Novel anti-tumor two-dimensional photo-thermal material and preparation method thereof

Abstract

The invention discloses a novel anti-tumor two-dimensional photothermal material and a preparation method thereof.The preparation method comprises the steps that after a BP nanosheet is prepared through a liquid stripping technology, Fe < 3 + > is introduced, FeBPs is prepared through an in-situ biomimetic mineralization method, BP is subjected to in-situ biomimetic mineralization through metal ions Fe < 3 + >, and the black phosphorus nanosheet-(FeBPs) with the surface modified by an iron-phosphoric acid organic coordination layer is constructed. After the FeBPs enter tumor cells, dissociation of a mineralization layer is initiated under the synergistic effect of GSH, Fe < 3 + > is reduced into Fe < 2 + >, a large amount of GSH in the cells is consumed, the GPX4 protein level is lowered, ROS accumulation is caused, and tumor cell ferroptosis is induced. Meanwhile, by means of the excellent photo-thermal performance of BP after dissociation of the mineralization layer, under irradiation of near-infrared light 808 nm, the temperature of tumor tissue is increased, tumor ablation is caused, then photo-thermal / ferroptosis collaborative treatment is achieved, and a new treatment strategy is provided for tumor treatment research.

Description

TECHNICAL FIELD

[0001] The present application relates to a novel two-dimensional photothermal material for resisting tumors and a preparation method thereof. BACKGROUND

[0002] Cancer has always been a threat to human health and life with high morbidity and mortality. Cancer treatment has become a major challenge in the current medical research field in China. Surgery, radiotherapy and chemotherapy are the main treatment methods for cancer, but all have certain limitations, such as: radiotherapy and chemotherapy have greater toxic side effects on the whole body, and while killing tumor cells, normal tissue cells are often also damaged, causing the body's immune function to be low and hematopoietic function to be inhibited; surgical treatment often cannot be treated in time due to the patient's physical weakness or poor tumor location, and when the tumor infiltrates multiple organs or is adhered to important tissues, surgical resection also has great risks. Therefore, in order to improve the efficiency of cancer patient treatment and reduce toxic side effects, there is an urgent need in the clinic to develop new methods with small trauma, low toxic side effects and obvious treatment effects to improve the success rate of cancer patient treatment.

[0003] Photothermal therapy (PTT) as a new method of cancer treatment has great development potential in tumor treatment due to its minimally invasive, short recovery time and low incidence of complications. Its principle is based on the absorption of light energy by exogenous photothermal conversion materials under near-infrared light source irradiation to generate heat, which increases the temperature of the lesion site, leading to cell membrane damage. At the same time, high temperature also increases the damage of genetic material deoxyribonucleic acid and inhibits its repair ability, causing tissue necrosis, inducing cell apoptosis, and achieving the purpose of tumor treatment. In the field of tumor treatment, commonly used photothermal conversion materials mainly include: noble metal nanoparticles, carbon materials, metal and non-metal compounds, and organic dye substances. However, these photothermal materials have problems such as poor biocompatibility, physiological instability and poor targeting, which affect their further application.

[0004] In recent years, a new two-dimensional photothermal material, black phosphorus (BP), has been widely used in tumor photothermal therapy, photodynamic therapy, and anti-tumor drug delivery due to its excellent near-infrared photothermal effect (NIR: 808 nm), good biocompatibility and biodegradability, tunable direct band gap, and ultra-large specific area. Since phosphorus is an essential element for the human body, BP can degrade the phosphate (PO43-) necessary for the human body. Therefore, BP has an unparalleled advantage in tumor treatment compared to other inorganic two-dimensional photothermal materials. However, BP is sensitive to oxygen, water, and light in the surrounding environment, leading to easy oxidation and chemical degradation of its composition and physical properties, which affects its further application in tumor treatment. To solve the problem of easy oxidation and degradation of BP, the current strategy is to coat it with relatively stable polymers such as polydopamine (PDA), polylactic acid-glycolic acid (PLGA), polyethyleneimine (PEI), or liposomes, in-situ gels, and biomimetic cell membranes to improve its stability. Based on this, a nano-composite system of BP has been developed, which has certain clinical application potential in tumor treatment. Although these strategies for modifying BP can complement each other to improve the stability of BP while effectively improving the anti-tumor effect, the complex construction strategy of these composite nano-systems and the compatibility between nano-materials and BP pose challenges.

[0005] Biomineralization is the process of forming minerals at the interface between organic macromolecules and inorganic ions through interaction. Various inorganic minerals produced by biomineralization, including calcium phosphate, calcium carbonate, iron oxide, and silicon minerals, have been applied in the biomedical field. Compared to traditional composite nanomaterials, biomineralized materials have the advantages of simple preparation, easy modification, good biocompatibility, targeting, and multifunctionality. In addition, the degradation products of biomineralized materials are ubiquitous in the body, so they have low immunogenicity. Biomineralized materials have many excellent properties, so advanced functional materials with similar properties have been constructed in vitro by mimicking biomineralization reactions. Research results have shown that BP, as a phosphorus source donor and mineralization substrate, has achieved in-situ mineralization of BP by calcium ions, artificially synthesizing calcium phosphate-modified black phosphorus nanosheets (CaBPs). Compared to unmodified BP, BP mineralized by calcium ions not only retains the intrinsic properties of BP, but also has PH-responsive degradation, which can degrade in the acidic conditions of tumor cells and release a large amount of calcium ions, causing calcium ion levels in tumor cells to overload and trigger the death of tumor cells. Therefore, the strategy of biomineralizing new photothermal material BP with metal ions provides a new idea for the field of tumor treatment.

[0006] Ferroptosis is a new way of cell death discovered in recent years, which is caused by the accumulation of reactive oxygen species (ROS) on the membrane lipid under the induction of small molecules, leading to the failure of membrane lipid repair enzyme glutathione peroxidase (GPX4), and it has iron ion dependence. Studies have shown that the consumption of intracellular glutathione (GSH) can induce the down-regulation of GPX4 protein, causing ROS accumulation to induce ferroptosis. In addition, when the cell is iron overload and antioxidant capacity is insufficient, free iron ions can directly catalyze lipid peroxides through Fenton reaction to produce a large number of hydroxyl radicals, causing strong oxidative stress and a large amount of ROS, inducing ferroptosis. At present, the anti-tumor treatment based on ferroptosis mainly plays a role through chemotherapeutic drugs with potential ferroptosis mechanism or synergistic ferroptosis inducer (Erastin), but the toxic side effects of chemotherapeutic drugs and drug resistance after drug use are still the problems faced by ferroptosis anti-tumor treatment. SUMMARY

[0007] In view of the above problems in the prior art, the present application provides a novel anti-tumor two-dimensional photothermal material and a preparation method thereof. 3+ The BP is in-situ biomimetic mineralized to construct a black phosphorus nanosheet modified with an iron-phosphoric acid organic coordination layer (FeBPs). After entering the tumor cells, the FeBPs dissociate the mineralized layer under the synergistic action of GSH, Fe 3+ is reduced to Fe 2+ and consumes a large amount of intracellular GSH, down-regulates the GPX4 protein level, causes ROS accumulation, and induces tumor cell ferroptosis. At the same time, the BP has excellent photothermal performance after the dissociation of the mineralized layer, and under near-infrared light 808nm irradiation, the temperature of the tumor tissue is increased to cause tumor ablation, thereby realizing the synergistic treatment of photothermal / ferroptosis and providing a new treatment strategy for tumor treatment research.

[0008] In order to achieve the above purpose, the technical scheme of the present application is as follows:

[0009] A preparation method of a novel anti-tumor two-dimensional photothermal material, which uses liquid exfoliation technology to prepare BP nanosheets, then introduces Fe 3+ and prepares FeBPs through in-situ biomimetic mineralization method.

[0010] Further, the preparation method of the BP nanosheet is as follows,

[0011] 20mg of BP powder is dispersed in 20mL of N-methyl pyrrolidone (NMP) solution, the solution is ultrasonically treated in an ice bath at a power of 1080W for 10h, then the solution is ultrasonically treated at a power of 300W for 10h, a brown suspension is obtained, and the residual unexfoliated particles are removed by centrifugation at 9000rpm for 10min, and the BPs supernatant is collected for use.

[0012] Further, the in-situ biomimetic mineralization preparation method is as follows,

[0013] Take 10ml of BPs standby solution and 1M 0.1% (V / V) ammonia solution, mix and stir at 40℃ water bath temperature for 2h, then add 1ml of 90mM FeCl3 solution, mix and stir at 300rpm for 12h in the dark, centrifugal treatment, clean with anhydrous ethanol and ultrapure water, then prepare FeBPs solution, and store at 4℃.

[0014] The application also discloses the FeBPs prepared by the preparation method of the anti-tumor novel two-dimensional photothermal material.

[0015] Further, the FeBPs prepared by the method are applied to anti-tumor, FeBPs enter the mineralization layer of tumor cells and are dissociated, GPX4 protein is induced to be down-regulated, ROS is accumulated to induce tumor cell ferroptosis, and the photothermal performance of BPs after the mineralization layer is dissociated is utilized, so that the temperature of tumor tissues is increased under the irradiation of infrared light 808nm, and tumor ablation is caused.

[0016] The application has the beneficial effects that: Fe 3+ The in-situ biomimetic mineralization is performed on the BP, and the black phosphorus nanosheet-(FeBPs) modified with a surface iron-phosphoric acid organic coordination layer is constructed. 3+ Fe 2+ is reduced into Fe 3+ , a large amount of GSH in cells is consumed, GPX4 protein level is down-regulated, ROS is accumulated to induce tumor cell ferroptosis. Meanwhile, the excellent photothermal performance of BPs after the mineralization layer is dissociated is utilized, so that the temperature of tumor tissues is increased under the irradiation of near-infrared light 808nm, and tumor ablation is caused, thereby realizing the synergistic treatment of photothermal / ferroptosis, and providing a new treatment strategy for tumor treatment research. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is a preparation route diagram of the novel mineralization material FeBPs;

[0018] Figure 2 is application of FeBPs in anti-tumor in vivo. DETAILED DESCRIPTION

[0019] The application will be further described in detail below in combination with specific embodiments and drawings.

[0020] A preparation method of an anti-tumor novel two-dimensional photothermal material, which utilizes liquid exfoliation technology to prepare BP nanosheet, then introduces Fe 3+FeBPs were prepared by in-situ biomimetic mineralization method.

[0021] Specifically, the preparation method of BP nanosheets is as follows,

[0022] 20 mg of BP powder was dispersed in 20 mL of N-methyl pyrrolidone (NMP) solution, and the solution was ultrasonically treated in an ice bath at a power of 1080 W for 10 h, and then ultrasonically treated at a power of 300 W for 10 h, to obtain a brown suspension. The residual unpeeled particles were removed by centrifugation at 9000 rpm for 10 min, and the BPs supernatant was collected for standby use.

[0023] The in-situ biomimetic mineralization preparation method is as follows,

[0024] 10 ml of the standby BPs solution was mixed with 1M 0.1% (V / V) ammonia solution, and after stirring at a water bath temperature of 40℃ for 2 h, 1 ml of 90 mM FeCl3 solution was added and mixed, and after stirring in the dark at a speed of 300 rpm for 12 h, centrifugal treatment was performed, and after washing with anhydrous ethanol and ultrapure water, FeBPs solution was prepared and stored at 4℃.

[0025] Fe 3+ FeBPs were prepared by in-situ biomimetic mineralization of BP, and the surface of FeBPs was modified with an iron-phosphoric acid organic coordination layer. After entering tumor cells, FeBPs dissociated the mineralization layer under the synergistic action of GSH, and Fe 3+ was reduced to Fe 2+ and consumed a large amount of intracellular GSH, down-regulated the GPX4 protein level, and caused ROS accumulation to induce tumor cell ferroptosis. At the same time, after the dissociation of the mineralization layer, the excellent photothermal performance of BP was utilized, and under near-infrared light 808 nm irradiation, the temperature of the tumor tissue was increased to cause tumor ablation, thereby realizing the synergistic treatment of photothermal / ferroptosis.

[0026] Parameter performance research on FeBPs:

[0027] 2.1. Material characterization

[0028] BPs and FeBPs samples were dissolved in distilled water, and an appropriate amount was added dropwise on a common carbon-supported copper mesh, which was placed in a vacuum drying box. After the water was evaporated, X-ray energy dispersive spectrometer (EDS), high-resolution transmission electron microscopy (HR-TEM), and atomic force microscopy (AFM) were used for observation.

[0029] 2.1.3 Material stability test

[0030] 2.1.3.1 Stability test of materials in different media

[0031] FeBPs samples were added into phosphate buffer solution (PBS), normal saline and serum-containing medium respectively, and stored at 37°C for 24 hours in a closed condition. A proper amount of the samples was dropped on a copper mesh supported by ordinary carbon, and placed in a vacuum drying oven. After the water was evaporated, EDS, HR-TEM and AFM were used for observation.

[0032] 2.1.3.2 Material storage stability test at different times

[0033] FeBPs samples were dissolved in distilled water and stored at 37°C in a closed condition. At 7d, 14d, 21d and 28d, a proper amount of the samples was dropped on a copper mesh supported by ordinary carbon, and placed in a vacuum drying oven. After the water was evaporated, EDS, HR-TEM and AFM were used for observation.

[0034] 2.1.4 Degradation behavior of the material under different GSH concentrations

[0035] 0.1 mg of BPs and FeBPs were added into GSH solutions with low, medium and high (2 mmol / L, 5 mmol / L and 10 mmol / L) concentrations respectively, and stored at 37°C in a closed condition. The absorbance value at 808 nm was detected by a UV spectrophotometer. The absorbance of the material in GSH was detected every 24 hours. The absorbance values at 0 hour, 24 hours, 48 hours, 72 hours and 96 hours were recorded. The degradation rate of the material was calculated by the following formula:

[0036] Degradation rate (%) = (1 - Ix / I0) x 100%

[0037] (in the formula, I0 is the absorbance value of the material at 0 hour; Ix is the absorbance value of the material at 24 hours, 48 hours, 72 hours and 96 hours)

[0038] 2.1.5 Study on the photothermal performance of the material

[0039] 2.1.5.1 Study on the material under different power illumination parameters

[0040] FeBPs sample solution was added into GSH solution, and stored at 37°C in a closed condition according to the time length for degradation of the material in GSH. An 808 nm laser was used for irradiation at a power density of 1 W / cm2, 2 W / cm2, 3 W / cm2, 4 W / cm2 and 5 W / cm2, and the irradiation time was 10 min. The temperature data were recorded every 30 s, and plotted with time as the horizontal coordinate and temperature as the vertical coordinate, so as to observe the warming effect of FeBPs under different power densities.

[0041] 2.1.5.2 Study on the selection of the optimal concentration of the material

[0042] First, the concentration of 25 μg / mL, 50 μg / mL, 100 μg / mL FeBPs solution was prepared. Respectively, 1 ml of different concentrations of FeBPs solution was added to the GSH solution, and the material was placed at 37°C for a certain period of time according to the degradation time of GSH. Then the 808 nm laser was used to irradiate the FeBPs dispersion solution with the optimal power density obtained by the test (10 min). Every 30 s, the temperature data was recorded, and the temperature was plotted as a function of time, and the temperature rise effect of different concentrations of FeBPs was observed.

[0043] 2.1.6 Hemolysis test of materials

[0044] First, prepare pure red blood cells, take the mouse blood and add sodium heparin anticoagulant, place it in a centrifuge tube, centrifuge at 2000 rpm and wash with saline solution until the supernatant is colorless. Mix 1 mL of 4% red blood cells (v / v) with water (positive control group), saline (negative control group) and the optimal concentration of FeBPs material (sample group).

[0045] Incubate the mixture at 37°C for 3 hours. After rotating the red blood cells, collect the supernatant and use the enzyme marker to record the absorbance at 540 nm. Use the formula to calculate the hemolysis percentage:

[0046] Hemolysis rate (%) = (A sample - A negative) / (A positive - A negative) x 100%

[0047] (Formula: sample group, negative group, positive group are the absorbance of FeBPs material solution, saline, water at 540 nm)

[0048] 2.2 In vitro study

[0049] 2.2.1 Cytotoxicity experiment

[0050] The in vitro cell experiment was detected by CCK-8 method. Normal lung epithelial cells (Beas-2B) and A549 lung cancer cells were selected to analyze the toxicity of FeBPs material. Cells were seeded in 96-well plates at a cell density of 1*105 / well, and the same volume of culture medium was added to the blank group, then placed in a 37°C constant temperature incubator containing 5% carbon dioxide for 24 hours. FeBPs was used to intervene cells at concentration gradient of 0, 25, 50, 100 μg / mL for 24, 48, 72 hours, and 5 parallel control wells were set in each group; 100 μL of 10% CCK-8 containing medium was added to each well, and the constant temperature incubator was continued for 1-2 hours; the absorbance value of each well at 450 nm was detected by enzyme marker, and the IC50 value was calculated, and the optimal intervention time was determined. Each group of experiments was repeated three times. According to the following formula, the survival rate of each group of cells was calculated.

[0051] Cell survival rate (%) = (ODsample-ODblank) / (ODcontrol-ODblank) * 100

[0052] (In the formula ODsample, ODcontrol, ODblank represent the experimental group, control group, CCK-8 itself at 450 nm absorption value respectively)

[0053] 2.2.2 Cell uptake study

[0054] 2.2.2.1 Study on the uptake of materials by different cells

[0055] Beas-2B cells and A549 cells were also selected to analyze the uptake of labeled FeBPs. Beas-2B cells and A549 cells in the logarithmic growth phase were inoculated into confocal dishes, and after overnight culture, Cy5-labeled FeBPs were added for co-culture (FeBPs concentration and culture time were determined according to the cell toxicity test in 2.2.1), washed with PBS for 3 times, added with 1 mL 4% paraformaldehyde (PFA) for room temperature fixation for 20 minutes, washed with PBS for 3 times, added with 100 ng / mL 4, 6-diamidino-2-phenylindole (DAPI) staining, and observed under a confocal microscope for the uptake of materials by cells and the localization of materials in cells, with each experiment repeated three times.

[0056] 2.2.2.2 Quantitative study on the uptake of materials by cells

[0057] A549 cells in the logarithmic growth phase were selected and inoculated into 6-well plates, and placed into a 37°C constant temperature incubator containing 5% carbon dioxide for 24 hours, then the fresh culture medium containing Cy5-labeled FeBPs materials (FeBPs concentration was configured according to the cell toxicity test in 2.2.1) was replaced, and cultured for 2, 4, 6, 8, 10, 12 and 24 hours; at each time point, the cells were washed and resuspended by centrifugation, and finally quantitatively detected by flow cytometry.

[0058] 2.2.2.3 Cell co-localization test

[0059] A549 cells in the logarithmic growth phase were selected and inoculated into confocal dishes, and after overnight culture, FeBPs materials were added for co-culture (FeBPs concentration and culture time were determined according to the cell toxicity test in 2.2.1), washed with PBS for 3 times, added with 100 ng / mL DAPI and lysosome dye for staining, and observed under a laser confocal microscope for the situation of materials in lysosomes.

[0060] 2.2.3 Cell ferroptosis test

[0061] 2.2.3.1 Study on GPX4 protein level in cells

[0062] A549 cells in the logarithmic growth phase were seeded in a 6-well plate at a density of 2.5*105 / well, and blank, BPs material, and FeBPs material groups were set. The BPs material group was given near-infrared light; the FeBPs material group was given near-infrared light, ferrostatin-1, and near-infrared light

[0063] Ferrostatin-1 three treatments; the blank group was not treated. The material concentration was configured according to the cell toxicity test in 2.2.1. The A549 cells in the above different treatment modes were placed in a 37°C incubator containing 5% carbon dioxide for 24 hours. The total protein was extracted by lysing the cells with RIPA lysis buffer containing protease inhibitors, and the protein content was determined by BCA method. The extracted total protein was mixed with 5x protein loading buffer, denatured at 95°C, and then loaded, polyacrylamide gel electrophoresis, membrane transfer, 1% bovine serum albumin (BSA) blocking PVDF membrane, and then incubated with anti-GPX4 primary antibody at 4°C overnight, and then incubated with the corresponding HRP-conjugated secondary antibody at room temperature. After incubation, the blot signal was detected by ECLPLUS reagent and Hyperfilm film (Amersham). Image J software was used for quantitative analysis.

[0064] 2.2.3.2 Study of intracellular ROS level

[0065] A549 cells in the logarithmic growth phase were seeded in a 6-well plate and placed in a 37°C incubator containing 5% carbon dioxide for 24 hours. Fresh culture medium containing FeBPs material (concentration configured according to the cell toxicity test in 2.2.1) was added, and the cells were cultured for another 12 hours. After PBS washing for 3 times, trypsin digestion was added, and the cells were transferred to an EP tube and centrifuged (300g, 5min) to collect the cells. After PBS washing for 2 times, 500μL of prepared 5μM H2DCFDA solution was added to resuspend the cells, and the cells were incubated in a 37°C incubator for 30min. After incubation, the cell suspension was centrifuged and the cells were washed twice, and finally the cells were resuspended with PBS. The cells were detected by flow cytometry, and each experiment was repeated three times.

[0066] 2.2.3 Study of intracellular photothermal

[0067] A549 cells in logarithmic growth phase were inoculated into 12-well plates and placed in a 37°C incubator with 5% CO2 for 24 hours. Fresh culture medium containing FeBPs (concentration configured according to the cell toxicity test in 2.2.1) was added, and the cells were cultured for another 12 hours. The cells were washed with PBS for 3 times, and then trypsinized and transferred into EP tubes for centrifugation (300g, 5 min). The cells in the bottom of the EP tubes were irradiated by an 808 nm laser (optimal power and irradiation parameters obtained from the test in 2.1.5.1), and the temperature data were recorded every 30 seconds. The temperature was plotted against time, and the photothermal effect of the material in the cells was observed. Each experiment was repeated three times.

[0068] 2.2.4 Study on synergistic killing of tumor cells in cells

[0069] Beas-2B and A549 cells in logarithmic growth phase were inoculated into 6-well plates at a concentration of 2.5*105 cells per well. Blank, FeBPs (1), and FeBPs (2) groups were set up, and the cells were placed in a 37°C incubator with 5% CO2 for 24 hours. Fresh culture medium containing FeBPs (concentration configured according to the cell toxicity test in 2.2.1) was added to each well, and the blank group was added with the same volume of culture medium. The cells were cultured for another 12 hours. The cells were washed with PBS for 3 times, and then the blank and FeBPs (1) groups were irradiated by an 808 nm laser (optimal power and irradiation parameters obtained from the test in 2.1.5.1) for 5 minutes. Note that the FeBPs (2) group was not irradiated and was cultured in the incubator for another 24 hours. The cells were trypsinized and transferred into EP tubes for centrifugation (300g, 5 min). The precipitate was collected and resuspended in 500 μL buffer. Annexin V-FITC and PI reagents were added, and the mixture was incubated on ice for 15 minutes. After incubation, 100 μL of the cell suspension was taken into a flow tube for detection of cell apoptosis in each group. Each experiment was repeated three times.

[0070] 2.3 In vivo study

[0071] 2.3.1 Construction of a transplanted tumor lung cancer model

[0072] SPF Balb / c nude mice, a total of 36 (half male and half female, 5-6 weeks old, 15-20 g), were raised in a SPF environment for 3-5 days. A549 cells in logarithmic growth phase were prepared into a cell suspension of 1*106 cells per 100 μL with pre-cooled PBS, and 100 μL of the cell suspension was inoculated into the back of each nude mouse to establish a transplanted tumor model. The tumor volume was calculated according to the following formula:

[0073] Tumor volume (mm3) = 1 / 2 x length x width2

[0074] 2.3.2 Grouping and intervention treatment

[0075] When the tumor volume reached about 100 mm3, the transplanted tumor lung cancer model was divided into 6 groups (n = 6 for each group): (1) saline, (2) NIR, (3) BPs, (4) Cy5-labeled FeBPs, (5) BPs + NIR, (6) Cy5-labeled FeBPs + NIR. Group (1) and group (2) of nude mice were injected with normal saline via the tail vein. For groups (3) to (6), the nude mice were injected with BPs and FeBPs (concentrations obtained from pre-experiments on nude mice) via the tail vein. The survival of the mice was recorded every day after treatment, and the tumor volume and body weight of the nude mice were recorded every other day to produce a tumor volume growth inhibition curve. After 14 days, the nude mice were sacrificed and the major organs (heart, liver, spleen, lung, kidney and tumor mass) were removed

[0076] 2.3.3 In vivo distribution of materials and time of maximum accumulation study

[0077] After treatment according to the grouping of 2.3.2, the in vivo distribution of FeBPs materials and the in vivo fluorescence intensity of groups (4) and (6) in the lung cancer model were recorded every day using a small animal live imaging instrument. The time point of the strongest fluorescence signal (maximum accumulation of in vivo materials) was found by plotting a curve, which was used as the light irradiation time point after administration in the in vivo anti-tumor activity test.

[0078] 2.3.4 Analysis of blood biochemical indicators in vivo

[0079] After treatment according to the grouping of 2.3.2, the nude mice in each group were anesthetized before being sacrificed 14 days later. The blood was then taken from the eyeball and centrifuged to obtain the supernatant. The blood biochemical indicators in the serum of the nude mice were analyzed using an automatic biochemical analyzer.

[0080] 2.3.5 Study of GPX4 protein levels in vivo

[0081] The tumor mass after different interventions was placed in a homogenizer tube and homogenized in a homogenizer. After homogenization, the liquid in the homogenizer tube was transferred to a low-temperature centrifuge and the supernatant was discarded. Then 500 μL of RIPA lysis buffer containing protease inhibitors was added and incubated on ice for 1 h. After lysis, the supernatant was obtained by high-speed centrifugation at low temperature, and the protein content was determined by BCA method.

[0082] The protein was mixed with 5x protein loading buffer, denatured at 95°C, and then loaded. Polyacrylamide gel electrophoresis was performed, the membrane was transferred, the PVDF membrane was blocked with 1% bovine serum albumin (BSA), and then incubated with anti-GPX4 primary antibody at 4°C overnight. Then, the corresponding secondary antibody conjugated with HRP was incubated at room temperature. After incubation, the blot signal was detected using -ECL PLUS reagent and Hyperfilm film (Amersham). Image J software was used for quantitative analysis.

[0083] 2.3.6 In vivo photothermal effect study

[0084] According to the grouping treatment of 2.3.2, the laser irradiation group (group (2), (5), (6)), wherein group (5), (6) is injected with material, according to the optimal light irradiation time point obtained in 2.3.3, is irradiated with 808 nm laser (taking the optimal power light irradiation parameter obtained in 2.1.5.1 experiment), and the temperature of the tumor site of each group of mice is recorded by the thermal imager during irradiation.

[0085] 2.3.7 In vivo tissue section study

[0086] According to the intervention treatment of 2.3.2, the nude mice are sacrificed after 14 days, and tissue sections of the main tissues (heart, liver, spleen, lung, kidney and tumor mass) are prepared, and the anti-tumor effect of FeBPs in vivo is further verified by TUNEL staining, H&E staining and immunohistochemistry.

[0087] After the above application research of FeBPs in anti-tumor proves that FeBPs enter tumor cells and cause the dissociation of the mineralization layer under the synergistic action of GSH, Fe 3+ is reduced to Fe 2+ and consumes a large amount of intracellular GSH, down-regulates the GPX4 protein level, causes ROS accumulation to induce tumor cell ferroptosis, and at the same time, the photothermal performance of BP after the dissociation of the mineralization layer is utilized to cause the temperature of the tumor tissue to rise under the irradiation of infrared light 808 nm, resulting in tumor ablation. Each research has good effect.

[0088] The technical solutions provided by the embodiments of the present application are described in detail above, and specific examples are applied in this paper to describe the principles and implementation modes of the embodiments of the present application. The above description of the embodiments is only applicable to help understand the principles of the embodiments of the present application; at the same time, for those skilled in the art, according to the embodiments of the present application, the specific implementation mode and application range will be changed, and the above description of the embodiments should not be understood as a limitation of the present application.

Claims

1. A preparation method of an anti-tumor novel two-dimensional photothermal material, characterized by comprising the following steps: After the BP nanosheets were prepared by liquid exfoliation technique, Fe was introduced 3+ and FeBPs were prepared by in-situ biomimetic mineralization method. ​ 2. The preparation method of the anti-tumor novel two-dimensional photothermal material according to claim 1, characterized in that: The preparation method of the BP nanosheet is as follows, 20 mg of BP powder was dispersed in 20 mL of N-methyl pyrrolidone (NMP) solution, and the solution was ultrasonically treated in an ice bath at a power of 1080 W for 10 h, and then ultrasonically treated at a power of 300 W for 10 h, to obtain a brown suspension. The residual unpeeled particles were removed by centrifugation at 9000 rpm for 10 min, and the BPs supernatant was collected for standby use.

3. The preparation method of the anti-tumor novel two-dimensional photothermal material according to claim 2, characterized in that: The in-situ biomimetic mineralization preparation method is as follows, 10 ml of the standby BPs solution was mixed with 1M 0.1% (V / V) ammonia solution, and stirred at a water bath temperature of 40°C for 2 h. Then, 1 ml of 90 mM FeCl3 solution was added, and the mixture was stirred at a speed of 300 rpm for 12 h in the dark. After centrifugal treatment, the FeBPs solution was prepared by washing with anhydrous ethanol and ultrapure water, and was stored at 4°C.

4. A novel two-dimensional photothermal material against tumors, characterized by: The FeBPs prepared according to the preparation method of the novel two-dimensional photothermal material for resisting tumors according to any one of claims 1-3.

5. The use of a novel two-dimensional photothermal material against tumors, characterized by: The FeBPs prepared according to the method of any one of claims 1-3 are used in resisting tumors. The FeBPs enter the mineralization layer of tumor cells and are dissociated, inducing down-regulation of GPX4 protein and accumulation of ROS, which induces ferroptosis of tumor cells. Meanwhile, the photothermal performance of the BP after dissociation of the mineralization layer is utilized, and under infrared light 808 nm irradiation, the temperature of the tumor tissue is increased, resulting in tumor ablation.

Images