Preparation method of targeted drug carrier for breast cancer treatment

Abstract

The invention provides a preparation method of a targeted drug carrier for breast cancer treatment, and the preparation method comprises the following steps: preparing a Fenton catalyst from raw materials including iron oxide, a manganese source, xanthan gum and a first inhibitor; a photo-thermal conversion agent is prepared from a gold source raw material; the preparation method comprises the following steps: preparing mesoporous silica from raw materials including a silicon source and a selenium source; the targeted drug carrier is prepared from raw materials including a Fenton catalyst, a photothermal conversion agent, mesoporous silica, a second inhibitor, paclitaxel, dopamine hydrochloride and a triple negative breast cancer cell membrane; wherein the first inhibitor comprises a GPX4 inhibitor, and the second inhibitor comprises one of a PD-1 inhibitor and a PD-L1 inhibitor. The invention provides a drug delivery system capable of combining chemotherapy, immunotherapy, photothermal therapy and chemodynamic therapy, so that multi-mode treatment means collaborative treatment of triple negative breast cancer is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor drugs, and in particular to a method for preparing a targeted drug carrier for treating breast cancer. Background Art

[0002] Cancer has always been a major public health problem in the world, and breast cancer is the most common cancer in women. Breast cancer (BRCA) is a disease that seriously endangers the health of women worldwide, and its incidence ranks first among female malignant tumors. Among them, triple-negative breast cancer (TNBC) accounts for about 15% of the total breast cancer population, and its progesterone receptor, estrogen receptor and HER2 expression are all negative. TNBC is highly malignant and is more prone to metastasis and recurrence. Due to the lack of targeted treatment methods, the 5-year survival rate of patients is only 12.2%.

[0003] For solid tumors, the main treatment methods currently are surgical removal of cancerous tissues or chemotherapy to kill cancerous cells. In addition to the above methods, the following new therapies have also gradually attracted the attention of technicians in this field.

[0004] Immunotherapy represented by immune checkpoint blockade (ICB) has brought innovation to the field of tumor treatment due to its genetic accuracy. Immunotherapy enhances the anti-cancer ability of immune cells by activating the autoimmune system, and demonstrates advantages in inhibiting metastasis and recurrence, and prolonging the survival of advanced patients. Currently, TNBC is clinically divided into four subtypes: luminal androgen receptor type, immunoregulatory type, basal-like immunosuppressive type, and interstitial type. Among them, patients with immunoregulatory TNBC have more activated immune microenvironment characteristics and benefit greatly from immunotherapy.

[0005] Photothermal therapy (PTT) has also been shown to be an effective treatment for breast cancer. Near-infrared (NIR) lasers of 700nm-900nm have deep tissue penetration. Photothermal therapy injects photothermal agents into tumor tissue. Under the excitation of 700nm-900nm near-infrared light, photothermal converters (PTAs) can absorb near-infrared light and convert it into heat energy to produce local high temperature, causing cancer cells to undergo necrosis or apoptosis, thereby achieving the purpose of cancer treatment. PTT treats local solid tumors through thermal ablation, which has the advantages of minimal invasion and low toxicity.

[0006] Chemodynamic therapy (CDT) relies on the metal-Fenton or Fenton-like reactions to produce reactive oxygen species (ROS) to treat tumors, which can catalyze endogenous hydrogen peroxide to produce reactive oxygen species. Excessive production of reactive oxygen species can lead to oxidative damage, cell apoptosis or necrosis. Due to the slightly acidic tumor microenvironment and the overexpression of hydrogen peroxide, chemodynamic therapy can be specifically triggered compared to normal cells, avoiding additional damage to normal tissues.

[0007] Relevant studies in the prior art have shown that chemotherapy drugs attack normal cells and tumor cells indiscriminately, have large side effects, and tumor cells can develop tolerance to chemotherapy drugs. Breast cancer has a limited response to immunotherapy. Immunosuppressant treatment has extraordinary clinical efficacy on some genes, but is completely ineffective on other genes. In short, the treatment effect of a single modality on triple-negative breast cancer is not satisfactory. Therefore, it is very necessary to enhance the anti-cancer treatment effect for triple-negative breast cancer through multimodal synergistic treatment.

[0008] The drug delivery system (DDS) is designed to deliver drugs accurately and effectively to the tumor site, thereby improving the therapeutic effect and reducing side effects. As mentioned above, for the treatment of triple-negative breast cancer, multimodal treatment methods need to work in synergy. Accordingly, how to improve a drug delivery system that can achieve the synergy of multimodal treatment methods is a technical problem that technicians in this field need to solve. Summary of the invention

[0009] One of the problems solved by the present invention is how to provide a drug delivery system that can combine chemotherapy, immunotherapy, photothermal therapy and chemodynamic therapy, so as to achieve synergistic treatment of triple-negative breast cancer with multimodal treatment methods.

[0010] In order to solve at least one of the above problems, the present invention provides a method for preparing a targeted drug carrier for treating breast cancer, the preparation method comprising: S100, preparing a Fenton catalyst using raw materials including iron oxide, a manganese source, xanthan gum and a first inhibitor; S200, preparing a photothermal conversion agent using raw materials including a gold source; S300, using raw materials including a silicon source and a selenium source to prepare mesoporous silica; S400, using raw materials including Fenton catalyst, photothermal conversion agent, mesoporous silica, second inhibitor, paclitaxel, dopamine hydrochloride and triple-negative breast cancer cell membrane to prepare targeted drug carrier; The first inhibitor includes a GPX4 inhibitor, and the second inhibitor includes one of a PD-1 inhibitor or a PD-L1 inhibitor.

[0011] In any of the above technical solutions, the iron oxide includes ferroferric oxide nanoparticles, and the manganese source includes potassium permanganate.

[0012] In any of the above technical solutions, the gold source includes chloroauric acid.

[0013] In any of the above technical solutions, the silicon source includes ethyl silicate, and the selenium source includes sodium selenite.

[0014] In any of the above technical solutions, S100 specifically includes: S110, mixing cardanol, iron oxide, xanthan gum, polyvinyl alcohol and water, and performing a hydrothermal treatment at a temperature of 140° C. to 150° C. and a pressure of 2 MPa to 3 MPa for 1.5 h to 2 h to obtain a first mixture; S120, mixing a manganese source, polyacrylamine hydrochloride and water to obtain a second mixture; S130, adding the second mixture dropwise to the first mixture while stirring simultaneously, and after the addition is complete, adding sodium citrate and performing ultrasonic dispersion to obtain an aqueous phase in a suspension state; S140, mixing the first inhibitor, polylactic acid polyethylene glycol polylactic acid triblock copolymer, dichloromethane and ethyl acetate to obtain an oil phase; S150, adding Tween 80 to the aqueous phase, and then dropping the oil phase while stirring simultaneously, ultrasonically emulsifying after the addition is complete, centrifugally separating the solids after standing and washing to obtain a Fenton catalyst.

[0015] In any of the above technical solutions, in S110, the mass ratio of cardanol: iron oxide: xanthan gum: polyvinyl alcohol: water is (1-2): (6-10): (2-4): (8-12): 50; and / or in S120, the mass ratio of manganese source: polyacrylamine hydrochloride: water is (2-3): (4-6): 50; and / or in S130, the mass ratio of sodium citrate: first mixture: second mixture is (10-12): (80-120): 100; and / or in S140, the mass ratio of the first inhibitor: polylactic acid poly(ethylene glycol) is (10-12): (80-120): 100. Diol polylactic acid triblock copolymer: dichloromethane: ethyl acetate = (4-6): (16-20): 40: 60; and / or in S150, by mass ratio, Tween 80: aqueous phase: oil phase = (2-4): (80-120): 100; and / or in S130, the power of ultrasonic dispersion is 400W to 600W, and the time of ultrasonic dispersion is 20min to 30min; and / or in S150, the power of ultrasonic emulsification is 1600W to 1800W, the time of ultrasonic emulsification is 20min to 30min, and the standing time is 3h to 4h.

[0016] In any of the above technical solutions, S200 specifically includes: S210, firstly mix the chelating agent, the gold source and the water in a mass ratio of chelating agent: sodium citrate: gold source: water = (2-8): (6-12): (4-6): 100, then dropwise add the sodium hydroxide aqueous solution and stir until the pH value reaches 9-10, finally dropwise add the sodium citrate and stir, and continue stirring for 20 to 30 minutes after the dropwise addition is completed to obtain a third mixture; S220, after the third mixture is allowed to stand for 0.5 h to 1 h, the solid matter is separated by centrifugation and washed to obtain a photothermal conversion agent.

[0017] In any of the above technical solutions, S300 specifically includes: S310, mixing and dispersing the selenium source, sodium nitrate, magnesium nitrate, calcium nitrate and silicon source in water to obtain a fourth mixture, and dispersing phosphorus pentoxide in ethanol to obtain a fifth mixture; S320, adding the fourth mixture dropwise into the fifth mixture while stirring simultaneously, adding a phosphoric acid aqueous solution after the addition is complete to adjust the pH value to 4 to 5, and continuing stirring for 3 to 4 hours to obtain a sol; S330, subjecting the sol to a hydrothermal treatment at a temperature of 180° C. to 190° C. and a pressure of 5 MPa to 6 MPa for 2 h to 2.5 h to obtain a gel; S340, using carbon dioxide as a medium, supercritically drying the gel to obtain mesoporous silica.

[0018] In any of the above technical solutions, in S310, the mass ratio of selenium source: sodium nitrate: magnesium nitrate: calcium nitrate: silicon source: phosphorus pentoxide: ethanol: water = (10-12): (10-12): (6-8): (38-40): (35-45): (42-44): (150-200): (80-120).

[0019] In any of the above technical solutions, S400 specifically includes: S410, mixing and dispersing the Fenton catalyst, the photothermal conversion agent, the mesoporous silica, the second inhibitor, the paclitaxel and the dopamine hydrochloride in a Tris-HCl buffer solution in a mass ratio of Fenton catalyst: photothermal conversion agent: mesoporous silica: the second inhibitor: paclitaxel: dopamine hydrochloride = (12-16): (12-16): (24-28): (16-20): (18-24): (2-4) to obtain a sixth mixture; S420, adding triple-negative breast cancer cell membrane to the sixth mixture, and incubating at a temperature of 36.5° C. to 37.5° C. for 20 h to 24 h; S430, after the incubation, the solids are collected by extrusion through a polycarbonate porous membrane with gradually decreasing pore sizes, thereby obtaining a targeted drug carrier.

[0020] Beneficial Effects The present invention provides a method for preparing a targeted drug carrier for treating breast cancer. The preparation method first adopts raw materials including iron oxide, a manganese source, xanthan gum and a first inhibitor to prepare a Fenton catalyst; then adopts raw materials including a gold source to prepare a photothermal converter; subsequently adopts raw materials including a silicon source and a selenium source to prepare mesoporous silica; finally, adopts raw materials including a Fenton catalyst, a photothermal converter, mesoporous silica, a second inhibitor, paclitaxel, dopamine hydrochloride and a triple-negative breast cancer cell membrane to prepare a targeted drug carrier; wherein the first inhibitor includes a GPX4 inhibitor, and the second inhibitor includes one of a PD-1 inhibitor or a PD-L1 inhibitor.

[0021] A series of metal ions (Fe ions, Mn ions, Cu ions, etc.) can exhibit Fenton catalytic activity. Among them, iron oxide nanoparticle magnetic materials are currently the most studied metal oxide nanozymes widely used in chemodynamic therapy. When iron oxide nanoparticles are co-cultured with macrophages, studies in the prior art have found more than 10 times the increase in hydrogen peroxide and hydroxyl radicals. Iron oxide nanoparticles can enter cells, increase the production of intracellular reactive oxygen species, lead to lipid peroxidation and cell membrane damage, and thereby induce cell apoptosis (also known as "ferroptosis"). Therefore, the present invention chooses to add a Fenton catalyst containing iron oxide to a targeted drug carrier to achieve chemodynamic therapy.

[0022] Gold nanoparticles with strong localized surface plasmon resonance (LSPR) properties can be used as photothermal conversion agents (PTAs). In photothermal therapy, PTAs do not need to enter the cancer cells, but gather around the tumor tissue and convert light energy into heat energy under the irradiation of an external light source to kill cancer cells.

[0023] Mesoporous silica has voids and a high specific surface area, and can be used to load drug molecules. It also exhibits good compatibility and stability in vivo, and is a good drug carrier. By encapsulating anticancer agents or drugs in mesoporous silica, the drug loading can be increased and side effects can be reduced. The present invention dopes selenium into the mesoporous silica material. Selenium has many effects in tumor treatment, including inhibiting tumor cell growth, anti-oxidation, enhancing immune function, reducing the side effects of chemotherapy drugs, and improving the quality of life.

[0024] Paclitaxel is a highly effective, low-toxic, broad-spectrum natural anticancer drug that can be used to exert chemotherapy and anticancer effects. GPX4 inhibitors can aggravate and promote the process of lipid peroxidation, thereby accelerating the occurrence of ferroptosis. Immune checkpoint inhibitors of PD-1 and PD-L1 can block the binding of PD-1 and PD-L1, thereby relieving the immunosuppressive state of T cells and restoring their killing function.

[0025] Therefore, the technical solution of the present invention can provide a drug delivery system that combines chemotherapy, immunotherapy, photothermal therapy and chemodynamic therapy, thereby achieving synergistic treatment of triple-negative breast cancer with multimodal treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 These are the cytotoxicity test results of targeted drug carrier samples 1 to 6 prepared in the examples of the present invention on triple-negative breast cancer cells. DETAILED DESCRIPTION

[0027] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the following is a detailed description of the specific embodiments of the present invention.

[0028] Unless otherwise specified, the reagents and raw materials used in the present invention can be purchased through commercial channels. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product specifications.

[0029] The present invention provides a method for preparing a targeted drug carrier for treating breast cancer, the preparation method comprising: S100, preparing a Fenton catalyst using raw materials including iron oxide, a manganese source, xanthan gum and a first inhibitor; S200, preparing a photothermal conversion agent using raw materials including a gold source; S300, using raw materials including a silicon source and a selenium source to prepare mesoporous silica; S400, using raw materials including Fenton catalyst, photothermal conversion agent, mesoporous silica, second inhibitor, paclitaxel, dopamine hydrochloride and triple-negative breast cancer cell membrane to prepare targeted drug carrier; The first inhibitor includes a GPX4 inhibitor, and the second inhibitor includes one of a PD-1 inhibitor and a PD-L1 inhibitor. The triple-negative breast cancer cell membrane can be extracted using a cell membrane protein extraction kit using 4T1 triple-negative breast cancer cells as a tumor model.

[0030] One of the purposes of the present invention is to provide a drug delivery system that combines chemotherapy, immunotherapy, photothermal therapy and chemodynamic therapy, thereby achieving synergistic multimodal treatment of triple-negative breast cancer.

[0031] In order to achieve the above-mentioned purpose, the present invention uses mesoporous silica as a carrier, and loads anticancer agents and drugs on mesoporous silica. The porous characteristics of mesoporous silica are utilized to achieve loading and delivery of agents and drugs. Among them, the targeted drug carrier of the present invention can be directed to the affected part of the patient by injection. Mesoporous silica has good biocompatibility, is safe and reliable, and has no side effects. Among them, the mesoporous silica of the present invention is preferably prepared by a sol-gel method using ethyl silicate as a raw material. The advantage of the sol-gel method is that it can prepare nanoscale silica with a smaller particle size and uniform particle size distribution.

[0032] The anticancer agents and drugs used in the present invention include Fenton catalysts, photothermal converters, GPX4 inhibitors, and one of PD-1 inhibitors or PD-L1 inhibitors. Specifically, the Fenton catalyst of the present invention is an iron-manganese composite Fenton catalyst, and the photothermal converter of the present invention is a nano-gold photothermal converter. Accordingly, the iron oxide of the present invention includes ferroferric oxide nanoparticles, the manganese source includes potassium permanganate, and the gold source of the present invention includes chloroauric acid.

[0033] The role of ferroferric oxide nanoparticles is to provide iron ions and induce ferroptosis through reactive oxygen and free radicals. In addition, in addition to inducing ferroptosis, due to its unique magnetic properties, ferroferric oxide nanoparticles can also be targeted to specific parts of the human body under the action of an external magnetic field. Considering that manganese oxide also has a Fenton-like effect, and it can produce oxygen and consume glutathione in the tumor environment to alleviate the hypoxic environment in the tumor and avoid excessive consumption of hydroxyl radicals by glutathione. The present invention uses potassium permanganate to prepare manganese oxide to carry out composite modification of iron oxide.

[0034] The present invention adopts chloroauric acid to prepare nano-gold particles through chemical reaction. As an inorganic photothermal conversion agent, the nano-gold particles have small systemic toxicity and side effects and good biological safety, and have strong local surface plasmon resonance, so they have high photothermal conversion efficiency, can effectively convert light energy into heat energy, and achieve good therapeutic effect.

[0035] Paclitaxel is a highly effective, low-toxic, broad-spectrum natural anticancer drug that mainly acts on the interphase and mitotic phases of cells. Paclitaxel promotes tubulin polymerization and inhibits depolymerization, which can maintain tubulin stability, thereby inhibiting the mitosis of tumor cells and exerting an anti-tumor effect. In addition, paclitaxel can also induce the expression of tumor necrosis factor-related genes, further exerting its anti-cancer effect.

[0036] As mentioned above, lipid peroxidation is one of the core characteristics of ferroptosis. During ferroptosis, reactive oxygen species in cells generate hydroxyl radicals to attack polyunsaturated fatty acids on cell membranes, leading to the accumulation of lipid peroxides. The present invention can further aggravate and promote the above-mentioned lipid peroxidation process by adding GPX4 inhibitors to the Fenton catalyst. GPX4 (glutathione peroxidase 4) is an antioxidant enzyme in cells that uses glutathione as a reducing agent to reduce lipid peroxides to corresponding alcohols, thereby protecting cells from damage by lipid peroxides. Since GPX4 inhibitors can covalently bind to GPX4 and inhibit the activity of GPX4, GPX4 inhibitors can aggravate and promote the above-mentioned lipid peroxidation process, so that the level of lipid peroxides is further increased, thereby accelerating the occurrence of ferroptosis. Exemplarily, the GPX4 inhibitors in the prior art are mainly RSL3.

[0037] PD-1 (Programmed Death-1) and PD-L1 (Programmed Death Ligand-1) are key molecules in the immune regulation process. PD-1 is a type I transmembrane protein composed of 288 amino acid residues, also known as CD279. It belongs to the CD28 family and is widely expressed on a variety of immune cells, such as activated T cells, B cells, NK cells, and DC cells. PD-L1 is the ligand of PD-1, also known as CD274 or surface antigen differentiation factor 274. It is a type I transmembrane protein encoded by the CD274 gene. When PD-1 binds to its ligand PD-L1, it can transmit immunosuppressive signals, thereby inhibiting the body's anti-tumor immune response. Immune checkpoint inhibitors targeting PD-1 / PD-L1 have become an important means of tumor immunotherapy. This type of immune checkpoint inhibitor drug can block the binding of PD-1 to PD-L1, thereby relieving the immunosuppressive state of T cells and restoring their killing function. Illustratively, the PD-1 inhibitors in the prior art include Nivolumab (Drug O) and Keytruda (Drug K), and the PD-L1 inhibitors in the prior art include Atezolizumab (Tecentriq), Avelumab (Bavencio) and Durvalumab (Imfinzi), etc.

[0038] Therefore, the technical solution of the present invention can provide a drug delivery system that combines chemotherapy, immunotherapy, photothermal therapy and chemodynamic therapy, thereby achieving synergistic treatment of triple-negative breast cancer with multimodal treatment methods.

[0039] In order to further promote ferroptosis and improve the anti-cancer therapeutic effect, the present invention prepares a Fenton catalyst by the following steps: S110, mixing cardanol, iron oxide, xanthan gum, polyvinyl alcohol and water in a mass ratio of cardanol: iron oxide: xanthan gum: polyvinyl alcohol: water = (1-2): (6-10): (2-4): (8-12): 50, and performing a hydrothermal treatment at a temperature of 140° C. to 150° C. and a pressure of 2 MPa to 3 MPa for 1.5 h to 2 h to obtain a first mixture; S120, mixing the manganese source, polyacrylamine hydrochloride and water in a mass ratio of manganese source: polyacrylamine hydrochloride: water = (2-3): (4-6): 50 to obtain a second mixture; S130, adding the second mixture dropwise to the first mixture in a mass ratio of sodium citrate: first mixture: second mixture = (10-12): (80-120): 100 and stirring simultaneously, and after the dropwise addition, adding sodium citrate and ultrasonically dispersing at a power of 400 W to 600 W for 20 min to 30 min to obtain an aqueous phase in a suspension state; S140, mixing the first inhibitor, polylactic acid polyethylene glycol polylactic acid triblock copolymer, dichloromethane and ethyl acetate in a mass ratio of the first inhibitor: polylactic acid polyethylene glycol polylactic acid triblock copolymer: dichloromethane: ethyl acetate = (4-6): (16-20): 40: 60 to obtain an oil phase; S150. Add Tween 80 to the aqueous phase at a mass ratio of Tween 80: aqueous phase: oil phase = (2-4): (80-120): 100, and then drop the oil phase while stirring simultaneously. After the dropwise addition is completed, perform ultrasonic emulsification at a power of 1600 W to 1800 W for 20 min to 30 min. After standing for 3 h to 4 h, centrifuge to separate the solids and wash to obtain Fenton catalyst.

[0040] Through the above S110, cardanol, iron oxide, and xanthan gum can be mixed in a dispersion system of polyvinyl alcohol and water, and hydrothermal treatment under high temperature and high pressure is used to load an appropriate amount of cardanol and xanthan gum on the surface of the iron oxide. The present invention preferably uses xanthan gum with a molecular weight of more than 1.2 million.

[0041] Through the above S120, the manganese source can be dispersed in the water dispersion system. Through the above S130, the first mixture containing iron oxide and the second mixture containing the manganese source can be mixed with each other. In the process of adding the second mixture to the first mixture and stirring simultaneously, the manganese source can be uniformly loaded on the surface of the iron oxide particles. Furthermore, under the reduction effect of sodium citrate and the ultrasonic dispersion effect, manganese dioxide can be uniformly precipitated on the surface of the iron oxide, thereby achieving the loading of manganese.

[0042] Through the above S140, the GPX4 inhibitor as the first inhibitor can be uniformly dispersed in an organic solvent. Subsequently, through S150, the oil phase containing the GPX4 inhibitor can be gradually mixed into the aqueous phase in the suspension state containing the nano iron oxide particles under the emulsification effect of Tween 80 to form oil-in-water microcapsules. On the one hand, as described above, the GPX4 inhibitor can aggravate and promote the above-mentioned lipid peroxidation process, thereby promoting the nano iron oxide particles to play a better role in chemodynamic therapy and accelerate the occurrence of ferroptosis. On the other hand, polylactic acid polyethylene glycol polylactic acid triblock copolymer is a block polymer formed by connecting polylactic acid and polyethylene glycol through a chemical synthesis method. The present invention utilizes the good biocompatibility of polylactic acid polyethylene glycol polylactic acid triblock copolymer to coat and encapsulate nano iron oxide particles in order to reduce its side effects and improve its biocompatibility. The present invention preferably uses polylactic acid polyethylene glycol polylactic acid triblock copolymer with a molecular weight of more than 5000.

[0043] Since cells have an active iron excretion mechanism, the amount of iron ions that can enter cancer cells is limited, and the limited iron concentration is one of the key factors that limit the effectiveness of chemodynamic therapy. To this end, the present invention modifies ferroferric oxide nanoparticles by using cardanol and xanthan gum, thereby making it easier for them to enter cells.

[0044] Specifically, xanthan gum is a microbial extracellular polysaccharide produced by fermentation engineering of Xanthomonas campestris using carbohydrates as the main raw material, which is composed of D-glucose, D-mannose and D-glucuronic acid. As a polysaccharide polymer compound, xanthan gum can act as a carrier and transporter of iron ions. The polysaccharide molecules of xanthan gum are coated with nano-iron oxide to form a ribbon-shaped spiral copolymer, which is adsorbed and suspended around the cell membrane, thereby helping more iron ions to enter cancer cells and improve the efficiency of iron ions entering cancer cells. Thus, the iron concentration in cancer cells is increased in a short time, thereby accelerating ferroptosis and promoting apoptosis of cancer cells. In addition, xanthan gum can remain stable in the acidic environment of the tumor and is not affected by the photothermal conversion effect. Cardanol is a compound extracted from natural cashew nut shell oil, which includes a benzene ring structure, a polar hydroxyl group, and a carbon 15 straight chain containing an unsaturated double bond in the meta position. This structure gives cardanol good thermal stability, which is also not affected by the photothermal conversion effect, and the phenolic hydroxyl group of cardanol can help promote redox imbalance in tumor cells, thereby enhancing the effect of ferroptosis.

[0045] In addition, after the polyacrylamine hydrochloride as a polyelectrolyte contacts with polyvinyl alcohol, the amine groups in the polyacrylamine hydrochloride structural unit and the hydroxyl groups in the polyvinyl alcohol form hydrogen bonds and cross-link with each other, so that the manganese source can be uniformly loaded on the surface of the iron oxide particles. Therefore, the present invention can make the surface of the iron oxide nanoparticles precipitate and load manganese oxide uniformly.

[0046] Finally, the oil-in-water microcapsule structure can load the GPX4 inhibitor and can also coat and encapsulate the nano-iron oxide particles, thereby reducing the side effects of the nano-iron oxide particles and improving their biocompatibility.

[0047] Therefore, the Fenton catalyst prepared by the above steps can play a better role in chemodynamic therapy, reduce side effects and improve treatment effects.

[0048] In some embodiments of the present invention, the photothermal conversion agent is prepared by the following steps: S210, firstly mix the chelating agent, the gold source and the water in a mass ratio of chelating agent: sodium citrate: gold source: water = (2-8): (6-12): (4-6): 100, then dropwise add the sodium hydroxide aqueous solution and stir until the pH value reaches 9-10, finally dropwise add the sodium citrate and stir, and continue stirring for 20 to 30 minutes after the dropwise addition is completed to obtain a third mixture; S220, after the third mixture is allowed to stand for 0.5 h to 1 h, the solid matter is separated by centrifugation and washed to obtain a photothermal conversion agent.

[0049] Preferably, the chelating agent is alanine or lysine or glycine. In the above step, chloroauric acid is used as the gold source, sodium citrate is used as the reducing agent, and the gold source solution is adjusted to be alkaline and then the reducing agent is added to obtain the nano-gold particle photothermal converter.

[0050] In order to further improve the anti-cancer therapeutic effect, the present invention prepares mesoporous silica by the following steps: S310, according to the mass ratio of selenium source: sodium nitrate: magnesium nitrate: calcium nitrate: silicon source: phosphorus pentoxide: ethanol: water = (10-12): (10-12): (6-8): (38-40): (35-45): (42-44): (150-200): (80-120), the selenium source, sodium nitrate, magnesium nitrate, calcium nitrate and silicon source are mixed and dispersed uniformly in water to obtain a fourth mixture, and the phosphorus pentoxide is dispersed uniformly in ethanol to obtain a fifth mixture; S320, adding the fourth mixture dropwise into the fifth mixture while stirring simultaneously, adding a phosphoric acid aqueous solution after the addition is complete to adjust the pH value to 4 to 5, and continuing stirring for 3 to 4 hours to obtain a sol; S330, subjecting the sol to a hydrothermal treatment at a temperature of 180° C. to 190° C. and a pressure of 5 MPa to 6 MPa for 2 h to 2.5 h to obtain a gel; S340, using carbon dioxide as a medium, supercritically drying the gel to obtain mesoporous silica.

[0051] In the above steps, organic silicon such as ethyl silicate or methyl silicate can be used as a silicon source, preferably ethyl silicate as a silicon source. The silicon source can form a silica sol in a mixture of water and alcohol with an inorganic acid as a catalyst. In tumor treatment, the acidic environment of the tumor is potentially closely related to the growth, invasion and metastasis of the tumor. The prior art generally neutralizes the acidic environment of the tumor site by directly injecting an alkaline solution such as sodium bicarbonate into the tumor site. This method has shown a certain therapeutic effect in mouse experiments, but has shown strong irritation and side effects in human trials, and its action time is short and lacks a sustained release effect. For this reason, in the process of preparing silica sol, the present invention adds a selenium source, sodium nitrate, magnesium nitrate, calcium nitrate and phosphorus pentoxide. Among them, the selenium source preferably includes sodium selenite. The purpose of the present invention is to prepare a precursor of alkaline glass by a sol-gel method using sodium magnesium calcium as a raw material. The precursor of selenium and alkaline glass is uniformly blended with silica sol, and a silica gel containing selenium and alkaline glass can be obtained by hydrothermal treatment under heating and pressure. Through supercritical drying, the organic matter in the silica gel is extracted by carbon dioxide, thereby obtaining mesoporous silica uniformly blended with selenium and alkaline glass. Selenium can inhibit the growth and reproduction of tumor cells, and achieve the purpose of adjuvant therapy to inhibit tumors by blocking the proliferation signal pathway of tumor cells and interfering with their growth cycle. Compared with the injection of alkaline solution, the sustained-release alkaline glass can gently and long-term regulate the acidic environment of the tumor site, which helps to improve the activity of chemotherapy drugs and immune cells, enhance the efficacy of chemotherapy drugs and immunotherapy drugs and reduce side effects.

[0052] Finally, the present invention mixes the Fenton catalyst, the photothermal conversion agent, the chemotherapeutic drug and the immunosuppressant through the following steps to prepare a targeted drug carrier: S410, mixing and dispersing the Fenton catalyst, the photothermal conversion agent, the mesoporous silica, the second inhibitor, the paclitaxel and the dopamine hydrochloride in a Tris-HCl buffer solution in a mass ratio of Fenton catalyst: photothermal conversion agent: mesoporous silica: the second inhibitor: paclitaxel: dopamine hydrochloride = (12-16): (12-16): (24-28): (16-20): (18-24): (2-4) to obtain a sixth mixture; S420, adding triple-negative breast cancer cell membrane to the sixth mixture, and incubating at a temperature of 36.5° C. to 37.5° C. for 20 h to 24 h; S430, after the incubation, the solids are collected by extrusion through a polycarbonate porous membrane with gradually decreasing pore sizes, thereby obtaining a targeted drug carrier.

[0053] Among them, dopamine hydrochloride is used to improve the hydrophilicity of Fenton catalysts, photothermal conversion agents, chemotherapy drugs and immunosuppressants. In order to improve the targeting and therapeutic effect of carriers containing anticancer agents and drugs, the present invention mixes mesoporous silica loaded with anticancer agents and drugs with triple-negative breast cancer cell membranes, and incubates the triple-negative breast cancer cell membranes to wrap the carriers, so that the carriers can selectively interact with tumor cells while reducing toxic side effects on normal cells.

[0054] Embodiment 1 In this example, a series of targeted drug carrier samples 1 to 4 for the treatment of breast cancer were prepared by using the raw material ratios shown in Table 1 through the following steps: S1, mixing cardanol, iron oxide, xanthan gum, polyvinyl alcohol and water, sending the mixture into a reactor, and performing a hydrothermal treatment for 2 hours at a temperature of 145° C. and a pressure of 2.5 MPa. After the hydrothermal treatment, the mixture is cooled, depressurized and cooled to obtain a first mixture; S2, stirring and mixing potassium permanganate, polyacrylamine hydrochloride and water in a beaker at room temperature to obtain a second mixture; S3, adding the second mixture dropwise to the first mixture and stirring synchronously with a stirring rod, gradually adding sodium citrate dropwise after the addition is complete and continuing to stir, and then ultrasonically dispersing at a power of 400 W for 20 min to obtain an aqueous phase in a suspension state; S4, stirring and mixing the GPX4 inhibitor, polylactic acid-polyethylene glycol-polylactic acid triblock copolymer, dichloromethane and ethyl acetate in a beaker at room temperature to obtain an oil phase; S5, adding Tween 80 to the aqueous phase, and then dropping the oil phase and stirring simultaneously, after the dropwise addition, ultrasonic emulsification was performed at a power of 1600 W for 30 min, and the solid was separated by centrifugation after standing for 3 h, and washed twice with ethanol, and dried with hot air at 85° C. to obtain a Fenton catalyst; S6, firstly mix alanine, chloroauric acid and water, then dropwise add 8wt% sodium hydroxide aqueous solution and stir until the pH value reaches 9, finally dropwise add sodium citrate and stir, and continue stirring for 20 minutes after the dropwise addition is completed to obtain a third mixture; S7, after the third mixture is allowed to stand for 0.5 h, the solid is separated by centrifugation and washed twice with water, and dried with hot air at 85° C. to obtain a photothermal conversion agent; S8, mixing and dispersing sodium selenite, sodium nitrate, magnesium nitrate, calcium nitrate and ethyl silicate in water to obtain a fourth mixture, and dispersing phosphorus pentoxide in ethanol to obtain a fifth mixture; S9, adding the fourth mixture dropwise into the fifth mixture while stirring simultaneously, and after the addition is complete, adding an aqueous phosphoric acid solution with a concentration of 85 vol% to adjust the pH value to 5, and continuing stirring for 3 hours to obtain a sol; S10, subjecting the sol to a hydrothermal treatment at a temperature of 180° C. and a pressure of 5.2 MPa for 2 h to obtain a gel; S11, using carbon dioxide as a medium, supercritically drying the gel at a temperature of 32° C. and a pressure of 7.38 MPa for 2 h, cooling and releasing the pressure after the supercritical drying, extracting the solid, washing it with ethanol twice, and drying it with hot air at 85° C. to obtain mesoporous silica; S12, mixing and dispersing the Fenton catalyst, the photothermal conversion agent, the mesoporous silica, the second inhibitor, paclitaxel and dopamine hydrochloride in a Tris-HCl buffer solution with a pH value of 7.5 and a mass that is 10 times the mass of the mesoporous silica to obtain a sixth mixture; S13, adding triple-negative breast cancer cell membranes having a mass equal to that of the mesoporous silica to the sixth mixture, and incubating at 37° C. for 24 h; S14. After the incubation, the mixture was first squeezed through a polycarbonate porous membrane with a pore size of 300 nm, and then squeezed again through a polycarbonate porous membrane with a pore size of 100 nm. The solids were collected by centrifugation and washed twice with PBS, and dried with hot air at 85°C to obtain a targeted drug carrier.

[0055] Table 1 Comparative Example 1 In this comparative example, a targeted drug carrier comparative sample 1 for the treatment of breast cancer was prepared by the following steps: S1. Mixing iron oxide, polyvinyl alcohol and water in a mass ratio of iron oxide: polyvinyl alcohol: water = 6:8:50 to obtain a first mixture; The steps from S2 to S14 and the raw material proportions are the same as those of sample 1.

[0056] Comparative Example 2 In this comparative example, a targeted drug carrier comparative sample 2 for the treatment of breast cancer was prepared by the following steps: The steps from S1 to S7 and the raw material ratio are the same as those of sample 1; S8. Dispersing ethyl silicate in a mixture of ethanol and water in a mass ratio of ethyl silicate: ethanol: water = 40:150:100 to obtain a precursor; S9, adding an aqueous phosphoric acid solution with a concentration of 85 vol% to the precursor to adjust the pH value to 5, and continuing stirring for 3 hours to obtain a sol; The steps from S10 to S14 and the raw material proportions are the same as those of sample 1.

[0057] Treatment effect test 1 4T1 triple-negative breast cancer cells were inoculated in a 96-well plate and divided into 6 groups after the cell confluence reached 75%. Targeted drug carrier samples 1 to 4, as well as comparative samples 1 and 2 were added to each group in turn at a concentration of 100 μg / ml. After the targeted drug carrier and 4T1 triple-negative breast cancer cells were incubated at 37°C for 24 hours, the 808nm, power of 0.5W / cm 2 The cells were irradiated with NIR laser for 5 min and washed twice with PBS to obtain test groups 1 to 6.

[0058] 40 mg of thiazolyl blue tetrazolium salt was added to test groups 1 to 6 in sequence, and the cells were cultured at 37°C in the dark for 6 h. After removing the culture medium, 100 μl of dimethyl sulfoxide was added, and the cells were shaken for 20 min. The absorbance at 570 nm was detected by an ELISA reader, and compared with the blank control group. The cell survival rate was evaluated accordingly. The test results of samples 1 to 4, and comparative samples 1 and 2 are shown in the following table. Figure 1 shown. Figure 1 The test results show that the number of surviving 4T1 triple-negative breast cancer cells treated with samples 1 to 4 is relatively low.

[0059] Treatment effect test 2 Four-week-old female BALB / c mice were used. Before the experiment, the mice were adaptively raised under SPF conditions for one week. 4T1 triple-negative breast cancer cells were dispersed in PBS to prepare a cell suspension. After mixing evenly, 1×10 7 The tumor model was constructed by subcutaneous injection into the mouse axilla.

[0060] When the tumor volume grew to about 100 cubic millimeters, the mice were randomly divided into 6 groups and the treatment began. The treatment method was: 20 μg of targeted drug carrier samples 1 to 4, as well as comparative samples 1 and 2 were mixed in PBS and injected into the armpits of mice. 808nm, power 0.3W / cm 2 The NIR laser was irradiated for 3 minutes. After the treatment of the 6 groups of mice, the tumor volume of the mice was measured every 3 days using a vernier caliper, and the average value of each group of mice was calculated. Tumor volume = tumor long axis × tumor short axis 2×0.5, the test results of samples 1 to 4, and the corresponding test results of comparative samples 1 and 2 are shown in Table 2. The test results in Table 2 show that after being injected with the targeted drug carriers of samples 1 to 4 and irradiated with near-infrared light, the relative tumor volume of mice within 20 days was smaller than that of comparative samples 1 and 2. In addition, the relative tumor volume of mice injected with comparative sample 1 was larger in the early stage (5 days and 10 days) than that of mice injected with comparative sample 2, but showed the opposite trend in the later stage (15 days and 20 days). This result shows that compared with mice injected with comparative sample 2, the growth of tumor cells in mice injected with comparative sample 1 was better inhibited.

[0061] Table 2 Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. A method for preparing a targeted drug carrier for the treatment of breast cancer, characterized in that: The preparation method comprises: S100, preparing a Fenton catalyst using raw materials including iron oxide, a manganese source, xanthan gum and a first inhibitor; S200, preparing a photothermal conversion agent using raw materials including a gold source; S300, using raw materials including a silicon source and a selenium source to prepare mesoporous silica; S400, preparing the targeted drug carrier by using raw materials including the Fenton catalyst, the photothermal conversion agent, the mesoporous silica, the second inhibitor, paclitaxel, dopamine hydrochloride and triple-negative breast cancer cell membrane; Wherein, the first inhibitor includes a GPX4 inhibitor, and the second inhibitor includes one of a PD-1 inhibitor or a PD-L1 inhibitor.

2. The preparation method according to claim 1, characterized in that: The iron oxide includes ferrosoferric oxide nanoparticles, and the manganese source includes potassium permanganate.

3. The preparation method according to claim 1, characterized in that: The gold source includes chloroauric acid.

4. The preparation method according to claim 1, characterized in that: The silicon source includes ethyl silicate, and the selenium source includes sodium selenite.

5. The preparation method according to any one of claims 1 to 4, characterized in that: S100 specifically includes: S110, mixing cardanol, the iron oxide, the xanthan gum, polyvinyl alcohol and water, and performing a hydrothermal treatment at a temperature of 140° C. to 150° C. and a pressure of 2 MPa to 3 MPa for 1.5 h to 2 h to obtain a first mixture; S120, mixing the manganese source, polyacrylamine hydrochloride and water to obtain a second mixture; S130, adding the second mixture dropwise to the first mixture while stirring simultaneously, and after the addition is complete, adding sodium citrate for ultrasonic dispersion to obtain an aqueous phase in a suspension state; S140, mixing the first inhibitor, polylactic acid polyethylene glycol polylactic acid triblock copolymer, dichloromethane and ethyl acetate to obtain an oil phase; S150, adding Tween 80 to the aqueous phase, then dropping the oil phase and stirring simultaneously, ultrasonically emulsifying after the dropping is completed, centrifuging and washing the solids after standing, to obtain the Fenton catalyst.

6. The preparation method according to claim 5, characterized in that: In S110, by mass ratio, cardanol: iron oxide: xanthan gum: polyvinyl alcohol: water = (1-2): (6-10): (2-4): (8-12): 50; and / or In S120, the mass ratio of manganese source: polyacrylamine hydrochloride: water = (2-3): (4-6): 50; and / or In S130, in terms of mass ratio, sodium citrate: first mixture: second mixture = (10-12): (80-120): 100; and / or In S140, the mass ratio of the first inhibitor: polylactic acid polyethylene glycol polylactic acid triblock copolymer: dichloromethane: ethyl acetate = (4-6): (16-20): 40: 60; and / or In S150, the mass ratio of Tween 80:water phase:oil phase = (2-4): (80-120): 100; and / or In S130, the power of ultrasonic dispersion is 400W to 600W, and the time of ultrasonic dispersion is 20min to 30min; and / or In S150, the power of ultrasonic emulsification is 1600W to 1800W, the time of ultrasonic emulsification is 20min to 30min, and the standing time is 3h to 4h.

7. The preparation method according to any one of claims 1 to 4, characterized in that: S200 specifically includes: S210, firstly mixing the chelating agent, the gold source and the water in a mass ratio of chelating agent: sodium citrate: gold source: water = (2-8): (6-12): (4-6): 100, then dropping a sodium hydroxide aqueous solution and stirring until the pH value reaches 9-10, finally dropping the sodium citrate and stirring, and continuing to stir for 20 min to 30 min after the dropping is completed, to obtain a third mixture; S220, after the third mixture is allowed to stand for 0.5 h to 1 h, the solid matter is separated by centrifugation and washed to obtain the photothermal conversion agent.

8. The preparation method according to any one of claims 1 to 4, characterized in that: S300 specifically includes: S310, mixing and dispersing the selenium source, sodium nitrate, magnesium nitrate, calcium nitrate and the silicon source in water to obtain a fourth mixture, and dispersing phosphorus pentoxide in ethanol to obtain a fifth mixture; S320, adding the fourth mixture dropwise into the fifth mixture while stirring simultaneously, adding a phosphoric acid aqueous solution after the addition is complete to adjust the pH value to 4 to 5, and continuing stirring for 3 to 4 hours to obtain a sol; S330, subjecting the sol to a hydrothermal treatment at a temperature of 180° C. to 190° C. and a pressure of 5 MPa to 6 MPa for 2 h to 2.5 h to obtain a gel; S340, using carbon dioxide as a medium, supercritically drying the gel to obtain the mesoporous silica.

9. The preparation method according to claim 8, characterized in that: In S310, by mass ratio, selenium source: sodium nitrate: magnesium nitrate: calcium nitrate: silicon source: phosphorus pentoxide: ethanol: water = (10-12): (10-12): (6-8): (38-40): (35-45): (42-44): (150-200): (80-120).

10. The preparation method according to any one of claims 1 to 4, characterized in that: S400 specifically includes: S410, mixing and dispersing the Fenton catalyst, the photothermal conversion agent, the mesoporous silica, the second inhibitor, the paclitaxel and the dopamine hydrochloride in a Tris-HCl buffer solution in a mass ratio of Fenton catalyst: photothermal conversion agent: mesoporous silica: second inhibitor: paclitaxel: dopamine hydrochloride = (12-16): (12-16): (24-28): (16-20): (18-24): (2-4) to obtain a sixth mixture; S420, adding the triple-negative breast cancer cell membrane to the sixth mixture, and incubating at a temperature of 36.5° C. to 37.5° C. for 20 h to 24 h; S430, after the incubation is completed, the solid matter is squeezed through a polycarbonate porous membrane with gradually decreasing pore sizes, and centrifuged to collect the solid matter to obtain the targeted drug carrier.

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