Gallium-indium alloy nanodispersion system, preparation thereof and application thereof in radio frequency sensitization

Abstract

The application discloses a gallium-indium alloy nanodispersion system and a preparation method and application thereof in radio frequency sensitization, and belongs to the technical field of cross disciplines of chemistry, medicine and the like. Raw materials of the gallium-indium alloy nanodispersion system include gold nanoparticles prepared by a glutathione reduction method and nanodroplets of liquid metal gallium-indium alloy after ultrasonic treatment; the gold nanoparticles prepared by the glutathione reduction method are electrochemically replaced in the process of contacting the liquid metal nanodroplets and are adsorbed on the surface of the nanodroplets of the liquid metal gallium-indium alloy after ultrasonic treatment. The gallium-indium alloy nanodispersion system can generate various active oxygen free radicals under the action of radio frequency electromagnetic waves, rapidly heats the irradiation part, cooperatively kills tumor cells, activates the whole-body anti-tumor immune response and effectively inhibits tumor recurrence and metastasis. In addition to the fact that the gold nanoparticles can significantly improve the stability of the nanodispersion system, the gold nanoparticles also have catalase activity, and further enhance the anti-tumor activity.

Description

Technical Field

[0001] This application belongs to the interdisciplinary technical field of chemistry, medicine, etc., and relates to a gallium-indium alloy nanodispersion system, its preparation method, and its application. Background Technology

[0002] Tumor kinetic therapy refers to the conversion of different forms of energy (such as infrared radiation, X-rays, ultrasound, and chemical energy) into reactive oxygen species (ROS), inducing the accumulation of excessive ROS within the tumor, leading to intratumoral oxidative stress that causes tumor cell apoptosis and necrosis. Furthermore, studies have shown that ROS can trigger an immune response in tumors. Compared to traditional cancer treatments (such as chemotherapy and radiotherapy), tumor kinetic therapy effectively avoids drug resistance and systemic toxicity, reducing side effects and invasive damage. Based on the different forms of energy that stimulate tumor cells, stimuli can be divided into endogenous and exogenous stimuli. Exogenous stimuli include photodynamic therapy (PDT), radiodynamic therapy (RDT), and sonodynamic therapy (SDT). Although these methods can effectively oxidatively damage tumor cell DNA and proteins with low invasiveness to some extent, their inherent limitations—limited tissue penetration depth, unstable photosensitizers / soundsensitizers, and non-selective killing—result in low tumor treatment efficiency. Conversely, endogenous stimulation, including chemodynamic therapy (CDT), leads to uncertainty in treatment efficacy due to the dynamic and complex nature of the tumor microenvironment (TME) and insufficient amounts of endogenous stimulation, resulting in a completely spontaneous response.

[0003] Radiofrequency (RF) devices generate electromagnetic waves with long wavelengths and low energy, making them ideal for efficient tissue penetration in clinical practice and leading to their widespread application. Numerous tumor treatment strategies have been developed based on RF electromagnetic waves as an energy source. Radiofrequency ablation (RFA) is one of the most widely used thermotherapy methods in clinical practice. It uses electrode needles to deliver high-frequency alternating current to the tumor lesion site, generating heat through ion vibration to cause thermal damage to the tumor. However, it has inherent drawbacks such as trauma, insufficient ablation area, and non-selective killing. Therefore, RF-responsive nanomaterials (such as gold nanoparticles, graphene, and carbon nanotubes) have attracted widespread attention. These materials utilize the electromagnetic waves generated by RF devices to achieve efficient tissue penetration and spatiotemporal selectivity, potentially improving thermal deposition in deep tumor tissues. Furthermore, studies have shown that thermotherapy can induce immunogenic cell death (ICD) in tumor cells, thereby promoting a systemic anti-tumor immune response. However, due to the low energy of the electromagnetic waves generated by RF devices, there are currently no reports in the literature on using RF electromagnetic waves as a stimulus to generate ROS-induced tumor cell necrosis and apoptosis, or even to induce ICD effects in cancer cells.

[0004] In recent years, gallium-based liquid metals (LMs, defined as gallium-based alloys with melting points below room temperature, such as GaIn and GaInSn) have been widely used in energy, electronics, and biomedical engineering due to their excellent biocompatibility and thermal and electrical conductivity. In particular, due to their good deformability, pH-responsive degradation, and microwave (MW) / light / magnetic field responsiveness, LMs have been used in research to treat tumors through methods such as heat generation, ROS generation, and improving the efficacy of chemotherapy. Due to the size effect of nanomaterials, liquid metal micro / nanodroplets have even wider applications. However, because LMs have high surface energy and high surface tension, they exhibit poor colloidal stability in biological media. Therefore, the preparation of LM nanodroplets often employs methods stabilizing them with functional inorganic / organic ligand molecules. Under the influence of sonochemistry, these molecules enable LM nanodroplets to achieve a core-shell structure (with liquid metal as the core) at a nanoscale while also possessing surface multifunctionality. For example, lipid polyethylene glycol (DSPE-PEG) and immunomodulators such as imiquimod (IMIQ), thio(2-hydroxypropyl)-β-cyclodextrin (MUA-CD), doxorubicin (DOX), glucose oxidase (GOX), and platinum (Pt) can be encapsulated in LM nanodroplets using methods such as electrostatic / physical adsorption, chemical coupling, and electrochemical displacement to achieve tumor immunotherapy, chemotherapy, starvation, thermotherapy, and kinetic therapy. Therefore, developing "stabilizers" that can stabilize LM nanodroplets while also fulfilling their own and the functions of the LM nanodroplets is of profound significance for tumor treatment. Summary of the Invention

[0005] The purpose of this application is to provide a gallium-indium alloy nanodispersion system, its preparation, and its application in radiofrequency sensitization. By using gold nanoparticles prepared via glutathione reduction as an emulsifier, the system achieves stability of the gold nanoparticles in the ultrasonicated droplets of liquid gallium-indium alloy while also enabling the gold nanoparticles and the ultrasonicated droplets to function under radiofrequency stimulation. The ultrasonicated droplets of liquid gallium-indium alloy exhibit radiofrequency-responsive reactive oxygen species (ROS) generation, effectively solving the technical problems of limited tissue penetration depth and spatiotemporal selectivity in tumor dynamics therapy, while expanding the application scope of radiofrequency to achieve radiofrequency dynamics therapy for tumors. The gold nanoparticles possess radiofrequency-responsive thermogenic properties, which can synergistically enhance radiofrequency dynamics for tumor treatment. Furthermore, the gold nanoparticles also possess catalase activity, effectively decomposing endogenous hydrogen peroxide in the tumor microenvironment to generate oxygen and promote ROS production, significantly improving the hypoxic tumor microenvironment.

[0006] To achieve the above objectives, this application provides a gallium-indium alloy nanodispersion system. The raw materials of the gallium-indium alloy nanodispersion system include gold nanoparticles prepared by glutathione reduction and nanodroplets of liquid gallium-indium alloy after ultrasonication. During the contact process between the gold nanoparticles prepared by glutathione reduction and the nanodroplets of liquid gallium-indium alloy after ultrasonication, electrochemical displacement occurs, and the gold nanoparticles are adsorbed on the surface of the nanodroplets.

[0007] This application provides a method for preparing the above-mentioned gallium-indium alloy nano-dispersion system, comprising the following preparation steps:

[0008] Step 1: Chloroauric acid and reduced glutathione are mixed and heated to react, followed by ultrafiltration and washing to obtain gold nanoparticles prepared by the glutathione reduction method.

[0009] Step 2: Take out the nanodroplets of the liquid gallium indium alloy after ultrasonication and mix them with the gold nanoparticles prepared by the glutathione reduction method obtained in Step 1, and ultrasonicate them to obtain the gallium indium alloy nanodispersion system.

[0010] Preferably, in step one, the molar ratio of chloroauric acid to reduced glutathione is 1:1.5, and the mixture is carried out at 20-30°C; the temperature of the heating reaction is raised to 65-75°C, the heating reaction time is 20-28 hours, and the number of ultrafiltration washings is 2-3 times.

[0011] Preferably, in step two, the ultrasonication time of the liquid gallium-indium alloy is not less than 30 minutes, and the ultrasonic power is 1000-2000W; the mixed ultrasonication is first performed in a non-contact cell disruptor for not less than 30 minutes with an ultrasonic power of 1000-2000W, and then in a contact cell disruptor for not less than 10 minutes with an ultrasonic power of 100-200W; the molar ratio of the gold nanoparticles prepared by the glutathione reduction method to the nanodroplets of the liquid gallium-indium alloy after ultrasonication is 1:2 to 2:1.

[0012] This application provides an application of the above-mentioned gallium-indium alloy nanodispersion system in radio frequency sensitization.

[0013] Preferably, the gallium-indium alloy nano-dispersion system is used as a sensitizer in a radio frequency field, where the frequency of the radio frequency electromagnetic wave is 13.56MHz, the power is 50W to 300W, and the action time is 0.5min to 10min.

[0014] Therefore, this application provides a gallium-indium alloy nano-dispersion system, its preparation, and its application in radio frequency sensitization, which has the following beneficial effects:

[0015] (1) The introduction of gold nanoparticles prepared by glutathione reduction method can significantly improve their stability compared with nanoparticles after ultrasonication of pure liquid metal gallium indium alloy, and effectively endow the surface of nanoparticles after ultrasonication of liquid metal gallium indium alloy with functionalization.

[0016] (2) The gallium-indium alloy nano-dispersion system provided in this application has the ability to generate active oxygen under radiofrequency stimulation, which successfully expands the application scope of radiofrequency therapy and achieves efficient tissue penetration and spatiotemporal selectivity in tumor treatment.

[0017] (3) The gallium-indium alloy nano-dispersion system provided in this application can be rapidly heated under a radio frequency field, and can be heated to above 40°C both in vivo and in vitro, thereby achieving tumor treatment through synergistic radio frequency dynamics.

[0018] (4) The gallium-indium alloy nano-dispersion system provided in this application has catalase-like activity, which can effectively decompose endogenous hydrogen peroxide in the tumor microenvironment to generate oxygen, promote the generation of reactive oxygen species, and improve the hypoxic microenvironment of the tumor.

[0019] (5) The gallium-indium alloy nano-dispersion system provided in this application can effectively activate anti-tumor immune response in vivo under the combined effects of thermotherapy and kinetic therapy, significantly inhibit the volume growth of breast cancer, and significantly inhibit the metastasis of breast cancer cells. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the LM@GNC fabrication process in an embodiment of this application;

[0021] Figure 2 The structure of the GNC prepared in Example 3 of this application as observed by transmission electron microscopy;

[0022] Figure 3 The valence state of gold in the GNC prepared in Example 3 of this application was detected by X-ray photoelectron spectroscopy.

[0023] Figure 4 The hydrated particle size of LM@GNC prepared in Examples 1-5 of this application was detected by a dynamic light scattering particle size analyzer;

[0024] Figure 5 The elemental energy spectrum of LM@GNC prepared in Example 3 of this application;

[0025] Figure 6The UV-Vis absorption and fluorescence spectra of gold nanoparticles GNC, LM nanodroplets, and LM@GNC obtained in Example 3 are shown below; where a is the UV-Vis absorption and fluorescence spectrum of GNC in Example 3; b is the UV-Vis absorption and fluorescence spectrum of LM in Example 3; and c is the UV-Vis absorption and fluorescence spectrum of LM@GNC in Example 3.

[0026] Figure 7 The images show the in vitro temperature rise curves of gold nanoparticles GNC and LM nanodroplets and LM@GNC obtained in Example 3; where a is the in vitro temperature rise curve of PBS, LM, GNC and LM@GNC; b is the highest temperature rise curve of GNC and LM@GNC at different gold concentrations under the same power radio frequency field; c is the in vitro temperature rise curve of LM@GNC in Example 3 under different power radio frequency fields; and d is the three heating-cooling cycle curve of LM@GNC in Example 3.

[0027] Figure 8 The curves showing the hydrogen peroxide decomposition capabilities of gold nanoparticles GNC and LM nanodroplets, as well as LM@GNC, obtained in Example 3 are shown.

[0028] Figure 9 Example 3 shows the determination of the ability of LM@GNC to generate reactive oxygen species under a radio frequency field; where a is the determination of the ability of LM@GNC to generate reactive oxygen species at different times under a radio frequency field of the same power; b is the determination of the ability of LM@GNC to generate reactive oxygen species under a radio frequency field of different power; c is the change trend of different types of reactive oxygen species of LM@GNC over time under a radio frequency field of the same power.

[0029] Figure 10 To observe the production of ROS in cancer cells under different treatments using laser confocal microscopy;

[0030] Figure 11 The figures show the extracellular ATP and HMGB-1 release levels of cancer cells; where a represents the extracellular ATP release levels of cancer cells under different treatments; and b represents the extracellular HMGB-1 release levels of cancer cells under different treatments.

[0031] Figure 12Figure 1 shows the results of the tumor suppression experiment. In this figure, a represents the timeline of the in vivo antitumor experiment; b represents the weight of the primary tumor in different groups on day 14; c represents the weight of the distal tumor in different groups on day 14; d represents the staining of tumors in different groups for TUNEL, Ki67, HIF-1α, Hypoxia, DCF, and H&E; e represents the normalized mean fluorescence intensity of TUNEL sections from different groups of tumors; f represents the normalized mean fluorescence intensity of Ki67 sections from different groups of tumors; g represents the normalized mean fluorescence intensity of HIF-1α sections from different groups of tumors; and h represents the normalized mean fluorescence intensity of Hypoxia sections from different groups of tumors.

[0032] Figure 13 The graph shows the results of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, urea, creatinine, and lactate dehydrogenase levels; where a represents alanine aminotransferase level; b represents aspartate aminotransferase level; c represents alkaline phosphatase level; d represents urea level; e represents creatinine level; and f represents lactate dehydrogenase level.

[0033] Figure 14 The graph shows the results for white blood cells, red blood cells, and hemoglobin; where a is the number of white blood cells; b is the number of red blood cells; c is the hemoglobin content; d is the hematocrit; e is the mean corpuscular volume; f is the mean corpuscular hemoglobin content; g is the mean corpuscular hemoglobin concentration; and h is the number of platelets.

[0034] Figure 15 Figure 1 shows the in vivo antitumor immune response evaluation of the 4T1 subcutaneous bilateral tumor model in BALB / c mice. Figure 2 shows the timeline of the in vivo antitumor immune response evaluation experiment; Figure 3 shows the maturation status of dendritic cells (DCs) in the right tumor of different groups; Figure 4 shows the maturation status of DCs in the right lymph nodes of different groups; Figure 5 shows the maturation status of DCs in the spleen of different groups; Figure 6 shows the maturation status of CD8+ cells in the right tumor of different groups. + T cell percentage; f represents CD8+ in the right tumor of different groups. + IFN-γ + T cell percentage; g represents CD8+ in the right tumor of different groups. + CD69 + T cell percentage; h represents CD8+ in the right tumor of different groups. + TNF-α + T cell percentage; i represents CD8+ in the right tumor of different groups. + GzmB + T cell percentage; j represents CD4 count in the spleen of different groups. + T cell percentage; k represents CD8+ in the spleen of different groups. +The percentage of T cells; l represents the percentage of polymorphonuclear myeloid-derived suppressor cells in tumors of different groups; m represents the percentage of CD8+ cells in the spleen of different groups. + The proportion of central memory T cells in T cells; n represents the proportion of CD4+ T cells in different groups of spleen. + The proportion of central memory T cells in T cells.

[0035] Figure 16 The figure shows the in vivo anti-tumor metastasis effect of the BALB / C mouse lung metastasis model; where a is the number of lung nodules in different groups of mice; b is the digital image of the lungs of different groups of mice and the corresponding H&E slices; and c is the survival curve of different groups of mice. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0037] This application provides a gallium-indium alloy nanodispersion system, which includes gold nanoparticles prepared by glutathione reduction and nanodroplets obtained by ultrasonication of liquid gallium-indium alloy; wherein, the gold nanoparticles prepared by glutathione reduction undergo electrochemical displacement during contact with the nanodroplets, adsorb onto the surface of the nanodroplets, and stabilize the nanodispersion system.

[0038] The preparation method of the gallium-indium alloy nano-dispersion system is as follows, including the following preparation steps:

[0039] Step 1: Chloroauric acid and reduced glutathione at a molar ratio of 1:1.5 are mixed at 20-30°C until the solution is clear and transparent. Then, the temperature is raised to 65-75°C and reacted for 20-28 hours to reduce chloroauric acid with glutathione. The mixture is then washed 2-3 times by ultrafiltration to obtain gold nanoparticles prepared by the glutathione reduction method.

[0040] Step 2: After ultrasonication, the liquid gallium-indium alloy is removed and mixed with the gold nanoparticles prepared by the glutathione reduction method obtained in Step 1 at a molar ratio of 1:2 to 2:1, and then ultrasonicated again to obtain the gallium-indium alloy nano-dispersion system. The ultrasonication time of the liquid metal is not less than 30 minutes, and the ultrasonic power is 1000-2000W; the mixing ultrasonication is first performed in a non-contact cell disruptor for not less than 30 minutes at an ultrasonic power of 1000-2000W, and then in a contact cell disruptor for not less than 10 minutes at an ultrasonic power of 100-200W.

[0041] This application provides an application of the above-mentioned gallium-indium alloy nanodispersion system in treating cancer and activating immune anti-tumor responses, wherein the cancer includes breast cancer. Under a radio frequency field, radio frequency dynamics cause the nanodroplets of liquid gallium-indium alloy to generate reactive oxygen species after ultrasonication, which excites the temperature of gold nanoparticles to rise, triggering the decomposition of endogenous hydrogen peroxide in the tumor microenvironment to produce oxygen, resulting in an increase in temperature and accumulation of reactive oxygen species at the tumor site, causing immunogenic cell death. The modulation of radio frequency causes the gallium-indium alloy nanodispersion system to induce a systemic anti-tumor immune response, inhibit the growth of breast cancer cells, and inhibit the metastasis of breast cancer cells.

[0042] The preparation method of the gold gallium indium alloy nano-dispersion system prepared by the glutathione reduction method in this application is simple, such as... Figure 1 As shown, firstly, liquid gallium indium alloy is ultrasonicated, and then a portion of the ultrasonicated liquid gallium indium alloy nanodroplets are taken out and placed into a gold nanoparticle solution for mixing and ultrasonication, so that the gold nanoparticles modify the nanodroplets, resulting in a stable nano-dispersion system with a nanoparticle size of approximately 100-500 nanometers.

[0043] This application provides a stable nanodispersion system containing gold nanoparticles and nanodroplets exhibiting radiofrequency-responsive behavior, which can be used for cancer treatment. This application utilizes gold nanoparticles prepared via glutathione reduction as an emulsifier to improve the stability of the nanodroplets. The nanodroplets possess the ability to generate reactive oxygen species (ROS) in a radiofrequency field; the gold nanoparticles exhibit catalase-like activity and also possess the ability to generate heat in a radiofrequency field. This nanodispersion system can improve ROS generation efficiency, expand the application scenarios of radiofrequency electromagnetic waves, and achieve multifunctional synergistic tumor treatment. Finally, animal experiments demonstrate that the synergistic effect of the two can induce immunogenic cell death, trigger a systemic anti-tumor immune response, and improve the anti-tumor therapeutic effect.

[0044] In this application, by selecting nanodroplets as the main nanoparticle and gold nanoparticles prepared by the glutathione reduction method as an emulsifier, the two are blended and ultrasonicated to prepare structurally stable nanoparticles with a particle size of approximately 100-500 nanometers. Compared with nanodroplets obtained by ultrasonicating only liquid metal, these nanoparticles significantly improve the stability of the nanodroplets. Furthermore, due to the radiofrequency responsive behavior of the gold nanoparticles, they exhibit better antitumor effects in a mouse model of breast cancer.

[0045] The nano-dispersion system provided in this application utilizes radiofrequency radiation to rapidly raise the temperature of the tumor site and generate reactive oxygen species, while simultaneously inducing immunogenic cell death in cancer cells, thus achieving a combined treatment of radiofrequency thermotherapy, radiofrequency kinetic therapy, and immunotherapy, thereby enhancing the anti-tumor efficacy.

[0046] Example 1

[0047] (1) Chloroauric acid and reduced glutathione were reacted at 25°C for 15 min at a molar ratio of 1:1.5, and then the temperature was raised to 70°C and reacted for 24 hours. The gold nanoparticles prepared by the glutathione reduction method were obtained by ultrafiltration and washing.

[0048] (2) Take an appropriate amount of LM and put it into a non-contact cell disruptor to obtain the mother liquor of liquid metal gallium indium alloy nanodroplets by ultrasonication. The ultrasonication time is 30 minutes and the ultrasonic power is 1440W.

[0049] (3) Gold nanoparticles (GNC) and LM nanodroplets were mixed and sonicated in an ice-water bath at a molar ratio of 1:2. The mixing and sonication were first performed in a non-contact cell disruptor for 30 minutes at a power of 1440W, and then in a contact cell disruptor for 10 minutes at a power of 150W. Finally, LM@GNC was purified by gentle centrifugation.

[0050] Example 2

[0051] (1) Chloroauric acid and reduced glutathione were reacted at 25°C for 15 min at a molar ratio of 1:1.5, and then the temperature was raised to 70°C and reacted for 24 hours. The gold nanoparticles prepared by the glutathione reduction method were obtained by ultrafiltration and washing.

[0052] (2) Take an appropriate amount of LM and put it into a non-contact cell disruptor to obtain the mother liquor of liquid metal gallium indium alloy nanodroplets by ultrasonication. The ultrasonication time is 30 minutes and the ultrasonic power is 1440W.

[0053] (3) Gold nanoparticles (GNC) and LM nanodroplets were mixed and sonicated in an ice-water bath at a molar ratio of 1:1.5. The mixing and sonication were first performed in a non-contact cell disruptor for 30 minutes at a power of 1440W, and then in a contact cell disruptor for 10 minutes at a power of 150W. Finally, LM@GNC was purified by gentle centrifugation.

[0054] Example 3

[0055] (1) Chloroauric acid and reduced glutathione were reacted at 25°C for 15 min at a molar ratio of 1:1.5, and then the temperature was raised to 70°C and reacted for 24 hours. The gold nanoparticles prepared by the glutathione reduction method were obtained by ultrafiltration and washing.

[0056] Its structure was observed using a transmission electron microscope, and the results are as follows: Figure 2 As shown, the results indicate that the average particle size is approximately 1.5 nm, and the interplanar spacing is 0.29 nm. X-ray photoelectron spectroscopy was used to determine the valence state of the gold element, and the results are as follows. Figure 3As shown, the detection of its bivalent (0-valent and 1-valent) results indicates the successful synthesis of GNC.

[0057] (2) Take an appropriate amount of LM and put it into a non-contact cell disruptor to obtain the mother liquor of liquid metal gallium indium alloy nanodroplets by ultrasonication. The ultrasonication time is 30 minutes and the ultrasonic power is 1440W.

[0058] (3) Gold nanoparticles GNC and LM nanodroplets were mixed and sonicated in an ice-water bath at a molar ratio of 1:1. The mixing and sonication were first performed in a non-contact cell disruptor for 30 minutes at a power of 1440W, and then in a contact cell disruptor for 10 minutes at a power of 150W. Finally, LM@GNC was purified by gentle centrifugation.

[0059] Example 4

[0060] (1) Chloroauric acid and reduced glutathione were reacted at 25°C for 15 min at a molar ratio of 1:1.5, and then the temperature was raised to 70°C and reacted for 24 hours. The gold nanoparticles prepared by the glutathione reduction method were obtained by ultrafiltration and washing.

[0061] (2) Take an appropriate amount of LM and put it into a non-contact cell disruptor to obtain the mother liquor of liquid metal gallium indium alloy nanodroplets by ultrasonication. The ultrasonication time is 30 minutes and the ultrasonic power is 1440W.

[0062] (3) Gold nanoparticles (GNC) and LM nanodroplets were mixed and sonicated in an ice-water bath at a molar ratio of 1.5:1. The mixing and sonication were first performed in a non-contact cell disruptor for 30 minutes at a power of 1440W, and then in a contact cell disruptor for 10 minutes at a power of 150W. Finally, LM@GNC was purified by gentle centrifugation.

[0063] Example 5

[0064] (1) Chloroauric acid and reduced glutathione were reacted at 25°C for 15 min at a molar ratio of 1:1.5, and then the temperature was raised to 70°C and reacted for 24 hours. The gold nanoparticles prepared by the glutathione reduction method were obtained by ultrafiltration and washing.

[0065] (2) Take an appropriate amount of LM and put it into a non-contact cell disruptor to obtain the mother liquor of liquid metal gallium indium alloy nanodroplets by ultrasonication. The ultrasonication time is 30 minutes and the ultrasonic power is 1440W.

[0066] (3) Gold nanoparticles (GNC) and LM nanodroplets were mixed and sonicated in an ice-water bath at a molar ratio of 2:1. The mixing and sonication were first performed in a non-contact cell disruptor for 30 minutes at a power of 1440W, and then in a contact cell disruptor for 10 minutes at a power of 150W. Finally, LM@GNC was purified by gentle centrifugation.

[0067] The average particle size of LM@GNC obtained in Examples 1-5 was measured using a dynamic light scattering particle size analyzer, and the results are as follows: Figure 4 As shown, the hydrated particle size of the nanoparticles in several ratios ranges from 100 to 500 nanometers. Considering the stability, heat generation, and reactive oxygen species performance of the nanoparticles, the optimal ratio is 1:1. The elemental distribution results are shown in the figure. Figure 5 As shown, this illustrates its core-shell structure.

[0068] I. UV-Vis absorption and fluorescence spectra of gold nanoparticles (GNC), LM nanodroplets, and LM@GNC obtained in Example 3.

[0069] Gold nanoparticles (GNC), LM nanodroplets, and LM@GNC obtained in Example 3, along with ultrapure aqueous solutions, were used as references. The absorption spectra of the three samples were measured using a UV-Vis spectrophotometer with a scanning wavelength range of 200-850 nm and a scanning step size of 1 nm. The excitation and emission spectra of the three samples were measured using a time-resolved fluorescence spectrometer. The results are shown below. Figure 6 As shown.

[0070] The results show that LM has no Stokes displacement property, while GNC has a large Stokes displacement. LM@GNC also has a large Stokes displacement, indicating that GNC has successfully adhered to the LM surface.

[0071] II. In vitro temperature rise of different concentrations and samples

[0072] Take the gold nanoparticles GNC and LM nanodroplets and LM@GNC obtained in Example 3, wherein the Au molar concentration is 1 mmol / L. -1 The molar concentration of LM is 1 mmol / L. -1In addition, PBS was used as a control group. 1.6 mL of the above solution was placed in a liquid chromatography vial and irradiated with a radiofrequency instrument at 100 W. The temperature change of the liquid in the vial was detected using a thermal imager, and the temperature was recorded every 30 seconds for 300 seconds. Separately, 1.6 mL of the LM@GNC solution from Example 3 was irradiated with a radiofrequency instrument at 50, 75, 100, 150, and 200 W, respectively. The temperature change of the liquid in the vial was detected using a thermal imager, and the temperature was recorded every 30 seconds for 300 seconds. Finally, the LM@GNC solution was subjected to three heating-cooling cycles, and the temperature change of the liquid in the vial was detected using a thermal imager, with the temperature recorded every 30 seconds. The results are as follows: Figure 7 Contents a, b, c, and d are shown.

[0073] The results showed that LM had no radio frequency (RF) heating effect, while GNC and LM@GNC did. The concentration of Au in the solution and the power of the RF field had a significant impact on the RF thermal effect of both GNC and LM@GNC. Furthermore, LM@GNC exhibited good thermal stability, with the maximum temperature remaining essentially the same throughout the three thermal cycles.

[0074] III. Catalase activity in different samples

[0075] The gold nanoparticles GNC, LM nanodroplets, and LM@GNC obtained in Example 3 were analyzed using an oxygen-specific probe ([Ru(dpp)3]Cl2). Weaker fluorescence intensity of [Ru(dpp)3]Cl2 indicated a higher concentration of oxygen in the solution. Results are as follows: Figure 8 As shown, the fluorescence intensity of the LM@GNC and GNC groups decreased continuously within 10 minutes, while that of the ultrapure water and LM groups remained basically unchanged. After 10 minutes, the fluorescence intensity of LM@GNC and GNC was similar and lower than that of the ultrapure water and LM groups, indicating that GNC has catalase-like activity and can decompose hydrogen peroxide to produce oxygen.

[0076] IV. RF Dynamics of LM@GNC

[0077] Total ROS generation was detected using a DCFH-DA probe. 400 μL of LM@GNC from Example 3 (LM to GNC molar ratio 1 / 1) was mixed with 20 μL of DCFH-DA (20 mM) and treated at a radio frequency field (100 W) for 1, 2, 3, 4, and 5 min, respectively, followed by centrifugation (12000 rpm, 5 min). The supernatant was then analyzed using a Fluro Max+ spectrometer. To investigate the effect of radio frequency field power on reactive oxygen species generation, LM@GNC from Example 3 was mixed with 20 μL of DCFH-DA (20 mM) and treated at different radio frequency fields (50 W, 75 W, 100 W, 150 W, 200 W) for 5 min, followed by centrifugation (12000 rpm, 5 min), and finally analyzed using a Fluro Max+ spectrometer. To detect the types of ROS generated under a radio frequency field, hydroxyl radical-specific probes (HPF), superoxide anion-specific probes (BES-SO), and singlet oxygen-specific probes (DPBF) were used to detect the changes in these three different reactive oxygen species over time in LM@GNC under the same radio frequency field (100W). The results are as follows: Figure 9 Content a, content b, and content c are shown.

[0078] The results showed that as the radio frequency time increased and the radio frequency field power increased, LM@GNC generated more reactive oxygen species in the radio frequency field. These reactive oxygen species mainly included hydroxyl radicals, superoxide anions, and singlet oxygen.

[0079] V. Studying the intracellular ROS content of cells after different treatments using confocal fluorescence microscopy

[0080] Intracellular ROS production was measured under CLSM using a 2',7'-dichlorofluorescein diacetate (DCFH-DA) probe. 20,000 4T1 cells were seeded in 6-well plates using gold nanoparticles (GNC), LM nanodroplets, and LM@GNC obtained in Example 3. After 12 hours, the cells were co-cultured under different treatment conditions (G1:PBS, G2:LM, G3:LM+RF, G4:GNC, G5:GNC+RF, G6:LM@GNC, G7:LM@GNC+RF). After another 4 hours of culture, the cells were digested with trypsin to remove the substances. The reactive oxygen species assay was then performed according to the instructions. Finally, fluorescence was detected under CLSM. The results are shown below. Figure 10 As shown

[0081] The results showed that ROS was generated in cells only when LM was present and placed in a radio frequency field.

[0082] VI. Studying in vitro cell immunogenicity death after different treatments using an enzyme-linked immunosorbent assay (ELISA) reader

[0083] The gold nanoparticles GNC, LM nanodroplets, and LM@GNC obtained in Example 3 were used to seed 50,000 4T1 cells in 24-well plates. After 12 hours, the cells were co-cultured under different treatment conditions (G1:PBS, G2:LM, G3:LM+RF, G4:GNC, G5:GNC+RF, G6:LM@GNC, G7:LM@GNC+RF). After another 4 hours of culture, the extracellular HMGB-1 and ATP levels were measured using the cell culture supernatant according to the operating procedures of the HMGB-1 enzyme-linked immunosorbent assay kit and the enhanced chemiluminescent ATP assay kit. The results are as follows: Figure 11 Content a and content b are shown.

[0084] The results showed that the cells in group G7 released the most ATP and HMGB-1, which was higher than other radiofrequency groups (G5, G3), indicating that heat and ROS have a significant synergistic effect in promoting the ICD effect.

[0085] VII. Establishment of a 4T1 subcutaneous bilateral tumor model in BALB / c mice: 1 million 4T1 cells were subcutaneously inoculated into the right hind limb of mice, and 330,000 4T1 cells were inoculated into the left hind limb. When the average tumor volume of the right hind limb increased to 100 cubic millimeters, gold nanoparticles (GNC), LM nanodroplets, and LM@GNC obtained in Example 3 were used. Mice were randomly divided into 7 groups: G1 (PBS), G2 (LM), G3 (LM+RF), G4 (GNC), G5 (GNC+RF), G6 (LM@GNC), and G7 (LM@GNC+RF). After injecting the materials into the right tumor of the mice, the mice were treated with a radiofrequency field of 300W for 10 minutes. The volume of the left and right tumors was recorded every two days, calculated using the following formula: Volume = Length × Width 2 / 2. The result is as follows: Figure 12 As shown in content b and content c. Mice were euthanized on day fourteen after treatment, and the tumors on both sides were dissected. The tumors were then fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned for staining. The staining indicators included: H&E, Ki67, TUNEL, HIF-1α, and Hypoxia. The results are as follows. Figure 12 As shown in d. Simultaneously, three mice from each group were sampled for whole blood and serum collection to determine their complete blood count and blood biochemical parameters. The results are shown in d. Figure 13 and 14 As shown in the figure. For the determination of intratumoral ROS content, mouse tumors were pretreated with LM@GNC, and then DCFH-DA was injected into the mouse tumors. After 0.5 h, the mice were subjected to radiofrequency field treatment. Subsequently, the mice were euthanized, the tumors were removed and sectioned, and observed using CLSM. The results are shown in the figure. Figure 12As shown in d. Immunofluorescence sections of TUNEL and Ki67 and H&E results indicated that mice treated with LM, GNC, and LM@GNC all exhibited varying degrees of tumor cell apoptosis and inhibition of cell proliferation. Among them, LM@GNC+RF showed the strongest tumor cell killing effect. DCF fluorescence section results showed that mice treated with LM and LM@GNC produced ROS under radiofrequency. HIF-1α and Hypoxia fluorescence section results showed that mice treated with materials containing GNC exhibited reduced fluorescence in their corresponding fluorescence sections, indicating a good effect in alleviating tumor hypoxia.

[0086] The blood routine indicators of the sample were measured using a hematology analyzer. Whole blood was collected by centrifugation before testing. The results are as follows: Figure 13 and 14 As shown. Figure 13 Content a represents the alanine aminotransferase content; Figure 13 Content b is the aspartate aminotransferase content; Figure 13 Content c represents the alkaline phosphatase content; Figure 13 Content d represents the urea content; Figure 13 Content e represents creatinine content; Figure 13 Content f represents the lactate dehydrogenase content. Figure 14 Content a represents the number of white blood cells; Figure 14 Content b is the number of red blood cells; Figure 14 Content c represents the hemoglobin content; Figure 14 Content d represents hematocrit; Figure 14 Content e represents the mean corpuscular volume (MCV). Figure 14 Content f represents the mean corpuscular hemoglobin level; Figure 14 Content g represents the mean corpuscular hemoglobin concentration; Figure 14 The content h represents the platelet count. Results showed that, due to the poor response to tumor treatment, the PBS group mice exhibited elevated white blood cell counts, decreased platelet counts, and impaired liver, kidney, and cardiac function in their hematological examinations. In contrast, the LM@GNC+RF group mice showed no significant hematological or hepatic / renal / cardiac abnormalities, demonstrating good biocompatibility.

[0087] 9. After constructing a 4T1 subcutaneous bilateral tumor model in BALB / C mice, we evaluated the in vivo antitumor immune effect of LM@GNC+RF.

[0088] Following administration of the drug and radiofrequency ablation to establish a 4T1 subcutaneous bilateral tumor model in BALB / c mice, tumors, lymph nodes, and spleens were harvested on day 4 post-treatment to assess DC cell maturation and enhance CD8 expression. + Detection of T cell function and improvement of the immunosuppressive microenvironment. For example... Figure 15As shown, the LM@GNC+RF group had significantly higher levels of mature dendritic cells in tumors, lymph nodes, and spleen compared to other groups, and also exhibited enhanced CD8 activity. + T cell function (CD8) + IFN-γ + CD8 + CD69 + CD8 + TNF-α + CD8 + GzmB + CD8 + The LM@GNC+RF group showed the strongest ability to enhance T cell levels and improve the immunosuppressive microenvironment (reduced PMN-MDSCs cell levels). Furthermore, the LM@GNC+RF group also exhibited the strongest central memory T cell levels. Therefore, these results indicate that LM@GNC+RF-induced thermotherapy and kinetic therapy can significantly enhance tumor-specific immune responses.

[0089] 10. After constructing a 4T1 lung metastasis model in BALB / C mice, evaluate the in vivo anti-tumor metastasis effect of LM@GNC+RF.

[0090] Fluorescent 4T1 cells were injected into the tail vein of mice with subcutaneous bilateral 4T1 tumors. Twenty-four hours later, the mice were treated with medication and radiofrequency ablation. Lung tissue was harvested on day 30 post-treatment, and the number of nodules was counted and H&E staining was performed. Figure 16 As shown, the results indicate that mice treated with LM@GNC+RF had the lowest number of lung nodules and exhibited the longest survival, demonstrating a good anti-tumor metastasis effect.

[0091] Therefore, this application provides a gallium-indium alloy nanodispersion system and its preparation, as well as its application in radiofrequency sensitization. The introduction of gold nanoparticles prepared by the glutathione reduction method can significantly improve the stability of the nanodroplets and endow the nanodispersion system with catalase activity, significantly improving the hypoxic microenvironment of tumors. Radiofrequency stimulation can rapidly raise the temperature of the tumor site and generate reactive oxygen species, while inducing a systemic anti-tumor immune response, achieving synergistic treatment of tumor tissue.

[0092] In the description of this specification, the references to terms such as "an experimental example," "example," "specific example," etc., mean that a specific feature, structure, material, or characteristic described in connection with that experimental example or example is included in at least one experimental example or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same experimental example or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more experimental examples or examples.

[0093] Finally, it should be noted that the above experimental examples are only used to illustrate the technical solutions of this application and not to limit them. Although this application has been described in detail with reference to preferred experimental examples, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of this application, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of this application.

Claims

1. A gallium-indium alloy nano-dispersion system, characterized in that, The raw materials of the gallium-indium alloy nanodispersion system include gold nanoparticles prepared by glutathione reduction and nanodroplets of liquid gallium-indium alloy after ultrasonication. During the contact process between the gold nanoparticles prepared by glutathione reduction and the nanodroplets of liquid gallium-indium alloy after ultrasonication, electrochemical displacement occurs, and the gold nanoparticles are adsorbed on the surface of the nanodroplets of liquid gallium-indium alloy after ultrasonication.

2. A method for preparing a gallium-indium alloy nano-dispersion system, characterized in that, The preparation steps of the gallium-indium alloy nano-dispersion system are as follows: Step 1: Chloroauric acid and reduced glutathione are mixed and heated to react, followed by ultrafiltration and washing to obtain gold nanoparticles prepared by the glutathione reduction method. Step 2: Take out the nanodroplets of the liquid gallium indium alloy after ultrasonication and mix them with the gold nanoparticles prepared by the glutathione reduction method obtained in Step 1, and ultrasonicate them to obtain the gallium indium alloy nanodispersion system.

3. The method for preparing a gallium-indium alloy nano-dispersion system according to claim 2, characterized in that, In step one, the molar ratio of chloroauric acid to reduced glutathione is 1:1.5, and the mixture is carried out at 20~30℃; the temperature of the heating reaction is raised to 65~75℃, the heating reaction time is 20~28 hours, and the ultrafiltration washing is performed 2~3 times.

4. The method for preparing a gallium-indium alloy nano-dispersion system according to claim 2, characterized in that, In step two, the ultrasonic treatment of the liquid gallium-indium alloy is for no less than 30 minutes and the ultrasonic power is 1000~2000W; the mixed ultrasonic treatment is first performed in a non-contact cell disruptor for no less than 30 minutes and the ultrasonic power is 1000~2000W, and then in a contact cell disruptor for no less than 10 minutes and the ultrasonic power is 100~200W; the molar ratio of the gold nanoparticles prepared by the glutathione reduction method to the nanodroplets of the liquid gallium-indium alloy after ultrasonic treatment is 1:2~2:

1.

5. The application of the gallium-indium alloy nanodispersion system as described in claim 1 in the preparation of radio frequency sensitizers.

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