Gallium-indium alloy nano-dispersion system, preparation thereof and application of gallium-indium alloy nano-dispersion system in radio frequency sensibilization

Abstract

The invention discloses a gallium-indium alloy nano dispersion system, preparation thereof and application of the gallium-indium alloy nano dispersion system in radio frequency sensibilization, and belongs to the cross technical field of chemistry, medicine and other subjects. Raw materials of the gallium-indium alloy nanometer dispersion system comprise gold nanoparticles prepared by a glutathione reduction method and nanometer liquid drops obtained after ultrasonic treatment of liquid metal gallium-indium alloy, and electrochemical replacement occurs in the process that the gold nanoparticles prepared by the glutathione reduction method make contact with the liquid metal nanometer liquid drops. And adsorbing on the surface of the nano liquid drop of the liquid metal gallium-indium alloy after ultrasonic treatment. The gallium-indium alloy nano dispersion system can generate various reactive oxygen free radicals under the action of radio-frequency electromagnetic waves, enables the temperature of an irradiated part to rise quickly, cooperatively kills tumor cells, activates whole-body anti-tumor immune response, and effectively inhibits tumor recurrence and metastasis. The gold nanoparticles not only can significantly improve the stability of the nano-dispersion, but also endow the nano-dispersion with catalase activity, and further enhance the antitumor activity of the nano-dispersion.

Description

Technical Field

[0001] This application belongs to the cross-disciplinary technical field of chemistry, medicine, etc., and relates to a gallium-indium alloy nano-dispersion system and its preparation method and application. Background Art

[0002] Tumor dynamic therapy refers to the conversion of different forms of energy (such as infrared, x-rays, ultrasound, chemical energy, etc.) into reactive oxygen species (ROS), inducing the accumulation of excessive ROS in the tumor, leading to oxidative stress in the tumor and causing apoptosis and necrosis of tumor cells. In addition, studies have shown that ROS can induce an immune response in tumors. Compared with traditional cancer treatments (such as chemotherapy and radiotherapy), tumor dynamic therapy can effectively avoid drug resistance and systemic toxicity, reduce side effects and invasive damage. According to the different energy forms that stimulate tumor cells, the stimulus sources can be divided into endogenous and exogenous stimuli. Among them, exogenous stimuli include photodynamic therapy (PDT), radiodynamic therapy (RDT), sonodynamic therapy (SDT), etc. Although they can effectively oxidize and damage tumor cell DNA and proteins in a low-invasive manner to a certain extent, the above-mentioned therapies have inherent limitations such as limited tissue penetration depth, instability of photosensitizers / sonosensitizers, and non-selective killing, resulting in low tumor treatment efficiency. In contrast, endogenous stimulation, including chemodynamic therapy (CDT), is completely spontaneous, leading to uncertainty in therapeutic efficacy due to the dynamics and complexity of the tumor microenvironment (TME) and the insufficient amount of endogenous stimulation.

[0003] The electromagnetic waves generated by radiofrequency devices have the characteristics of long wavelength and low energy. They are easy to achieve efficient tissue penetration in clinical practice and are currently widely used. Many tumor treatment strategies have been developed based on radiofrequency electromagnetic waves as energy sources. Radiofrequency ablation (RFA) is one of the thermal therapies widely used in clinical practice. It uses electrode needles to deliver high-frequency alternating current to the tumor lesion site, and generates heat through ion vibration to cause thermal damage to the tumor site. However, it has inherent trauma, insufficient ablation area, and non-selective killing. Therefore, nanomaterials based on radiofrequency responsiveness (such as gold nanoparticles, graphene, carbon nanotubes, etc.) have received widespread attention. They use the electromagnetic waves generated by radiofrequency devices to achieve efficient tissue penetration and spatiotemporal selectivity, and are expected to improve the thermal deposition of deep tumor tissues. At the same time, studies have shown that hyperthermia can induce immunogenic cell death (ICD) of tumor cells to promote systemic anti-tumor immune response. However, due to the low energy of electromagnetic waves generated by radiofrequency devices, there is no literature reporting the use of radiofrequency electromagnetic waves as a stimulus to generate ROS to induce tumor cell necrosis and apoptosis or even cause the ICD effect of cancer cells.

[0004] In recent years, gallium-based liquid metals (LM, defined as gallium-based alloys with melting points below room temperature, such as GaIn and GaInSn, etc.) have been widely used in energy, electronics, biomedical engineering and other fields due to their good biocompatibility and thermal and electrical conductivity. In particular, due to LM's good deformability, pH-responsive degradation, and microwave (MW) / light / magnetic field responsiveness, studies have used it to treat tumors by generating heat, producing ROS, and improving chemotherapy effects. Due to the size effect of nanomaterials, liquid metal micro / nano droplets have a wider range of uses. However, due to the high surface energy and high surface tension of LM, it has poor colloidal stability in biological media, so the preparation of LM nanodroplets mostly adopts the method of stabilization by functional inorganic / organic ligand molecules. Under the action of sonochemistry, these molecules enable LM nanodroplets to achieve a nano-sized core-shell structure (liquid metal as the core) while also having surface multifunctionality. For example, lipid polyethylene glycol (DSPE-PEG) and immunomodulator imiquimod (IMIQ), thio(2-hydroxypropyl)-β-cyclodextrin (MUA-CD) and doxorubicin (DOX), glucose oxidase (GOX), platinum (Pt) and the like are encapsulated in LM by electrostatic / physical adsorption, chemical coupling and electrochemical replacement to achieve tumor immunity, chemotherapy, starvation, heat and dynamic therapy. Therefore, the development of a "stabilizer" that can stabilize LM nanodroplets while exerting its own and LM nanodroplet functions has far-reaching significance for tumor treatment. Summary of the invention

[0005] The purpose of the present application is to provide a gallium-indium alloy nano-dispersion system and its preparation and application in radio frequency sensitization. By using gold nanoparticles prepared by glutathione reduction method as emulsifiers, the gold nanoparticles are stabilized on the nanodroplets of liquid metal gallium-indium alloy after ultrasound, and the gold nanoparticles and the nanodroplets of liquid metal gallium-indium alloy after ultrasound can also play the functions of gold nanoparticles and liquid metal gallium-indium alloy after ultrasound under radio frequency stimulation. Among them, the nanodroplets of liquid metal gallium-indium alloy after ultrasound have the performance of radio frequency responsive production of reactive oxygen, which effectively solves the technical problems of limited tissue penetration depth and uncertainty of spatiotemporal selection in the field of tumor dynamics treatment, and at the same time expands the application scope of radio frequency to realize radio frequency dynamics treatment of tumors. Gold nanoparticles have radio frequency responsive heat production performance, which can cooperate with radio frequency dynamics to achieve tumor treatment. In addition, gold nanoparticles also have catalase activity, which can effectively decompose endogenous hydrogen peroxide in the tumor microenvironment to produce oxygen to promote the production of reactive oxygen, and significantly improve the hypoxic microenvironment of the tumor.

[0006] To achieve the above-mentioned purpose, the present application provides a gallium-indium alloy nano-dispersion system. The raw materials of the gallium-indium alloy nano-dispersion system include gold nanoparticles prepared by glutathione reduction method and nano-droplets of liquid metal gallium-indium alloy after ultrasound. The gold nanoparticles prepared by glutathione reduction method undergo electrochemical replacement during contact with the nano-droplets of liquid metal gallium-indium alloy after ultrasound, and are adsorbed on the surface of the nano-droplets of liquid metal gallium-indium alloy after ultrasound.

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

[0008] Step 1, mixing chloroauric acid and reduced glutathione, heating the mixture for reaction, and ultrafiltration and washing to obtain gold nanoparticles prepared by glutathione reduction method;

[0009] Step 2: Take out the nano droplets of the liquid metal gallium-indium alloy after ultrasonic treatment and mix them with the gold nanoparticles prepared by glutathione reduction method obtained in step 1 and ultrasonically obtain the gallium-indium alloy nano-dispersion system.

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

[0011] Preferably, in step 2, the ultrasonic time of the liquid metal gallium indium alloy is not less than 30 minutes, and the ultrasonic power is 1000-2000W; the mixed ultrasound is first ultrasonicated in a non-contact cell disruptor for not less than 30 minutes, and the ultrasonic power is 1000-2000W, and then ultrasonicated in a contact cell disruptor for not 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 metal gallium indium alloy after ultrasound is 1:2-2:1.

[0012] The present 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, the frequency of the radio frequency electromagnetic wave is 13.56 MHz, the power is 50 W to 300 W, and the action time is 0.5 min to 10 min.

[0014] Therefore, the present application provides a gallium-indium alloy nano-dispersion system and its preparation and 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 the stability of nanoparticles after ultrasound treatment of pure liquid metal gallium-indium alloy, and effectively endow the surface of nanoparticles after ultrasound treatment of liquid metal gallium-indium alloy with functionalization.

[0016] (2) The present application provides a gallium-indium alloy nanodispersion system, which has the ability to generate reactive oxygen under radio frequency stimulation, successfully expanding the application scope of radio frequency treatment and achieving efficient tissue penetration and spatiotemporal selectivity in tumor treatment.

[0017] (3) The gallium-indium alloy nanodispersion system provided in the present application can rapidly heat up under a radio frequency field, and can heat up to above 40°C both inside and outside the body, thereby achieving tumor treatment through coordinated radio frequency dynamics.

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

[0019] (5) The gallium-indium alloy nanodispersion system provided in the present application can effectively activate anti-tumor immune response in vivo under the combined action of thermal therapy and dynamic therapy, significantly inhibit the volume growth of breast cancer, and significantly inhibit the metastasis of breast cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A schematic diagram of the process for preparing LM@GNC in the embodiment of the present application;

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

[0022] Figure 3 The valence state of gold element detected by X-ray photoelectron spectroscopy technique for GNC prepared in Example 3 of the present application;

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

[0024] Figure 5 Elemental spectrum of LM@GNC prepared in Example 3 of the present application;

[0025] Figure 6The UV-visible absorption spectra and fluorescence spectra of the gold nanoparticles GNC, LM nanodroplets and LM@GNC obtained in Example 3; wherein a is the UV-visible absorption spectrum and fluorescence spectrum of GNC in Example 3; b is the UV-visible absorption spectrum and fluorescence spectrum of LM in Example 3; c is the UV-visible absorption spectrum and fluorescence spectrum of LM@GNC in Example 3;

[0026] Figure 7 The in vitro temperature rise curves of the gold nanoparticles GNC, LM nanodroplets and LM@GNC obtained in Example 3; wherein, a is the in vitro temperature rise curves of PBS, LM, GNC and LM@GNC; b is the maximum 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 powers radio frequency fields; d is the three heating-cooling cycle curves of LM@GNC in Example 3.

[0027] Figure 8 The ability curves of the gold nanoparticles GNC, LM nanodroplets and LM@GNC obtained in Example 3 to decompose hydrogen peroxide are measured;

[0028] Fig. 9 The figure is the measurement of the ability of LM@GNC to produce active oxygen under a radio frequency field in Example 3; wherein, a is the measurement of the ability of LM@GNC to produce active oxygen at different times in a radio frequency field of the same power; b is the measurement of the ability of LM@GNC to produce active oxygen under radio frequency fields of different powers; c is the change trend of different types of active oxygen over time in a radio frequency field of the same power of LM@GNC.

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

[0030] Fig.11 Figure 1 is a graph showing the extracellular ATP and HMGB-1 release of cancer cells; a is the measurement of the extracellular ATP release of cancer cells under different treatments; b is the measurement of the extracellular HMGB-1 release of cancer cells under different treatments;

[0031] Fig.12Figure 1 is the result of the tumor inhibition experiment; wherein, a is the time plan of the in vivo anti-tumor experiment; b is the weight of the main end tumor of different groups on the fourteenth day; c is the weight of the distal tumor of different groups on the fourteenth day; d is the staining of TUNEL, Ki67, HIF-1α, Hypoxia, DCF, and H&E of tumors in different groups; e is the normalized mean fluorescence intensity of TUNEL sections of tumors in different groups; f is the normalized mean fluorescence intensity of Ki67 sections of tumors in different groups; g is the normalized mean fluorescence intensity of HIF-1α sections of tumors in different groups; h is the normalized mean fluorescence intensity of Hypoxia sections of tumors in different groups.

[0032] Fig.13 It is a result graph of alanine aminotransferase, aspartate aminotransferase content, alkaline phosphatase, urea, creatinine and lactate dehydrogenase content; wherein, a is the alanine aminotransferase content; b is the aspartate aminotransferase content; c is the alkaline phosphatase content; d is the urea content; e is the creatinine content; f is the lactate dehydrogenase content;

[0033] Fig.14 The result graph of white blood cells, red blood cells and hemoglobin; among them, 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] Fig.15 The following is an analysis diagram of the in vivo anti-tumor immune effect evaluation of the 4T1 subcutaneous bilateral tumor model in BALB / C mice; a is the experimental time plan for evaluating the in vivo anti-tumor immune response; b is the maturation of DCs cells in the right tumor of different groups; c is the maturation of DCs cells in the right lymph nodes of different groups; d is the maturation of DCs cells in the spleen of different groups; e is the CD8 + T cell ratio; f is the CD8 + IFN-γ + T cell ratio; g is the CD8 + CD69 + T cell ratio; h is CD8 in the right tumor of different groups + TNF-α + T cell ratio; i is the CD8 + G JZ + T cell ratio; j is the CD4 + T cell ratio; k is the CD8 +The proportion of T cells; l is the proportion of polymorphonuclear myeloid suppressor cells in tumors of different groups; m is the proportion of CD8 + The proportion of central memory T cells in T cells; n is the spleen of different groups, in CD4 + The proportion of central memory T cells in T cells.

[0035] Fig.16 This is an analysis diagram for evaluating the in vivo anti-tumor metastasis effect in the lung metastasis model of BALB / C mice; wherein, a is the number of lung nodules in different groups of mice; b is the digital images of the lungs of different groups of mice and the corresponding H&E sections; c is the survival curves of different groups of mice. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0037] The present application provides a gallium-indium alloy nano-dispersion system, which includes gold nanoparticles prepared by glutathione reduction method, and also includes nano-droplets obtained after ultrasonic treatment of liquid metal gallium-indium alloy; wherein the gold nanoparticles prepared by glutathione reduction method undergo electrochemical replacement during contact with the nano-droplets, adsorb on the surface of the nano-droplets, and stabilize the nano-dispersion system.

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

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

[0040] Step 2: ultrasonically treat the liquid metal gallium-indium alloy and then take it out and ultrasonically mix it with the gold nanoparticles prepared by the glutathione reduction method obtained in step 1 at a molar ratio of 1:2 to 2:1 to obtain the gallium-indium alloy nanodispersion system. The liquid metal ultrasonic time is not less than 30 minutes, and the ultrasonic power is 1000 to 2000W; the mixed ultrasonic is first ultrasonicated in a non-contact cell disruptor for not less than 30 minutes, and the ultrasonic power is 1000 to 2000W, and then ultrasonicated in a contact cell disruptor for not less than 10 minutes, and the ultrasonic power is 100 to 200W.

[0041] The present application provides an application of the above-mentioned gallium-indium alloy nanodispersion system in treating cancer and activating immune anti-tumor response, wherein the cancer includes breast cancer. Under a radio frequency field, radio frequency dynamics cause the nanodroplets of liquid metal gallium-indium alloy after ultrasound to produce reactive oxygen, which stimulates the temperature of gold nanoparticles to rise, triggers the decomposition of endogenous hydrogen peroxide in the tumor microenvironment to produce oxygen, leads to temperature rise and accumulation of reactive oxygen at the tumor site, causes immunogenic cell death, and radio frequency modulation causes the gallium-indium alloy nanodispersion system to induce systemic anti-tumor immune response, inhibits the volume growth of breast cancer, and inhibits 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 of the present application is simple, such as Figure 1 As shown, the liquid metal gallium indium alloy is first ultrasonicated, and then a portion of the liquid metal gallium indium alloy nanodroplets after ultrasonication are taken out according to a certain ratio and put into a gold nanoparticle solution for mixed ultrasonication, so that the gold nanoparticles modify the nanodroplets, and the obtained nanoparticle size is about 100-500 nanometers and a stable nanodispersion system.

[0043] The stable nano-dispersion system provided by the present application contains gold nanoparticles and nano-droplets with radio frequency responsive behavior, which can be used for cancer treatment. The present application uses gold nanoparticles prepared by glutathione reduction method as an emulsifier to improve the stability of nano-droplets. Among them, the nano-droplets have the ability to produce reactive oxygen in the radio frequency field; the gold nanoparticles have catalase-like activity and the ability to generate heat in the radio frequency field. The nano-dispersion system can improve the efficiency of ROS production, expand the application scenarios of radio frequency electromagnetic waves, and realize multifunctional synergistic tumor treatment. Finally, it was proved in animal experiments that the synergistic effect of the two can cause cell immunogenic death, induce systemic anti-tumor immune response, trigger anti-tumor immune response, and improve anti-tumor efficacy.

[0044] In the present application, nanodroplets are selected as the main body of nanoparticles, gold nanoparticles prepared by glutathione reduction method are selected as emulsifiers, and the two are blended and ultrasonicated to prepare nanoparticles with a particle size of about 100-500 nanometers and a stable structure. Compared with nanodroplets obtained by ultrasonicating liquid metal alone, the nanoparticles significantly improve the stability of nanodroplets. At the same time, due to the radio frequency responsive behavior of gold nanoparticles, better anti-tumor effects are shown in breast cancer mouse models.

[0045] The nano-dispersion system provided in the present application can rapidly increase the temperature of the tumor site and generate reactive oxygen species by irradiating the tumor site with radio frequency, while causing immunogenic cell death of cancer cells, thereby achieving combined treatment of radio frequency thermal therapy, radio frequency dynamic therapy and immunity, and enhancing the anti-tumor efficacy.

[0046] Example 1

[0047] (1) Chloroauric acid and reduced glutathione were reacted at a molar ratio of 1:1.5 at 25° C. for 15 min, then heated to 70° C. for 24 h, and ultrafiltered and washed to obtain gold nanoparticles prepared by the glutathione reduction method.

[0048] (2) taking an appropriate amount of LM and placing it in a non-contact cell disruptor to obtain a mother solution of liquid metal gallium indium alloy nanodroplets by ultrasonication, wherein the ultrasonication time is 30 minutes and the ultrasonic power is 1440 W;

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

[0050] Example 2

[0051] (1) Chloroauric acid and reduced glutathione were reacted at a molar ratio of 1:1.5 at 25° C. for 15 min, then heated to 70° C. for 24 h, and ultrafiltered and washed to obtain gold nanoparticles prepared by the glutathione reduction method.

[0052] (2) taking an appropriate amount of LM and placing it in a non-contact cell disruptor to obtain a mother solution of liquid metal gallium indium alloy nanodroplets by ultrasonication, wherein the ultrasonication time is 30 minutes and the ultrasonic power is 1440 W;

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

[0054] Example 3

[0055] (1) Chloroauric acid and reduced glutathione were reacted at a molar ratio of 1:1.5 at 25° C. for 15 min, then heated to 70° C. for 24 h, and ultrafiltered and washed to obtain gold nanoparticles prepared by the glutathione reduction method.

[0056] The structure was observed using a transmission electron microscope. Figure 2 The results show that the average particle size is about 1.5nm and the inter-crystalline spacing is 0.29nm. The X-ray photoelectron spectroscopy technique is used to detect the valence state of the gold element. The results are as follows Figure 3As shown, its bivalent (0-valent and 1-valent) results were detected, indicating the successful synthesis of GNC.

[0057] (2) taking an appropriate amount of LM and placing it in a non-contact cell disruptor to obtain a mother solution of liquid metal gallium indium alloy nanodroplets by ultrasonication, wherein the ultrasonication time is 30 minutes and the ultrasonic power is 1440 W;

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

[0059] Example 4

[0060] (1) Chloroauric acid and reduced glutathione were reacted at a molar ratio of 1:1.5 at 25° C. for 15 min, then heated to 70° C. for 24 h, and ultrafiltered and washed to obtain gold nanoparticles prepared by the glutathione reduction method.

[0061] (2) taking an appropriate amount of LM and placing it in a non-contact cell disruptor to obtain a mother solution of liquid metal gallium indium alloy nanodroplets by ultrasonication, wherein the ultrasonication time is 30 minutes and the ultrasonic power is 1440 W;

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

[0063] Example 5

[0064] (1) Chloroauric acid and reduced glutathione were reacted at a molar ratio of 1:1.5 at 25° C. for 15 min, then heated to 70° C. for 24 h, and ultrafiltered and washed to obtain gold nanoparticles prepared by the glutathione reduction method.

[0065] (2) taking an appropriate amount of LM and placing it in a non-contact cell disruptor to obtain a mother solution of liquid metal gallium indium alloy nanodroplets by ultrasonication, wherein the ultrasonication time is 30 minutes and the ultrasonic power is 1440 W;

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

[0067] The average particle size of LM@GNC obtained in Examples 1-5 was measured by a dynamic light scattering particle size analyzer. Figure 4 As shown in Figure 1, the hydrated particle size of nanoparticles in several ratios is between 100-500 nanometers. Considering the stability, heat generation and active oxygen performance of the nanoparticles, the ratio is preferably 1:1. Figure 5 As shown, it shows its core-shell structure.

[0068] 1. UV-Vis absorption and fluorescence spectra of gold nanoparticles GNC, LM nanodroplets and LM@GNC obtained in Example 3

[0069] The gold nanoparticles GNC, LM nanodroplets and LM@GNC obtained in Example 3 were taken, and ultrapure aqueous solution was used as a reference. The absorption spectra of the three samples were measured by UV-visible spectrophotometer, with a scanning wavelength range of 200-850nm and a scanning step of 1nm. The excitation and emission spectra of the three samples were measured by time-resolved fluorescence spectrometer. The results are as follows: Figure 6 shown.

[0070] The results show that LM has no Stokes shift property, GNC has a large Stokes shift, and LM@GNC also has a large Stokes shift, indicating that GNC is successfully attached to the LM surface.

[0071] 2. In vitro temperature rise of different samples at different concentrations

[0072] The gold nanoparticles GNC, LM nanodroplets and LM@GNC obtained in Example 3 were prepared, wherein the Au molar concentration was 1 mmol L -1 LM molar concentration is 1 mmol L -1. In addition, PBS was used as the control group. Take 1.6 mL of the above solution in a liquid phase injection bottle, irradiate it with a radio frequency instrument at 100 W, use a thermal imager to detect the temperature change of the liquid in the liquid phase injection bottle, record the temperature every 30 seconds, and record the temperature change continuously for 300 seconds. In addition, take 1.6 mL of the LM@GNC solution of Example 3 and irradiate it with a radio frequency instrument at 50, 75, 100, 150, and 200 W, respectively, use a thermal imager to detect the temperature change of the liquid in the liquid phase injection bottle, record the temperature every 30 seconds, and record the temperature change continuously for 300 seconds. Finally, the LM@GNC solution was subjected to three heating-cooling cycles, and the temperature change of the liquid in the liquid phase injection bottle was detected with a thermal imager, and the temperature was recorded every 30 seconds. The results are as follows. Figure 7 Content a, content b, content c, content d are shown.

[0073] The results show that LM has no RF heating effect, but GNC and LM@GNC have RF heating effect. The concentration of Au in the solution and the power of the RF field have a significant impact on the RF thermal effect of GNC and LM@GNC. In addition, LM@GNC has good thermal stability, and the highest temperature is basically the same in three thermal cycles.

[0074] 3. Catalase activity of different samples

[0075] The gold nanoparticles GNC, LM nanodroplets and LM@GNC obtained in Example 3 were detected using an oxygen-specific probe ([Ru(dpp)3]Cl2). The weaker the fluorescence intensity of [Ru(dpp)3]Cl2, the more oxygen there is in the solution. The results are shown in Figure 2. Figure 8 As shown in the figure, the fluorescence intensity of the LM@GNC and GNC groups continued to decrease within 10 minutes, but 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] 4. RF dynamic behavior of LM@GNC

[0077] The generation of total ROS was detected using a DCFH-DA probe. 400 μL of LM@GNC (the molar ratio of LM to GNC was 1 / 1) from Example 3 was mixed with 20 μL of DCFH-DA (20 mM), treated for 1, 2, 3, 4, and 5 min under a radio frequency field (100 W), then centrifuged (12000 rpm, 5 min), and the supernatant was detected using a Fluro Max+ spectrometer. In order to detect the effect of radio frequency field power on the generation of reactive oxygen species, the LM@GNC from Example 3 was mixed with 20 μL of DCFH-DA (20 mM), treated for 5 min in different radio frequency fields (50 W, 75 W, 100 W, 150 W, 200 W), then centrifuged (12000 rpm, 5 min), and finally detected in a Fluro Max+ spectrometer. In order to detect the types of ROS produced under the RF field, the hydroxyl radical specific probe (HPF), superoxide anion specific probe (BES-SO), and singlet oxygen specific probe (DPBF) were used to detect the change trends of the three different reactive oxygen species over time in LM@GNC under the same RF field (100W). Fig. 9 Content a, content b, content c.

[0078] The results show that with the increase of RF time and the enhancement of RF field power, LM@GNC produces more reactive oxygen in the RF field, which mainly includes hydroxyl radicals, superoxide anions, and singlet oxygen.

[0079] 5. Using confocal fluorescence microscopy to study the ROS content in in vitro cells after different treatments

[0080] The generation of intracellular ROS was measured under CLSM using a 2',7'-dichlorofluorescein diacetate (DCFH-DA) probe. The gold nanoparticles GNC, LM nanodroplets and LM@GNC obtained in Example 3 were taken, and 20,000 4T1 cells were inoculated in a 6-well plate. After 12 hours, the cells were co-cultured with different treatment conditions (G1: PBS, G2: LM, G3: LM+RF, G4: GNC, G5: GNC+RF, G6: LM@GNC, G7: LM@GNC+RF). After continuing to culture for 4 hours, the above substances were digested and removed by trypsin. Then follow the instructions for the reactive oxygen species test kit. Finally, the fluorescence was detected under CLSM. The results are shown in the figure. Fig.10 Shown

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

[0082] VI. Study of in vitro cell immunogenic death after different treatments using an ELISA

[0083] Take the gold nanoparticles GNC, LM nanodroplets and LM@GNC obtained in Example 3, and inoculate 50,000 4T1 cells in a 24-well plate. After 12 hours, the cells were co-cultured with different treatment conditions (G1: PBS, G2: LM, G3: LM+RF, G4: GNC, G5: GNC+RF, G6: LM@GNC, G7: LM@GNC+RF). After the above culture was continued for 4 hours, the extracellular HMGB-1 and ATP content were determined using the cell culture medium supernatant according to the operating procedures of the HMGB-1 enzyme-linked immunosorbent assay kit and the enhanced chemiluminescence ATP assay kit. The results are shown in Fig.11 Content a, content b as shown.

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

[0085] VII. Establishment of 4T1 subcutaneous bilateral tumor model in BALB / C mice: 1 million 4T1 cells were inoculated subcutaneously in the right hind limb of the mouse, and 330,000 4T1 cells were inoculated under the left hind limb of the mouse. When the average tumor volume of the right hind limb increased to 100 cubic millimeters, the gold nanoparticles GNC, LM nanodroplets and LM@GNC obtained in Example 3 were taken, and the mice were randomly divided into 7 groups: G1 (PBS), G2 (LM), G3 (LM+RF), G4 (GNC), G5 (GNC+RF), G6 (LM@GNC), G7 (LM@GNC+RF). After the material was injected into the right tumor of the mouse, the mouse was treated under a radio frequency field of 300W for 10 minutes. The volume of the left and right tumors was recorded every two days, and the calculation formula is as follows: Volume = length × width 2 / 2. The result is Fig.12 Content b and content c. On the 14th day after treatment, the mice were euthanized and the left and right tumors were removed. Then, the tumors were fixed with 4% paraformaldehyde, embedded in paraffin, and then sectioned and stained. The staining indicators included: H&E, Ki67, TUNEL, HIF-1α, Hypoxia, and the results were shown in Fig.12 d. At the same time, three mice in each group were taken to collect whole blood and serum for determination of blood routine and blood biochemical indicators. The results are shown in Fig.13 and 14 As shown. For the determination of intratumoral ROS content, the mouse tumor was pretreated with LM@GNC, and then DCFH-DA was injected into the mouse tumor. After 0.5h, the mice were treated with radiofrequency field. Subsequently, the mice were euthanized, the tumor was removed and sliced, and observed with CLSM. The results are shown in Fig.12d. According to the immunofluorescence sections of TUNEL and Ki67 and the H&E results, mice treated with LM, GNC and LM@GNC all showed different degrees of tumor cell apoptosis and inhibition of cell proliferation. Among them, LM@GNC+RF showed the strongest effect of killing tumor cells. According to the results of DCF fluorescence sections, mice treated with LM and LM@GNC produced ROS under radio frequency. According to the results of HIF-1α and Hypoxia fluorescence sections, the fluorescence in the corresponding fluorescence sections of mice treated with materials containing GNC was weakened, indicating its good effect in alleviating tumor hypoxia.

[0086] The blood routine index of the sample is measured by a blood cell analyzer. The whole blood is collected by centrifugation and then tested. The results are as follows: Fig.13 and 14 shown. Fig.13 Content a is the alanine aminotransferase content; Fig.13 Content b is the aspartate aminotransferase content; Fig.13 Content c is the alkaline phosphatase content; Fig.13 Content d is the urea content; Fig.13 Content e is the creatinine content; Fig.13 Content f is the lactate dehydrogenase content. Fig.14 Content a is the number of white blood cells; Fig.14 Content b is the number of red blood cells; Fig.14 Content c is the hemoglobin content; Fig.14 Content d is the hematocrit; Fig.14 Content e is the mean corpuscular volume; Fig.14 Content f is the mean corpuscular hemoglobin content; Fig.14 Content g is the mean corpuscular hemoglobin concentration; Fig.14 Content h is the number of platelets. The results showed that since the mice in the PBS group did not get a good tumor treatment effect, their hematological examination showed more white blood cells, fewer platelets, and impaired liver, kidney and heart functions. However, the mice in the LM@GNC+RF group did not have major hematological and liver, kidney and heart function abnormalities, showing good biocompatibility.

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

[0088] According to the BALB / C mouse 4T1 subcutaneous bilateral tumor model, drug administration and radiofrequency treatment were performed. On the 4th day after treatment, the tumor, lymph nodes and spleen of the mice were taken to analyze the DC cell maturation and enhance the CD8 + Detection of T cell function and improvement of immunosuppressive microenvironment. Fig.15As shown in Figure 2, the mature DC cells in the tumor, lymph nodes, and spleen of the LM@GNC+RF group were significantly higher than those in the other groups, and the CD8 + 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 the level of T cells) and improve the immunosuppressive microenvironment (weakened PMN-MDSCs cell levels). In addition, the LM@GNC+RF group also showed the strongest level of central memory T cells. Therefore, the above results indicate that the hyperthermia and dynamic therapy induced by LM@GNC+RF can significantly enhance tumor-specific immune responses.

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

[0090] Fluorescent 4T1 cells were injected into the tail vein of mice with bilateral 4T1 subcutaneous tumors, and drug administration and radiofrequency treatment were performed 24 hours later. Lung tissues were obtained from mice on the 30th day after administration, and the number of nodules in the lung tissues was counted and H&E staining was performed. Fig.16 As shown, the results showed that mice treated with LM@GNC+RF had the lowest number of lung nodules and displayed the longest survival period, indicating a good anti-tumor metastasis effect.

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

[0092] In the description of this specification, the description of reference terms such as "an experimental example", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the experimental example or example are included in at least one experimental example or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same experimental example or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more experimental examples or examples in a suitable manner.

[0093] Finally, it should be noted that the above experimental examples are only used to illustrate the technical solution of the present application rather than to limit it. Although the present application has been described in detail with reference to the preferred experimental examples, ordinary technicians in this field should understand that they can still modify or replace the technical solution of the present application with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present application.

Claims

1. A gallium-indium alloy nanodispersion system, characterized in that: The raw materials of the gallium-indium alloy nano-dispersion system include gold nanoparticles prepared by glutathione reduction method and nano-droplets of liquid metal gallium-indium alloy after ultrasound. The gold nanoparticles prepared by glutathione reduction method undergo electrochemical replacement during contact with the nano-droplets of liquid metal gallium-indium alloy after ultrasound, and are adsorbed on the surface of the nano-droplets of liquid metal gallium-indium alloy after ultrasound.

2. A method for preparing a gallium-indium alloy nano-dispersion system, characterized in that: The gallium-indium alloy nano-dispersion system comprises the following preparation steps: Step 1, mixing chloroauric acid and reduced glutathione, heating the mixture for reaction, and ultrafiltration and washing to obtain gold nanoparticles prepared by glutathione reduction method; Step 2: Take out the nano droplets of the liquid metal gallium-indium alloy after ultrasonic treatment and mix them with the gold nanoparticles prepared by glutathione reduction method obtained in step 1 and ultrasonically obtain the gallium-indium alloy nano-dispersion system.

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

4. The method for preparing a gallium-indium alloy nano-dispersion system according to claim 2, characterized in that: In step 2, the ultrasonic time of the liquid metal gallium indium alloy is not less than 30 minutes, and the ultrasonic power is 1000-2000W; the mixed ultrasound is first ultrasonicated in a non-contact cell disruptor for not less than 30 minutes, and the ultrasonic power is 1000-2000W, and then ultrasonicated in a contact cell disruptor for not 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 metal gallium indium alloy after ultrasound is 1:2-2:

1.

5. Use of a gallium-indium alloy nanodispersion system according to claim 1 in radio frequency sensitization.

6. The use of a gallium-indium alloy nano-dispersion system in radio frequency sensitization according to claim 5, characterized in that: The gallium-indium alloy nano-dispersion system is used as a sensitizer in a radio frequency field, the frequency of the radio frequency electromagnetic wave is 13.56 MHz, the power is 50 W to 300 W, and the action time is 0.5 min to 10 min.

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