CN115612902A - Magnesium-based alloy microparticles with synergistic TACE anti-liver cancer effect and preparation method - Google Patents

Abstract

The invention relates to magnesium-based alloy micro particles with a synergistic TACE anti-liver cancer effect and a preparation and application method thereof. The tumor tissue is locally implanted before TACE treatment, the negative effects of TACE are reversed in multiple modes such as alkalization of microenvironment by degradation products and antioxidation in the initial stage, and the negative effects are directly exerted in cells after the degradation products are degraded to nanoscale particles in the final stage and can be endocytosed. The invention provides a novel anti-tumor biodegradable material which can reverse the negative effect of TACE and enhance the anti-liver cancer curative effect of TACE for clinic, so that clinical malignant tumor patients can benefit from survival.

Description

Magnesium-based alloy microparticles with synergistic TACE anti-liver cancer effect and preparation method

technical field

The invention belongs to a medical functional alloy material and its preparation, and relates to a magnesium-based alloy micron particle synergistic with TACE's anti-liver cancer effect, and a preparation and application method. Use magnesium as a carrier to smelt other beneficial metal particles such as zinc, selenium and molybdenum that have anti-cancer effects, and optimize the smelting ratio of magnesium to other metals and the particle size of magnesium-based alloys to finally achieve the degradation of magnesium-based alloys in tumor tissues The rate is controllable. Utilize the chemical properties of the magnesium degradation process and the degradation products to exert direct/indirect anti-cancer effects such as alkalization of the tumor microenvironment and anti-oxidation to inhibit the proliferation, invasion and metastasis of liver cancer, reduce the "stemness" of liver cancer, and then play a synergistic TACE to fight and reverse liver cancer The role of TACE in the treatment of negative effects has strong pertinence in the treatment of liver cancer.

Background technique

According to the Barcelona Clinic Liver Cancer (BCLC) staging system, TACE is the treatment of choice for intermediate-stage HCC, including unresectable multinodular HCC without extrahepatic spread. The BCLC System also recommends that TACE should be used when other recommended treatments are not feasible or successful in the early stages of HCC. In Asian countries, TACE tends to be more widely recommended for various clinical situations. According to different staging systems, the clinical situation displayed by TACE is slightly different, but TACE is still a mature method for the treatment of advanced HCC, see literature: Young Chang, Soung Won Jeong, Jae Young Jang, et al.Recent Updates of Transarterial Chemoembolilzation in Hepatocellular Carcinoma. Int. J. Mol. Sci. 2020, 21, 8165. TACE plays an anti-liver cancer role mainly by blocking the blood supply of liver cancer, and at the same time combined with local injection of chemotherapy drugs (such as oxaliplatin). Although the imaging equipment and super-selection technology related to TACE treatment have been continuously improved in the past 10 years, the survival rate of patients with liver cancer The benefits are still very limited. Many studies have shown that TACE can accelerate the invasion and metastasis of liver cancer while resisting liver cancer, and eventually lead to treatment failure. See literature: Raoul JL, Gilabert M, Adhoute X: ToTACE or not to TACE? Lessons from a negative trial. Lancet Gastroenterol Hepatol 2017,2(8):541-543. The main reasons for the failure of TACE are: ①The hypoxic environment caused by the embolism of the tumor supplying artery will intensify the anaerobic glycolysis and acidification of the microenvironment of cancer cells, thereby promoting the invasion and metastasis of cancer cells; ②Repeated chemotherapy will aggravate the drug resistance of liver cancer cells; ③TACE The necrosis of carcinogenic tissue will also cause inflammatory stromal cells to infiltrate into cancer tissue, and this process also plays an important role in the metastasis, recurrence and drug resistance of liver cancer.

In order to enhance the clinical curative effect, people put forward the TACE combined treatment plan, and verified its curative effect and safety through a large number of clinical studies. However, studies have shown that TACE combined with radiofrequency ablation, radiation therapy, and systemic therapy did not bring significant benefits to patients. See literature: Strobel O, Buchler MW: Treatment effect of liver resection vs. RFA or TACE in hepatocellular carcinoma. Chirurg 2019. The proliferation, recurrence and metastasis of HCC are still clinically difficult problems. At present, there is no biodegradable material that can reverse the negative effects of TACE and enhance the efficacy of TACE against liver cancer. After searching domestic and foreign journals, literature, and patents, no relevant reports and related patent authorizations were found. Therefore, finding a new strategy that can effectively improve the acidification of the microenvironment of liver cancer and reverse chemotherapy resistance, and use it in the anti-liver cancer treatment with TACE, will be the key to improving the survival benefits of liver cancer patients.

Contents of the invention

technical problem to be solved

In order to improve the deficiencies of the prior art, the present invention proposes a magnesium-based alloy micron particle that cooperates with TACE in anti-liver cancer and its preparation and application method, using metal magnesium as a carrier to smelt other zinc, selenium and molybdenum that have anti-cancer effects Metal particles, by smelting a certain proportion of other metals with anti-cancer effects, improve the shortcoming of the rapid degradation of micron magnesium metal particles. ", reversing the negative effects of TACE treatment, and finally synergizing with TACE to play an anti-liver cancer effect. This will provide a new type of anti-liver cancer biodegradable material that can reverse the negative effects of TACE and enhance the efficacy of TACE against liver cancer, so as to benefit the survival of clinical liver cancer patients.

Technical solutions

A magnesium-based alloy micron particle that cooperates with TACE to resist liver cancer, is characterized in that metal magnesium is used as a carrier, and is smelted with other metal particles having an anticancer effect to prepare a new type of magnesium-based alloy micron particle; the metal magnesium is combined with other anticancer The mass ratio of metal particles with cancer effect is 85-99:1-15.

The metal particles with anticancer effect include but not limited to zinc, selenium or molybdenum metal particles.

A method for preparing the magnesium-based alloy microparticles synergistic with TACE for anti-liver cancer, characterized in that the steps are as follows:

Step 1: Put magnesium ingots and other metal ingots with anti-cancer effects into two pre-melting furnaces to vacuumize, set the temperature at 300°C, and use high-temperature inert gas argon to purge to remove the oxidative atmosphere gas adsorbed on the surface ;

Step 2: Heating the furnace body, the furnace body containing magnesium ingots is heated to 680°C, and the furnace body of other metal ingots with anti-cancer effects is heated to the melting temperature of the metal, and protected by argon to completely melt the metal Get liquid magnesium and other liquid metals that have cancer-fighting properties;

Step 3: Put liquid magnesium and other liquid metals with anti-cancer effects on the disc atomizer in the atomization furnace, mix the two liquid metals evenly on the disc atomizer, and spray them into Magnesium-based alloy droplets;

Step 4: rapidly condensing the magnesium-based alloy droplets to form a low-oxidation solid spherical magnesium-based alloy powder;

Step 5: Sieve the magnesium-based alloy powder for particle size classification, and store the spherical magnesium-based alloy micron particles with a size of 10-200 μm in distilled water/or absolute alcohol for long-term storage.

Vibrating sieves are used to carry out particle size classification of alloy powders, wherein, after sieving, they pass through 600-800 mesh sieves.

The step 2 uses the intermediate frequency heating ring to heat the furnace body.

In step 3, the mass ratio of the magnesium-based alloy micron particles is realized by controlling the respective flow rates of the two liquid metals on the disc atomizer passing into the atomization furnace.

In the step 3, the frequency of the atomizer and the rotational speed of the centrifuge are controlled to control the size of the metal droplets.

The nebulizer frequency is 55 Hz.

A method for using the magnesium-based alloy microparticles synergistically with TACE in anti-liver cancer, characterized in that: injecting into the hepatic artery through the femoral artery, along with the guide wire catheter, or superselecting into the blood supply artery of the liver segment, or the blood supply of the liver cancer focus The artery is implanted into the local tumor tissue, and according to the particle size of the magnesium-based alloy, stays in the capillaries of the cancer tissue, and cooperates with TACE treatment to exert an anti-hepatic effect.

When in use, magnesium-based alloy micro-particles are dissolved in normal saline, and 10 minutes before TACE treatment, the magnesium-based alloy micro-particles are locally implanted at the tumor site with the help of Taoism during TACE treatment.

Beneficial effect

The present invention proposes a magnesium-based alloy micron particle that cooperates with TACE in anti-liver cancer and its preparation and application method. The medical functional alloy material takes advantage of the natural corrosion degradation characteristics of magnesium metal and the anti-cancer potential of its degradation products, combined with zinc, The anti-cancer characteristics of molybdenum and selenium plasma optimize the best smelting ratio of metal elements, and use the high-temperature spray method to prepare micron-sized alloy particles with a degradation rate that meets the TACE treatment cycle, a controllable degradation rate, and mild action, which can be stored for a long time. Distilled water or absolute alcohol for use. During TACE treatment, magnesium-based alloy micro-particles are implanted into tumor tissue, and through various methods such as alkalinizing the microenvironment and anti-oxidation of its degradation products, they can exert synergistic anti-cancer effects and reverse the negative effects of TACE. It can be degraded into nano-sized particles in cancer tissue, and can further play a direct anti-hepatocarcinoma effect in cells after endocytosis.

Compared with the prior art, the present invention has the following effects:

1. Local implantation of metal magnesium to achieve slow release and controllable degradation in local liver cancer and synergistic anti-hepatic cancer

As a medical implantable alkaline metal, magnesium is not only rich in resources, low in price, but also has good biocompatibility and degradability. It is a new type of medical functional material with great clinical application prospects. Magnesium metal degradation products magnesium hydroxide, hydrogen and magnesium ions can directly or indirectly inhibit the malignant progression of liver cancer by alkalinizing the microenvironment and anti-oxidation, etc., but the degradation rate is fast and uneven, especially when prepared into micron particles The shortcoming of extremely fast post-degradation limits its clinical application. The present invention prepares micron-sized magnesium-based alloy particles that can be locally implanted in liver cancer tissue, degraded mildly and controllable, and the synergistic anti-liver cancer effect of local implantation is difficult to inhale, orally or intravenously inject metal magnesium particles and their degradation products. to achieve. The anti-tumor effect of metal magnesium in the present invention includes: first, implanting magnesium metal into the liver cancer tissue to degrade and release the alkaline substance magnesium hydroxide can precisely increase the local pH value of the cancer tissue and reverse the tumor cell damage caused by the acidic microenvironment. Dedifferentiation, enhancement of "stemness", and induction of epithelial-mesenchymal transition (EMT) and other pro-cancer effects play an effective anti-cancer role. Second, when the oxidation reaction is enhanced and/or the antioxidant capacity is impaired, the redox reaction is destroyed to generate oxidative stress, resulting in oxidative damage to lipids, proteins, and DNA, thereby causing/promoting the occurrence and development of tumors . Magnesium metal directly implanted into liver cancer tissue degrades and slowly releases hydrogen, which can precisely increase the local hydrogen concentration of liver cancer, and inhibit tumor progression through anti-oxidation. The traditional hydrogen therapy strategy reaches very little hydrogen content in the tumor tissue, and it is difficult to exert an effective anti-tumor effect. Third, the local release of magnesium ions can precisely increase the concentration of magnesium ions in the local cancer tissue and inhibit the metastasis and invasion of tumor cells. Patients with radiotherapy, chemotherapy, or molecular targeted therapy are often accompanied by a decrease in serum magnesium. Appropriate supplementation of magnesium ions can enhance the anti-cancer effect of DNA-damaging therapy and act as a sensitizer for anti-cancer therapy.

2. Accurately increase the safe concentration of zinc ions in the local liver cancer tissue, and play an anti-liver cancer effect by directly acting on the liver cancer itself and/or improving the microenvironment of the liver cancer

Zinc participates in the composition of various proteins, and can act as a signal molecule in the cell in the form of free ions to participate in the regulation of cell metabolism, activation of protein kinases and phosphatases, etc., and play an important role in tumor cell proliferation, apoptosis, differentiation and immune regulation . Zinc ions play a double-edged sword role in cancer patients. Zinc ion overload may lead to dyscrasia and even accelerate the death of patients, while insufficient concentration will not be able to exert effective anti-tumor effects. Therefore, increasing the concentration of zinc ions with systemic medication is safe for tumor patients. The windows are small. Therefore, only by accurately increasing the local zinc ion concentration in liver cancer tissue can it effectively exert its anti-tumor effect. The local implantation of the present invention can maximize the killing effect of metal zinc on liver cancer tissue, and enhance the utilization rate of zinc ions in the body. The anti-hepatic cancer effect of metal zinc in the present invention includes, first, inhibiting the release of a large number of tumor-related inflammatory factors, increasing the expression of two zinc finger proteins with anti-inflammatory effects, A20 and PPAR-a; second, inhibiting the intracellular Ras- The MAPK signaling pathway directly or synergistically inhibits the malignant progression of tumors, including inhibiting proliferation, inducing apoptosis, or enhancing the anticancer efficacy of radiotherapy and chemotherapy; thirdly, enhancing the activity of antioxidant proteins and enzymes, such as glutathione and peroxidase Catalase.

3. Selenium and molybdenum metal particles integrate diagnosis and treatment, combining physical therapy and chemical treatment

In this study, selenium, molybdenum particles and magnesium carriers were combined to integrate diagnosis and treatment. On the one hand, molybdenum-containing compounds can be used for tumor-targeted fluorescence imaging and photothermal therapy. Through the high permeability and retention effect, it can accumulate at the tumor site and reach the maximum amount, reducing the damage to the surrounding tissue. At the same time, its photothermal killing effect can damage the vascular endothelial cells of the tumor tissue. On the other hand, it can be used not only as a drug carrier, but also as a chemotherapeutic drug, a radiotherapy sensitizer, and can be designed to enhance drug efficacy. The present invention improves the toxicity and stability of the component metals while exerting the anti-tumor effects of selenium and molybdenum metals respectively, improves their targeting, biocompatibility and photothermal killing effect, and reduces the immune rejection effect of the body on the particles .

Therefore, the present invention focuses on the research and development of biomedical advanced functional materials, and synthesizes magnesium-based alloy micro-particles for local implantation of liver tumor tissues, which solves the clinical application problems of relatively active magnesium metal and uncontrollable degradation, and maximizes the use of magnesium. and other metal degradation products anti-hepatocarcinogenesis. At the same time, the present invention will provide an advanced functional material capable of reversing the negative effects of TACE and enhancing the curative effect of TACE against liver cancer, so as to benefit the survival of patients with clinical liver cancer.

Description of drawings

Figure 1: Cytotoxic effects of M-6Z, M-6S, and M-6M gradient time and gradient concentration on liver cancer cell lines, hepatic stellate cell lines, and normal liver cell lines

A: M-6Z; B: M-6S; C: M-6M

Figure 2: Effects of magnesium-based alloy microparticles on the proliferation and invasion of liver cancer cell lines

A: M-6Z, M-6S and M-6M inhibit the malignant proliferation of liver cancer cell lines;

B: M-6Z inhibits the invasion ability of liver cancer cell lines

Figure 3: Tumor growth of liver cancer in mice

Figure 4: Tumor growth after liver cancer treatment in mice

detailed description

Now in conjunction with embodiment, accompanying drawing, the present invention will be further described:

Magnesium-based alloy micron particles and preparation examples:

Embodiment 1: Magnesium-zinc alloy micron particles (6% zinc), prepared by high-temperature spraying method

(1) Put the magnesium ingots and zinc ingots into two pre-melting furnaces to vacuumize respectively, set the temperature at 300°C, and use high-temperature inert gas (argon) to purge to remove the gas containing oxidative atmosphere adsorbed on the surface;

(2) Use the intermediate frequency heating ring to heat the furnace body. The furnace body containing magnesium ingots is heated to 680°C, and the furnace body containing zinc ingots is heated to 650°C, and protected by argon to completely melt the metal;

(3) Feed liquid magnesium and liquid zinc into the disc atomizer in the atomization furnace, and realize the mass ratio of magnesium-based alloy micron particles (magnesium:zinc=94:6) by controlling their respective flow rates. Mix magnesium liquid and zinc liquid evenly on a disc atomizer, and spray them into small droplets after mixing. Control the frequency of the atomizer to 55Hz, and control the size of the metal droplets by controlling the speed of the centrifuge and other parameters;

(4) Quickly condense small alloy droplets to form a low-oxidation solid spherical magnesium-based alloy powder;

(5) Vibrating sieves are used to classify the alloy powders, wherein, after sieving, pass through a 600-800 mesh sieve, and the spherical magnesium-zinc alloy micron particles of 20 μm under the sieve are stored in distilled water.

Embodiment 2: Magnesium-zinc alloy micron particles (3% zinc), prepared by high-temperature spraying method

(1) Put the magnesium ingots and zinc ingots into two pre-melting furnaces to vacuumize respectively, set the temperature at 300°C, and use high-temperature inert gas (argon) to purge to remove the gas containing oxidative atmosphere adsorbed on the surface;

(2) Use the intermediate frequency heating ring to heat the furnace body. The furnace body containing magnesium ingots is heated to 680°C, and the furnace body containing zinc ingots is heated to 650°C, and protected by argon to completely melt the metal;

(3) Feed liquid magnesium and liquid zinc into the disc atomizer in the atomization furnace, and realize the mass ratio of magnesium-based alloy micron particles (magnesium:zinc=97:3) by controlling their respective flow rates. Mix magnesium liquid and zinc liquid evenly on a disc atomizer, and spray them into small droplets after mixing. Control the frequency of the atomizer to 55Hz, and control the size of the metal droplets by controlling the speed of the centrifuge and other parameters;

(4) Quickly condense small alloy droplets to form a low-oxidation solid spherical magnesium-based alloy powder;

(5) Vibrating sieves are used to classify the alloy powders, wherein, after sieving, pass through a 600-800 mesh sieve, and the spherical magnesium-zinc alloy micron particles of 20 μm under the sieve are stored in distilled water.

Embodiment 3: Magnesium-zinc alloy micron particles (12% zinc), prepared by high-temperature spraying method

(1) Put the magnesium ingots and zinc ingots into two pre-melting furnaces to vacuumize respectively, set the temperature at 300°C, and use high-temperature inert gas (argon) to purge to remove the gas containing oxidative atmosphere adsorbed on the surface;

(2) Use the intermediate frequency heating ring to heat the furnace body. The furnace body containing magnesium ingots is heated to 680°C, and the furnace body containing zinc ingots is heated to 650°C, and protected by argon to completely melt the metal;

(3) Feed liquid magnesium and liquid zinc into the disc atomizer in the atomization furnace, and realize the mass ratio of magnesium-based alloy micron particles (magnesium:zinc=88:12) by controlling their respective flow rates. Mix magnesium liquid and zinc liquid evenly on a disc atomizer, and spray them into small droplets after mixing. Control the frequency of the atomizer to 55Hz, and control the size of the metal droplets by controlling the speed of the centrifuge and other parameters;

(4) Quickly condense small alloy droplets to form a low-oxidation solid spherical magnesium-based alloy powder;

(5) Vibrating sieves are used to classify the alloy powders, wherein, after sieving, pass through a 600-800 mesh sieve, and the spherical magnesium-zinc alloy micron particles of 20 μm under the sieve are stored in distilled water.

Embodiment 4: Magnesium-zinc alloy micron particles (15% zinc), prepared by high-temperature spraying method

(1) Put the magnesium ingots and zinc ingots into two pre-melting furnaces to vacuumize respectively, set the temperature at 300°C, and use high-temperature inert gas (argon) to purge to remove the gas containing oxidative atmosphere adsorbed on the surface;

(2) Use the intermediate frequency heating ring to heat the furnace body. The furnace body containing magnesium ingots is heated to 680°C, and the furnace body containing zinc ingots is heated to 650°C, and protected by argon to completely melt the metal;

(3) Feed liquid magnesium and liquid zinc into the disc atomizer in the atomization furnace, and realize the mass ratio of magnesium-based alloy micron particles (magnesium: zinc = 85: 15) by controlling their respective flow rates. Mix magnesium liquid and zinc liquid evenly on a disc atomizer, and spray them into small droplets after mixing. Control the frequency of the atomizer to 55Hz, and control the size of the metal droplets by controlling the speed of the centrifuge and other parameters;

(4) Quickly condense small alloy droplets to form a low-oxidation solid spherical magnesium-based alloy powder;

(5) Vibrating sieves are used to classify the alloy powders, wherein, after sieving, pass through a 600-800 mesh sieve, and the spherical magnesium-zinc alloy micron particles of 20 μm under the sieve are stored in distilled water.

Embodiment five: Magnesium-selenium alloy microparticles (3% selenium), prepared by high-temperature spraying method

(1) Put the magnesium ingot and the selenium ingot into two pre-melting furnaces to vacuumize respectively, set the temperature at 300°C, and use high-temperature inert gas (argon) to purge to remove the gas containing the oxidizing atmosphere adsorbed on the surface;

(2) Use the intermediate frequency heating ring to heat the furnace body, the furnace body containing magnesium ingots is heated to 680 ° C, the furnace body containing selenium ingots is heated to 650 ° C, and protected by argon to completely melt the metal;

(3) Feed liquid magnesium and liquid selenium into the disc atomizer in the atomization furnace, and realize the mass ratio of magnesium-based alloy micron particles (magnesium:selenium=97:3) by controlling their respective flow rates. Mix the magnesium liquid and the selenium liquid on the disc atomizer evenly, and spray them into small droplets after mixing, control the atomizer frequency to 55Hz, and control the size of the metal droplets by controlling the centrifuge speed and other parameters;

(4) Quickly condense small alloy droplets to form a low-oxidation solid spherical magnesium-based alloy powder;

(5) Vibrating sieves are used to classify the alloy powders, wherein, after sieving, pass through a 600-800 mesh sieve, and the 20 μm spherical magnesium-selenium alloy micron particles under the sieve are stored in distilled water.

Embodiment 6: Magnesium-Selenium Alloy Micron Particles (6% Selenium), Prepared by High Temperature Spraying Method

(1) Put the magnesium ingot and the selenium ingot into two pre-melting furnaces to vacuumize respectively, set the temperature at 300°C, and use high-temperature inert gas (argon) to purge to remove the gas containing the oxidizing atmosphere adsorbed on the surface;

(2) Use the intermediate frequency heating ring to heat the furnace body, the furnace body containing magnesium ingots is heated to 680 ° C, the furnace body containing selenium ingots is heated to 650 ° C, and protected by argon to completely melt the metal;

(3) Feed liquid magnesium and liquid selenium into the disc atomizer in the atomization furnace, and realize the mass ratio of magnesium-based alloy micro-particles (magnesium:selenium=94:6) by controlling their respective flow rates. Mix the magnesium liquid and the selenium liquid on the disc atomizer evenly, and spray them into small droplets after mixing, control the atomizer frequency to 55Hz, and control the size of the metal droplets by controlling the centrifuge speed and other parameters;

(4) Quickly condense small alloy droplets to form a low-oxidation solid spherical magnesium-based alloy powder;

(5) Vibrating sieves are used to classify the alloy powders, wherein, after sieving, pass through a 600-800 mesh sieve, and the 20 μm spherical magnesium-selenium alloy micron particles under the sieve are stored in distilled water.

Example 7: Magnesium-Selenium Alloy Micron Particles (12% Selenium), Prepared by High Temperature Spraying

(1) Put the magnesium ingot and the selenium ingot into two pre-melting furnaces to vacuumize respectively, set the temperature at 300°C, and use high-temperature inert gas (argon) to purge to remove the gas containing the oxidizing atmosphere adsorbed on the surface;

(2) Use the intermediate frequency heating ring to heat the furnace body, the furnace body containing magnesium ingots is heated to 680 ° C, the furnace body containing selenium ingots is heated to 650 ° C, and protected by argon to completely melt the metal;

(3) Feed liquid magnesium and liquid selenium into the disc atomizer in the atomization furnace, and realize the mass ratio of magnesium-based alloy micron particles (magnesium:selenium=88:12) by controlling their respective flow rates. Mix the magnesium liquid and the selenium liquid on the disc atomizer evenly, and spray them into small droplets after mixing, control the atomizer frequency to 55Hz, and control the size of the metal droplets by controlling the centrifuge speed and other parameters;

(4) Quickly condense small alloy droplets to form a low-oxidation solid spherical magnesium-based alloy powder;

(5) Vibrating sieves are used to classify the alloy powders, wherein, after sieving, pass through a 600-800 mesh sieve, and the 20 μm spherical magnesium-selenium alloy micron particles under the sieve are stored in distilled water.

Embodiment 8: Magnesium-Selenium Alloy Micron Particles (15% Selenium), Prepared by High Temperature Spraying

(1) Put the magnesium ingot and the selenium ingot into two pre-melting furnaces to vacuumize respectively, set the temperature at 300°C, and use high-temperature inert gas (argon) to purge to remove the gas containing the oxidizing atmosphere adsorbed on the surface;

(2) Use the intermediate frequency heating ring to heat the furnace body, the furnace body containing magnesium ingots is heated to 680 ° C, the furnace body containing selenium ingots is heated to 650 ° C, and protected by argon to completely melt the metal;

(3) Feed liquid magnesium and liquid selenium into the disc atomizer in the atomization furnace, and realize the mass ratio of magnesium-based alloy micron particles (magnesium: selenium = 85: 15) by controlling their respective flow rates. Mix the magnesium liquid and the selenium liquid on the disc atomizer evenly, and spray them into small droplets after mixing, control the atomizer frequency to 55Hz, and control the size of the metal droplets by controlling the centrifuge speed and other parameters;

(4) Quickly condense small alloy droplets to form a low-oxidation solid spherical magnesium-based alloy powder;

(5) Vibrating sieves are used to classify the alloy powders, wherein, after sieving, pass through a 600-800 mesh sieve, and the 20 μm spherical magnesium-selenium alloy micron particles under the sieve are stored in distilled water.

Embodiment 9: Magnesium-molybdenum alloy micron particles (3% molybdenum), prepared by high-temperature spraying method

(1) Put the magnesium ingot and the molybdenum ingot into two pre-melting furnaces to vacuumize, set the temperature to 300°C, and use high-temperature inert gas (argon) to purge to remove the gas containing oxidative atmosphere adsorbed on the surface;

(2) Use the intermediate frequency heating ring to heat the furnace body, the furnace body containing magnesium ingots is heated to 680 ° C, the furnace body containing molybdenum ingots is heated to 2700 ° C, and protected by argon to completely melt the metal;

(3) Feed liquid magnesium and liquid molybdenum into the disc atomizer in the atomization furnace, and realize the mass ratio of magnesium-based alloy micron particles (magnesium:molybdenum=97:3) by controlling their respective flow rates. Mix magnesium liquid and molybdenum liquid evenly on a disc atomizer, and spray them into small droplets after mixing. Control the atomizer frequency to 55Hz, and control the size of metal droplets by controlling the centrifuge speed and other parameters;

(4) Quickly condense small alloy droplets to form a low-oxidation solid spherical magnesium-based alloy powder;

(5) Vibrating sieves are used to carry out particle size classification of the alloy powder, wherein, after sieving, pass through a 600-800 mesh sieve, and the spherical magnesium-molybdenum alloy micron particles of 20 μm under the sieve are stored in distilled water.

Embodiment 10: Magnesium-molybdenum alloy micron particles (6% molybdenum), prepared by high-temperature spraying method

(1) Put the magnesium ingot and the molybdenum ingot into two pre-melting furnaces to vacuumize, set the temperature to 300°C, and use high-temperature inert gas (argon) to purge to remove the gas containing oxidative atmosphere adsorbed on the surface;

(2) Use the intermediate frequency heating ring to heat the furnace body, the furnace body containing magnesium ingots is heated to 680 ° C, the furnace body containing molybdenum ingots is heated to 2700 ° C, and protected by argon to completely melt the metal;

(3) Pass liquid magnesium and liquid molybdenum into the disc atomizer in the atomization furnace, and realize the mass ratio of magnesium-based alloy micron particles (magnesium:molybdenum=94:6) by controlling their respective flow rates. Mix magnesium liquid and molybdenum liquid evenly on a disc atomizer, and spray them into small droplets after mixing. Control the atomizer frequency to 55Hz, and control the size of metal droplets by controlling the centrifuge speed and other parameters;

(4) Quickly condense small alloy droplets to form a low-oxidation solid spherical magnesium-based alloy powder;

(5) Vibrating sieves are used to carry out particle size classification of the alloy powder, wherein, after sieving, pass through a 600-800 mesh sieve, and the spherical magnesium-molybdenum alloy micron particles of 20 μm under the sieve are stored in distilled water.

Embodiment 11: Magnesium-molybdenum alloy microparticles (12% molybdenum), prepared by high-temperature spraying method

(1) Put the magnesium ingot and the molybdenum ingot into two pre-melting furnaces to vacuumize, set the temperature to 300°C, and use high-temperature inert gas (argon) to purge to remove the gas containing oxidative atmosphere adsorbed on the surface;

(2) Use the intermediate frequency heating ring to heat the furnace body, the furnace body containing magnesium ingots is heated to 680 ° C, the furnace body containing molybdenum ingots is heated to 2700 ° C, and protected by argon to completely melt the metal;

(3) Feed liquid magnesium and liquid molybdenum into the disc atomizer in the atomization furnace, and realize the mass ratio of magnesium-based alloy micron particles (magnesium:molybdenum=88:12) by controlling their respective flow rates. Mix magnesium liquid and molybdenum liquid evenly on a disc atomizer, and spray them into small droplets after mixing. Control the atomizer frequency to 55Hz, and control the size of metal droplets by controlling the centrifuge speed and other parameters;

(4) Quickly condense small alloy droplets to form a low-oxidation solid spherical magnesium-based alloy powder;

(5) Vibrating sieves are used to carry out particle size classification of the alloy powder, wherein, after sieving, pass through a 600-800 mesh sieve, and the spherical magnesium-molybdenum alloy micron particles of 20 μm under the sieve are stored in distilled water.

Embodiment 12: Magnesium-molybdenum alloy microparticles (15% molybdenum), prepared by high-temperature spraying method

(1) Put the magnesium ingot and the molybdenum ingot into two pre-melting furnaces to vacuumize, set the temperature to 300°C, and use high-temperature inert gas (argon) to purge to remove the gas containing oxidative atmosphere adsorbed on the surface;

(2) Use the intermediate frequency heating ring to heat the furnace body, the furnace body containing magnesium ingots is heated to 680 ° C, the furnace body containing molybdenum ingots is heated to 2700 ° C, and protected by argon to completely melt the metal;

(3) Pass liquid magnesium and liquid molybdenum into the disc atomizer in the atomization furnace, and realize the mass ratio of magnesium-based alloy micron particles (magnesium: molybdenum = 85: 15) by controlling their respective flow rates. Mix magnesium liquid and molybdenum liquid evenly on a disc atomizer, and spray them into small droplets after mixing. Control the atomizer frequency to 55Hz, and control the size of metal droplets by controlling the centrifuge speed and other parameters;

(4) Quickly condense small alloy droplets to form a low-oxidation solid spherical magnesium-based alloy powder;

(5) Vibrating sieves are used to carry out particle size classification of the alloy powder, wherein, after sieving, pass through a 600-800 mesh sieve, and the spherical magnesium-molybdenum alloy micron particles of 20 μm under the sieve are stored in distilled water.

Comparative example 1: Magnesium-aluminum alloy micro-particles (6% aluminum), prepared by high-temperature spraying method

(1) Put the magnesium ingot and the aluminum ingot into two pre-melting furnaces to vacuumize, set the temperature to 300°C, and use high-temperature inert gas (argon) to purge to remove the gas containing oxidative atmosphere adsorbed on the surface;

(2) Use the intermediate frequency heating ring to heat the furnace body, the furnace body containing magnesium ingots is heated to 680°C, the furnace body containing aluminum ingots is heated to 680°C, and protected by argon to completely melt the metal;

(3) Feed liquid magnesium and liquid aluminum into the disc atomizer in the atomization furnace, and realize the mass ratio of magnesium-based alloy micron particles (magnesium:aluminum=94:6) by controlling their respective flow rates. Mix the magnesium liquid and the aluminum liquid on the disc atomizer evenly, and spray them into small droplets after mixing. The frequency of the atomizer is controlled at 55Hz, and the size of the metal droplets is controlled by controlling the parameters such as the speed of the centrifuge;

(4) Quickly condense small alloy droplets to form a low-oxidation solid spherical magnesium-based alloy powder;

(5) Vibrating sieves are used to classify the alloy powders, wherein, after sieving, pass through a 600-800 mesh sieve, and the 20 μm spherical magnesium-aluminum alloy micron particles under the sieve are stored in distilled water.

Comparative example two: Magnesium-manganese alloy micron particles (6% manganese), prepared by high-temperature spraying method

(1) Put the magnesium ingots and manganese ingots into two pre-melting furnaces to vacuumize respectively, set the temperature at 300°C, and use high-temperature inert gas (argon) to purge to remove the gas containing oxidative atmosphere adsorbed on the surface;

(2) Use the intermediate frequency heating ring to heat the furnace body, the furnace body containing magnesium ingots is heated to 680 ° C, the furnace body containing manganese ingots is heated to 1300 ° C, and protected by argon to completely melt the metal;

(3) Feed liquid magnesium and liquid manganese into the disc atomizer in the atomization furnace, and realize the mass ratio of magnesium-based alloy micron particles (magnesium:manganese=94:6) by controlling their respective flow rates. Mix the magnesium liquid and the manganese liquid on the disc atomizer evenly, and spray them into small droplets after mixing. The frequency of the atomizer is controlled at 55Hz, and the size of the metal droplets is controlled by controlling the centrifuge speed and other parameters;

(4) Quickly condense small alloy droplets to form a low-oxidation solid spherical magnesium-based alloy powder;

(5) Vibrating sieves are used to classify the alloy powders, wherein, after sieving, pass through a 600-800 mesh sieve, and the spherical magnesium-manganese alloy micron particles of 20 μm under the sieve are stored in distilled water.

Table 1 Nutritional therapy program of the treatment group

1.1 Magnesium-based alloy microparticles have no direct cytotoxic effect on liver cancer cells, hepatic stellate cells and normal liver cells

Hepatoma cell lines, hepatic stellate cell lines, and normal liver cell lines were treated with gradient time and concentration of M-6Z, M-6S, and M-6M, and the expression of LDH in the supernatant of the cells was detected, indicating that with the increase of time and concentration, The expression of LDH in the supernatant tended to increase, but the overall increase was not obvious. And M-6Z reached plateau at 24h and 4mg. (see picture 1)

The cytotoxic effect of M-6Z, M-6S, M-6M gradient time and gradient concentration on liver cancer cell lines, hepatic stellate cell lines and normal liver cell lines. 1-A M-6Z; 1-B M-6S; 1-C M-6M.

1.2 Using two human liver cancer cell lines MHCC97H and Hep3B, CCK8 detection showed that with the gradual increase of the concentration of M-6Z, M-6S and M-6M, the inhibitory effect was significantly enhanced (see Figure 2).

Matrigel was added to the Transwell chamber to construct the in vitro invasion chamber, and the M-6Z 4mg/ml system was constructed in the lower chamber. The liver cancer cell lines MHCC97H (58.33±7.51vs.24.31±6.87p=0.0044) and Hep3B (65.00±6.51 vs.40.17±8.71p =0.0095) was significantly inhibited (see Figure 2).

Experimental Design II (Animal Experiments)

1.1 Purpose and object of the experiment

To investigate the anticancer effect and safety of the combined treatment scheme of the present invention and TACE.

75 8-week-old C57BL/6 healthy mice

1.2 Method

The 75 mice were randomly divided into 15 groups, with 5 cases in each group, and 14 treatment groups and 1 control group were given different treatment plans (see Table 2).

Construction of mouse orthotopic liver cancer model

The H22 cell line was cultured in 1640 medium containing 10% fetal bovine serum, and the cells in exponential growth phase were collected. After the mice were anesthetized and fixed in the supine position, the H22 liver cancer cell suspension (5×105/25μL) was injected under the capsule of the left outer lobe of the liver. A vesicle appeared at the injection site, which proved that the injection was successful.

Injection of magnesium-based alloy microparticles in mice with carcinoma in situ

Mice were treated with TACE on day 30 after inoculation with H22 cells. Routine disinfection of the skin, local anesthesia with 5mL 2% lidocaine hydrochloride injection. The first angiography determined the location of the lesion and the direction of the puncture needle, and explored the lesion in detail; the second angiography guided the puncture and injection needle to enter through the guide device and the skin, and passed through the abdomen and intercostals. After the lesion was clearly displayed, magnesium-based alloy microparticles were injected ( Dissolve 1mg in 5ml normal saline and mix well, inject slowly), after the injection is completed, quickly pull out the needle, and pressurize the puncture point to avoid bleeding.

Tumor ischemic surgery in mice 24 hours after local injection

Grouping: treatment group, control group

Mice were fasted for 12 h before operation and had free access to water.

Mouse anesthesia: intraperitoneal injection of pentobarbital sodium (100ul/20g), supine position after anesthesia, fixed limbs with tape, electric razor to remove abdominal hair, 1% iodophor disinfection, spread sterile towels.

Take a midline longitudinal incision into the abdomen, cut the skin layer and muscle layer respectively, go up to the xiphoid process and down to the bladder, use Alice forceps to pull apart and fix the muscle layers on both sides to expose the liver, wet the cotton swab and separate the porta hepatis , exposing the Glisson system of the left lobe and middle lobe of the liver.

5) Under a stereomicroscope, the main blood supply vessels of the tumor were peeled off with micro tweezers, and the blood supply of the tumor was blocked with a non-invasive vascular clip.

6) When the tumor tissue turns from reddish brown to pale, it means that the blood supply is successfully blocked. After the blood vessel is successfully blocked, the incision is sutured layer by layer in order and the incision is disinfected, placed on an electric blanket or a constant temperature board to keep warm until awake, and placed in a mouse cage covered with fine shavings, and soft food is placed in the cage. Each mouse Raise them separately, and pay attention to observe the survival status of the mice.

1.1 Results

1.3.1 Growth of mice

During the experiment period, the mice in the treatment group and the control group had normal activities, lustrous coat color, normal eating and drinking water, no abnormal changes in urine and urine, and no one died. Tumor tissues could be detected in mice in each group of liver cancer orthotopic model, and the tumor formation rate reached 100%.

1.3.2 Effect of magnesium-based alloy microparticles on tumor growth in experimental mice

An orthotopic model of human liver cancer in nude mice was constructed. After 4 weeks of hepatic cell injection, magnesium-zinc alloy microparticles were injected into the liver cancer lesion at multiple points. After 24 hours of injection, mice were given tumor ischemia intervention. After 1 week and 2 weeks, respectively, The mice were sacrificed and the tumor lesions were detected, and the tumor volume was significantly reduced. After 2 weeks of treatment, the tumor-inhibiting effect of magnesium-based alloy microparticles was better, and the effect of treatment group 1 was the best, followed by treatment groups 5 and 9, and the effect of treatment groups 13 and 14 was the worst. (See Figure 4).

1.3.3 Effect of magnesium-based alloy microparticles on organ coefficient of experimental mice

The organ coefficients of the organs in the treatment group were compared with those in the control group. The organ coefficients in groups 13 and 14 in the treatment group were significantly decreased (P<0.001), and there was no significant difference in the other groups compared with the control group (P> 0.05). The results are shown in Table 2.

Discussion of beneficial effects:

The local implantation of magnesium-based alloy micro-particles designed in conjunction with TACE anti-liver cancer treatment in the present invention takes advantage of the natural corrosion degradation characteristics of magnesium metal and the anti-cancer potential of its degradation products, combined with the anti-cancer properties of zinc, molybdenum, and selenium plasma, The synergistic anti-tumor effect of it and TACE is the best. Locally implanting magnesium-based alloy micro-particles 24 hours before TACE treatment, compared with simple TACE treatment, the overall effective rate, disease control rate, tumor volume, and survival were significantly improved, and 6% magnesium-zinc alloy had the best effect. Good, there was no statistical difference in the incidence of complications among the groups.

Zinc is an important trace element in the human body. It participates in the composition of various proteins, and can act as a signal molecule in cells in the form of free ions to participate in the regulation of cell metabolism, activation of protein kinases and phosphatases, etc., and plays a role in tumor cell proliferation, apoptosis, important role in differentiation and immune regulation. Tumor patients are often accompanied by a deficiency of zinc ions, and increasing the intake of zinc ions can reduce the incidence of tumors. Low zinc can induce the activation of NFκB signaling, thereby promoting the release of a large number of tumor-related inflammatory factors, such as TNF-a and IL-1b, and supplementing zinc ions can increase A20 and PPAR-a, two zinc finger proteins with anti-inflammatory effects. In addition, zinc ions can directly or synergistically inhibit the malignant progression of tumors by inhibiting the intracellular Ras-MAPK signaling pathway, such as inhibiting proliferation, inducing apoptosis, or enhancing the anticancer efficacy of radiotherapy and chemotherapy. In the present invention, magnesium-6% zinc alloy microparticles (Mg-6Zn) combined with TACE has the best anti-tumor effect.

Selenium compounds inhibit metastasis of mouse melanoma cells by inducing cell cycle arrest and cell death. In addition, selenium compounds can sensitize various cancer cells to a variety of widely used drugs. Selenium induces cell apoptosis or blocks cell cycle, inhibits cell proliferation, regulates redox state, detoxifies carcinogens, stimulates the immune system and inhibits angiogenesis and other mechanisms have been considered as important mechanisms of selenium's anti-tumor effect. The treatment has been increasingly researched. However, selenium compounds, as an organic selenium preparation, have high toxicity and poor targeting. Therefore, the present invention adopts nano-selenium material, which has higher bioavailability, stronger biological activity and lower toxicity than organic selenium and inorganic selenium. In addition, as a drug carrier, nano-selenium has the advantages of good biocompatibility, high loading rate, low toxicity, easy synthesis, and easy storage. Although nano-selenium particles can inhibit the growth of cancer cells for a long time, they cannot effectively eliminate solid tumors in a short time. Therefore, 3% magnesium-selenium alloy microparticles (Mg-3Se) as the optimal ratio, combined with TACE can quickly eliminate tumors and improve therapeutic efficacy.

Molybdenum-containing compounds can be used for tumor-targeted fluorescence imaging and photothermal therapy. Through high permeability and retention effect, it accumulates at the tumor site and reaches the maximum amount, causing little damage to surrounding tissues, and its photothermal killing effect can kill tumor tissue vascular endothelial cells.

To sum up, this special invention specifically enhances the effectiveness of TACE treatment and alleviates its drug resistance. Among them, magnesium-zinc alloy microparticles (Mg-6Zn), magnesium-selenium alloy microparticles (Mg-3Se), and magnesium-molybdenum alloy microparticles (Mg-3Mo) have good synergistic anti-tumor effects, and Mg-6Zn has the best effect. While bringing huge clinical benefits to patients, no serious adverse events occurred, and the safety and efficacy met the requirements.

Claims (9)

1. Magnesium-based alloy microparticles with synergistic TACE anti-liver cancer effect are characterized in that metal magnesium is used as a carrier, and the magnesium-based alloy microparticles are smelted with other metal particles with anti-cancer effect; the mass ratio of the metal magnesium to other metal particles with the anti-cancer effect is 85-97: 1-15.

2. The magnesium-based alloy microparticles with synergistic TACE anti-liver cancer effect of claim 1, wherein: the metal particles having an anticancer effect include, but are not limited to, zinc, selenium or molybdenum metal particles.

3. A method for preparing the magnesium-based alloy microparticles with the anti-liver cancer effect of TACE in cooperation with the TACE of claim 1 or 2, which is characterized by comprising the following steps:

step 1: respectively putting a magnesium ingot and other metal ingots with anticancer effect into two pre-melting furnaces, vacuumizing, setting the temperature at 300 ℃, and purging by adopting high-temperature inert gas argon to remove oxidizing atmosphere gas adsorbed on the surface;

step 2: heating the furnace body, heating the furnace body containing the magnesium ingot to 680 ℃, heating the furnace bodies of other metal ingots with anticancer effect to the melting temperature of the metal, and adopting argon gas for protection to completely melt the metal to obtain liquid magnesium and other liquid metals with anticancer effect;

and step 3: introducing liquid magnesium and other liquid metals with anticancer effect onto a disc atomizer in an atomizing furnace, uniformly mixing the two liquid metals on the disc atomizer, and spraying the mixture into magnesium-based alloy droplets;

and 4, step 4: rapidly condensing the magnesium-based alloy droplets to form low-oxidation solid spherical magnesium-based alloy powder;

and 5: sieving magnesium-based alloy powder, grading the granularity, sieving spherical magnesium-based alloy microparticles of 10-200um, and storing in distilled water or absolute alcohol.

And (3) grading the alloy powder by adopting a vibrating screen, wherein the alloy powder is sieved by a 600-800 mesh screen.

4. The method of claim 3, wherein: and 2, heating the furnace body by using the intermediate frequency heating ring.

5. The method of claim 3, wherein: and (4) in the step (3), controlling the flow rate of the two metal liquids on the disc-type atomizer introduced into the atomizing furnace to realize the mass ratio of the magnesium-based alloy microparticles.

6. The method of claim 3, wherein: and in the step 3, the frequency of the atomizer is controlled, and the rotating speed of the centrifugal machine is controlled to control the size of the metal liquid drops.

7. The method of claim 3, wherein: the atomizer frequency was 55Hz.

8. The use method of the magnesium-based alloy microparticles with synergistic TACE anti-liver cancer effect of claim 1, wherein the use method comprises the following steps: can be used for synergistic TACE antitumor therapy.

9. The method of claim 8, wherein: in application, magnesium-based alloy microparticles are locally implanted into tumor site 24 hr before TACE treatment after dissolving in physiological saline.

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