivity to radiotherapy or chemotherapy, and is especially suitable for adjunctive treatment of superficial tumors or postoperative residual lesions.

[0098] To facilitate understanding of the technical solution of this application, further explanation is provided below with reference to specific embodiments.

[0099] Example 1 (1) Preparation and post-treatment of microbubble suspension 0.59 g Span 60, 0.6 g NaCl, and 0.09 g PEG 4000 were mixed in 20 mL of PBS solution. Then, 0.4 mL of Tween 80 was added to the solution and stirred at room temperature until homogeneous. The mixture was then autoclaved at 121 °C for 12 min, and the temperature was lowered to 40–45 °C by stirring. Next, the mixture was sonicated for 2 min using an ultrasonic cell disruptor. The emulsified solution was then centrifuged, resulting in three layers, with the middle layer consisting of microbubbles. The middle layer microbubbles were resuspended, washed, and then sealed and stored at 4 °C. The average particle size of the obtained microbubbles was 3 μm; the microbubble concentration was 10. 8 per mL.

[0100] (2) Preparation of plasma-activated microbubble suspension Subsequently, 20 mL of purified microbubble suspension is added to a glass or quartz media container. The storage container is connected to the media container and the storage container via a liquid pipeline to form a circulating flow loop. A peristaltic pump is connected to the liquid pipeline to control the flow rate of the microbubble suspension. The flow rate is 20 mL / min. Driven by the pump, the microbubble suspension enters the media container and is activated in the electric field discharge region. It then returns to the storage container through the outlet pipe for recycling. The media container is located in the discharge region formed between the high-voltage electrode and the ground electrode, with a bottom thickness of 2 mm, a liquid layer thickness of 10 mm, and an electrode spacing of 15 mm. An AC high-voltage signal of 5 kV and a frequency of 90 kHz is applied to the high-voltage electrode for a processing time of 1 min. During the discharge process, the applied electric field acts on the microbubble core, the microbubble gas-liquid interface, and / or the liquid phase surrounding the microbubble, causing an in-situ plasma activation reaction in the microbubble system to generate a plasma-activated microbubble solution containing RONS.

[0101] After treatment, the resulting solution is collected, yielding the plasma-activated microbubble solution. The RONS content can be determined using a hydrogen peroxide detection kit or the Griess method; simultaneously, the average particle size and concentration of the microbubbles are measured using a microscope and a particle size analyzer.

[0102] Example 2 This embodiment provides a method for preparing plasma-activated microbubbles over a long period of time under circulating flow conditions.

[0103] First, the microbubble suspension was prepared and purified according to step (1) in Example 1. Then, the microbubbles were activated for 5 minutes according to step (2) in Example 1. During this process, the microbubble suspension was circulated by a drive pump system.

[0104] After treatment, the resulting solution is collected, yielding the plasma-activated microbubble solution. The RONS content can be determined using a hydrogen peroxide detection kit or the Griess method; simultaneously, the average particle size and concentration of the microbubbles are measured using a microscope and a particle size analyzer.

[0105] Compared with Example 1, this example extends the time required for plasma activation. Since the preparation method provided in this embodiment does not involve the preparation of microbubbles during the activation process, the plasma activation time can be extended, further increasing the concentration of RONS loaded in the microbubbles and resulting in better therapeutic effects.

[0106] Comparative Example 1 This comparative example is used to illustrate the role of microbubble carriers in RONS delivery.

[0107] Take the same volume of PBS solution as in Example 1, without adding microbubbles, and place it directly in the same medium container. Treat it under the same electric field discharge conditions to obtain a conventional plasma-activated solution. The RONS content can be determined using a hydrogen peroxide detection kit or the Griess method.

[0108] This comparative example demonstrates that although ordinary plasma-activated liquids can also generate RONS, they lack microbubble cores and gas-liquid interface storage structures, and cannot provide enhanced functions for microbubble cavitation, local release, and transmembrane delivery, thus limiting their therapeutic effect on cancer cells.

[0109] Comparative Example 2 This comparative example illustrates the advantages of the method for preparing plasma-activated microbubbles by in-situ discharge within microbubbles as described in this invention, compared to conventional plasma-activated microbubble preparation methods, in terms of the concentration of RONS loaded.

[0110] Take the same volume of microbubble raw material solution as in Example 1, place it in the same medium container, and seal the plasma discharge device from Example 1. Use the same discharge parameters as in Example 1 to generate plasma, and introduce the plasma-generated active gas into the microbubble raw material solution. Since continuous introduction of the plasma-generated active gas can cause foaming on the surface of the microbubble raw material solution and affect system stability, the introduction time is controlled to 1 minute. Simultaneously with the introduction of the plasma-generated active gas, use an ultrasonic cell disruptor to ultrasonically emulsify the microbubble raw material solution for 2 minutes. Then, centrifuge the emulsified solution; the solution separates into three layers, with the middle layer being the generated microbubbles. After resuspending and washing the middle layer microbubbles, seal the prepared microbubbles and store them in a 4°C refrigerator. The average particle size of the obtained microbubbles is 3 μm; the microbubble concentration is 10. 8 per mL.

[0111] After treatment, the resulting solution is collected, yielding a conventional plasma-activated microbubble solution. The RONS content can be determined using a hydrogen peroxide detection kit or the Griess method; simultaneously, the average particle size and concentration of the microbubbles are measured using a microscope and a particle size analyzer.

[0112] This comparative example demonstrates that although conventional plasma-activated microbubble methods can also generate RONS, the plasma discharge region is far from the microbubble, resulting in insufficient interaction between the discharge activation region and the microbubble. The interaction time is limited by the preparation process, the active material decays during transport, and subsequent centrifugation, layering, washing, and purification processes easily cause RONS diffusion, decay, or loss. Therefore, compared to conventional plasma-activated microbubbles, this application, through immediate in-situ discharge activation of pre-formed and purified microbubbles, can improve the generation efficiency and retention of RONS in the microbubble core, gas-liquid interface, and surrounding liquid phase.

[0113] The above embodiments and comparative examples show that by separating the microbubble preparation and purification process from the plasma activation process, and performing immediate in-situ discharge activation after microbubble formation, this application can increase the effective concentration of RONS in plasma-activated microbubbles and reduce their loss during post-processing.

[0114] Figure 5 The images show the microstructure of the microbubbles in Example 1 of this application before and after treatment with an electric field or plasma discharge. As can be seen from the images, the microbubbles are regularly spherical before and after treatment, exhibiting good overall dispersion. No obvious collapse, rupture, aggregation, or large particle impurities were observed. This indicates that the in-situ plasma activation conditions used in this application do not have a significant adverse effect on the basic morphology of the microbubbles, and the formed microbubbles can still maintain a relatively complete core-shell structure after plasma treatment.

[0115] Figure 6This figure shows the change in average particle size of microbubbles before and after plasma discharge activation in Example 1 of this application. The average particle size of microbubbles was measured before and after plasma discharge activation. As can be seen from the figure, the change in average particle size of microbubbles before and after activation is small, and both remain within the micrometer range. This indicates that the plasma discharge treatment conditions used in this application will not cause significant expansion, rupture, or uncontrolled particle size of microbubbles. The results show that the method of this application can activate formed microbubbles in situ while maintaining the basic structural stability of the microbubbles.

[0116] Figure 7 This figure shows the changes in microbubble concentration before and after plasma activation treatment in Example 1 of this application. The microbubble concentration in the microbubble suspension was measured before and after activation. As can be seen from the figure, the microbubble concentration did not decrease significantly after treatment with an electric field or plasma discharge, indicating that the microbubbles maintained good quantitative stability during activation. This result further demonstrates that the instantaneous in-situ activation method described in this application can achieve microbubble activation without significantly disrupting the concentration and dispersion state of the microbubble suspension, which is beneficial for subsequent delivery of reactive oxygen and nitrogen substances and for biological applications.

[0117] Figure 8 The figures show the detection results of hydrogen peroxide and nitrite concentrations in ordinary microbubbles and plasma-activated microbubbles of this application in Examples 1, 2, and Comparative Example 2 of this application. In the experiment, 1 mL of microbubble suspension was taken from each group. For the ultrasonic treatment group, an ultrasonic physiotherapy instrument with an ultrasonic frequency of 1 MHz, a sound intensity of 1.2 W / cm², and a duty cycle of 20% was used to treat the mixed microbubble suspension for 1 min, causing the microbubbles to oscillate, cavitate, or rupture, thereby promoting the release of the active substances loaded or enriched in the microbubbles into the liquid phase. After the ultrasonic treatment, the resulting solution was filtered through a 0.2 μm inorganic filter membrane, and the H₂O₂ and NO₂ in the filtrate were detected using a hydrogen peroxide detection kit and a nitrite detection kit, respectively. - content.

[0118] As can be seen from the figure, compared with before ultrasonic treatment, the H2O2 and NO2 in each microbubble group decreased after ultrasonic treatment. - The concentrations of H2O2 and NO2 increased, indicating that the microbubbles were able to release their loaded RONS after ultrasonic stimulation. Compared with the ordinary microbubbles in Comparative Example 2, the plasma-activated microbubbles prepared in Example 1 of this application showed higher concentrations of H2O2 and NO2. - The significantly increased concentration indicates that this application can improve the effective loading of RONS in the microbubble system through instant in-situ discharge activation of the formed microbubbles. Furthermore, the H2O2 and NO2 concentrations in the plasma-activated microbubbles obtained by cyclic activation in Example 2 are significantly higher. - The further increase in concentration indicates that by repeatedly passing the formed microbubbles through the discharge region, the interaction opportunities between the microbubbles and the discharge activation region can be enhanced, thereby further increasing the generation and enrichment of reactive oxygen and nitrogen substances.

[0119] Figure 9 This image shows the results of detecting the CCK-8 cell activity of HN-6 cancer cells using plasma-activated microbubbles in Examples 1 and 2 of this application, as well as the activation solution in Comparative Example 1 and ordinary microbubbles in Comparative Example 2. The experiment included Comparative Example 1 activation solution group, Comparative Example 2 ordinary microbubble group, Example 1 plasma-activated microbubble group, and Example 2 cyclically activated plasma-activated microbubble group. The effects of each group on the activity of HN-6 cancer cells before and after ultrasound stimulation were compared.

[0120] As can be seen from the figure, without ultrasound stimulation, the effects of each treatment group on the activity of HN-6 cancer cells were relatively limited. After ultrasound stimulation, the inhibitory effect on cancer cells in the activation solution group of Comparative Example 1 was not significant due to the lack of transmembrane delivery enhancement function of microbubbles. Both the ordinary microbubble group and the plasma-activated microbubble group of this application reduced the activity of HN-6 cells to varying degrees. Among them, the plasma-activated microbubble group in Example 1 of this application showed a more significant inhibitory effect on the activity of HN-6 cells, indicating that the plasma-activated microbubbles prepared in this application have a stronger inhibitory effect on cancer cells than ordinary microbubbles. Moreover, the cell activity reduction was most significant in the cyclically activated plasma-activated microbubble group in Example 2, indicating that by circulating the formed microbubbles through the plasma discharge area multiple times, the loading level of reactive oxygen and nitrogen substances in the microbubbles was further increased, thereby enhancing their local release effect and anti-tumor cell effect under ultrasound stimulation.

[0121] Although the invention has been described herein in conjunction with various embodiments, those skilled in the art will understand and implement other variations of the disclosed embodiments by reviewing the accompanying drawings, the disclosure, and the description of the drawings, in carrying out the claimed invention. In this specification, the word "comprising" does not exclude other components or steps, and "a" or "an" does not exclude multiple components. A single processor or other unit can implement several of the functions listed in the specification. While certain measures are described in different embodiments, this does not mean that these measures cannot be combined to produce good results.

[0122] Although the invention has been described in conjunction with specific features and embodiments, it is obvious that various modifications and combinations can be made therein without departing from the spirit and scope of the invention. Accordingly, this specification and drawings are merely illustrative of the invention and are considered to cover any and all modifications, variations, combinations, or equivalents within the scope of the invention. Clearly, those skilled in the art can make various alterations and modifications to the invention without departing from its spirit and scope. Thus, if such modifications and modifications fall within the scope of the invention and its equivalents, the invention is also intended to include such modifications and modifications.

Claims

1. A method for preparing plasma-activated microbubbles through in-situ discharge within microbubbles, characterized in that, Includes the following steps: S10. Obtain a microbubble suspension with uniform particle size distribution and a core and shell structure; S11. Place the microbubble suspension in a dielectric container within the electric field region; S12. An electric field is applied to the microbubble suspension in the medium container, causing the microbubble core, the microbubble gas-liquid interface and / or the liquid around the microbubble to undergo an in-situ activation reaction under preset conditions, generating a plasma-activated microbubble suspension containing active oxygen and nitrogen substances. S13. Collect plasma-activated microbubble suspensions.

2. The method for preparing plasma-activated microbubbles through in-situ discharge within microbubbles according to claim 1, characterized in that, The microbubble suspension preparation method is any one of ultrasonic emulsification, mechanical stirring, membrane emulsification, microfluidics, or other preparation methods capable of forming micron-sized bubbles; The microbubble suspension is subjected to at least one of the following post-treatments: centrifugation, static stratification, filtration, washing, sieving, concentration, or resuspension, to obtain a microbubble suspension with uniform particle size distribution. The gas in the microbubble core includes a rare gas or at least one of air, oxygen, and nitrogen. The microbubble shell material includes at least one of phospholipids, surfactants, proteins, and polymers. The liquid medium of the microbubble suspension includes at least one of water, physiological saline, phosphate buffer, culture medium, or liquid medium suitable for the delivery of reactive oxygen and nitrogen substances. The microbubble suspension contains microbubbles with an average particle size of 1~10 μm and a microbubble concentration of 10. 6 ~10 10 cells / mL; The volume of the microbubble suspension is 0.5~100mL.

3. The method for preparing plasma-activated microbubbles through in-situ discharge within microbubbles according to claim 2, characterized in that, The rare gases in the gas include helium, argon, or other rare gases, and the volume fraction of the rare gases is 50% to 100%. When the gas contains air, oxygen, and nitrogen, the total volume fraction of the air, oxygen, and nitrogen incorporated is 0.1% to 50%.

4. The method for preparing plasma-activated microbubbles through in-situ discharge within microbubbles according to claim 1, characterized in that, The applied electric field voltage is 1~20kV, the discharge frequency is 1~500kHz, and the discharge time is 10s~30min.

5. The method for preparing plasma-activated microbubbles through in-situ discharge within microbubbles according to claim 1, characterized in that, The preset conditions are that the liquid flow rate of the microbubble suspension is 0.1 to 100 mL / min, and the number of cycles of the microbubble suspension in the electric field region is 2 to 100.

6. An apparatus for preparing plasma-activated microbubbles through in-situ discharge within microbubbles, characterized in that, The method for preparing plasma-activated microbubble suspension according to any one of claims 1 to 5 specifically includes an excitation power supply, a high-voltage electrode, a grounding electrode, a dielectric container, a liquid pipeline, and a drive pump system. The excitation power source is at least one of AC power, pulse power, and radio frequency power; The high-voltage electrode and the ground electrode are respectively electrically connected to the excitation power supply. The excitation power supply applies an electric field to the plasma discharge structure formed by the high-voltage electrode and the ground electrode, generating an electric field region between the high-voltage electrode and the ground electrode, so that the microbubble core, microbubble gas-liquid interface and / or liquid phase around the microbubble in the microbubble suspension undergo in-situ activation reaction, generating and enriching active oxygen and nitrogen substances, thereby forming a plasma-activated microbubble suspension. The high-voltage electrode and the grounding electrode are arranged parallel to each other, with a spacing of 1~20mm; The high-voltage electrode can be suspended above the microbubble suspension or in contact with the microbubble suspension. The dielectric container is disposed in the electric field region formed by the high-voltage electrode and the grounding electrode. The dielectric container is placed on the high-voltage electrode or the grounding electrode. The dielectric container is used to carry the microbubble suspension and also serves as the dielectric layer of the plasma discharge structure. The liquid pipeline is connected to the drive pump system and the medium container for the circulation or collection of the microbubble suspension; The drive pump system includes at least one of a liquid storage container, a circulation pump, a diaphragm pump, a peristaltic pump, a syringe pump, and a flow control device, for causing the microbubble suspension to flow continuously or in a circulating manner through the electric field region.

7. The apparatus for preparing plasma-activated microbubbles by in-situ discharge within microbubbles according to claim 6, characterized in that, The high-voltage electrode is at least one of the following: plate electrode, needle electrode, ring electrode, mesh electrode, rod electrode, coaxial electrode, and thin film electrode; the grounding electrode is at least one of the following: plate electrode, needle electrode, ring electrode, mesh electrode, rod electrode, coaxial electrode, and thin film electrode.

8. The apparatus for preparing plasma-activated microbubbles by in-situ discharge within microbubbles according to claim 1 or claim 7, characterized in that, The medium container is made of insulating material and has a wall thickness of 0.1~5mm.

9. The apparatus for preparing plasma-activated microbubbles by in-situ discharge within microbubbles according to claim 8, characterized in that, The insulating material is at least one of glass, quartz, ceramic, polytetrafluoroethylene, polycarbonate, and polydimethylsiloxane.

10. An application of in-situ discharge within microbubbles to prepare plasma-activated microbubbles, characterized in that, The plasma-activated microbubbles are used to prepare adjuvant preparations for in vitro antibacterial disinfection, biofilm disruption, wound care, local delivery of reactive oxygen species, or local tumor treatment. When used, the adjuvant preparations are subjected to ultrasonic treatment, causing the plasma-activated microbubbles to oscillate, cavitate, or rupture, thereby promoting the local release or transmembrane delivery of reactive oxygen species.

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