Nanomedicine for inducing ferroptosis, preparation method and application thereof

Abstract

The present invention provides a nano drug for inducing ferroptosis, which is a nanoparticle formed by coordination self-assembly of an iron salt and a polyphenol drug, and a preparation method thereof comprises: dripping an organic solution containing an iron salt and a polyphenol drug into a liquid culture medium or a phosphate buffer solution to obtain a mixed solution; stirring the mixed solution to allow the iron salt and the polyphenol drug to self-assemble to obtain a nano drug. The nano drug provided by the present invention has a small size, good water solubility, and can be enriched in the tumor area, and can be used to prepare a drug for inducing ferroptosis of tumor cells. The present invention also provides a preparation method and application of the above-mentioned nano drug for inducing ferroptosis.

Description

Technical Field

[0001] The present invention relates to the field of nanomedicine, and in particular to a nanomedicine for inducing ferroptosis, a preparation method and application thereof. Background Art

[0002] Ferroptosis is a non-apoptotic cell death with special characteristics, which is different from traditional apoptosis, necrosis or autophagy. Ferroptosis is characterized by the iron-dependent accumulation of lipid peroxidation reaching lethal levels. It is a cell death mode regulated by cellular metabolism such as lipid, iron and amino acid metabolism. The main mechanism of ferroptosis is that under the action of divalent iron or esteroxygenase, unsaturated fatty acids on the cell membrane are catalyzed to cause lipid peroxidation, thereby inducing cell death; in addition, it is also manifested as a decrease in the activity of glutathione peroxidase 4 (GPX4) in the antioxidant system.

[0003] Inducing ferroptosis in tumor cells is an emerging treatment method. Inducing ferroptosis in tumors can effectively inhibit tumor growth and increase the sensitivity of chemotherapy-resistant cells. At the same time, when tumor cells undergo ferroptosis, they release pro-inflammatory factors and trigger inflammatory responses, thereby changing the living environment of the tumor and reducing the immune susceptibility of tumor cells. However, the induction of ferroptosis in tumors is limited by the antioxidant system in tumor cells, free iron ions in cells, and the content of polyunsaturated fatty acids.

[0004] At present, the relevant technologies are mainly based on iron nanoparticles, which catalyze the decomposition of hydrogen peroxide in the tumor microenvironment to produce toxic hydroxyl radicals, which then oxidize polyunsaturated fatty acids, induce lipid peroxidation in cells, and ultimately cause ferroptosis in tumor cells. However, the synthesis steps of iron-based nanoparticles are relatively complicated, and the catalytic activity of iron nanoparticles only comes from part of the iron ions released in the acidic environment, and the atomic utilization rate is low; the content of polyunsaturated fatty acids in the tumor is low, and the overexpressed glutathione inhibits the lipid peroxidation process. The defects in the relevant technologies make the efficiency of iron-based nanomedicines in inducing ferroptosis in tumor cells low. Summary of the invention

[0005] In view of this, the main purpose of the present invention is to provide a nanomedicine for inducing ferroptosis, a preparation method and application thereof, so as to at least partially solve the above technical problems.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] As a first aspect of the present invention, a nano drug for inducing ferroptosis is provided, which is a nanoparticle formed by coordination self-assembly of an iron salt and a polyphenol drug.

[0008] According to an embodiment of the present invention, the iron salt includes any one of the following: ferric chloride, ferric sulfate, ferric nitrate; the polyphenol drugs include any one of the following: rhein, dihydromyricetin, baicalein, mitoxantrone dihydrochloride.

[0009] According to an embodiment of the present invention, in the coordination self-assembly, the concentration of the iron salt is 1-10 mM; the concentration of the polyphenol drug is 4-40 mM.

[0010] According to an embodiment of the present invention, the coordination self-assembly is carried out in an aqueous solution environment with a pH of 7-9.

[0011] According to an embodiment of the present invention, the average particle size of the nano drug is 1 to 5 nm.

[0012] As a second aspect of the present invention, a method for preparing the above-mentioned nanomedicine is provided, comprising: dropping an organic solution containing iron salts and polyphenol drugs into a liquid culture medium or a phosphate buffer solution to obtain a mixed solution; stirring the mixed solution to allow the iron salts and polyphenol drugs to self-assemble to obtain the nanomedicine.

[0013] According to an embodiment of the present invention, the pH of the liquid culture medium or the phosphate buffer solution is 7-9.

[0014] According to an embodiment of the present invention, the concentration of the iron salt in the mixed solution is 1-10 mM; the concentration of the polyphenol drug in the mixed solution is 4-40 mM.

[0015] According to an embodiment of the present invention, the solvent of the organic solution includes any one of the following: dimethyl sulfoxide, N,N-dimethylformamide.

[0016] As a third aspect of the present invention, there is provided an application of the above-mentioned nanomedicine in the preparation of a drug for inducing ferroptosis of tumor cells, especially in the preparation of a drug for photothermal enhancement-induced ferroptosis of tumor cells.

[0017] Based on the above technical solution, the nanomedicine for inducing ferroptosis provided by the present invention has at least one or part of the following beneficial effects:

[0018] (1) According to the embodiments of the present invention, polyphenol drugs contain more phenolic hydroxyl groups in their structure, which are weakly acidic. They can be coordinated by free iron ions in iron salts and polyhydroxyl groups of polyphenol drugs, and then self-assembled to form nano-scale particles, which do not exist in the form of free salts, and are not easy to fail by chelating with transferrin during targeted tumor cell delivery. In addition, nano drugs improve the dispersibility and water solubility of polyphenol drugs, and are small in size. Due to the high permeability and retention effect (EPR) of solid tumors, they are conducive to enrichment in the tumor area, thereby enhancing the induction of ferroptosis.

[0019] (2) According to the embodiments of the present invention, the polyphenol drugs in the nanomedicine can promote the increase of polyunsaturated fatty acid content in cells. At the same time, iron ions can catalyze the Fenton reaction of overexpressed hydrogen peroxide in the tumor under the environment of tumor cells, produce highly toxic hydroxyl radicals, further oxidize polyunsaturated fatty acids, promote lipid peroxidation, and induce ferroptosis in tumor cells. Therefore, the polyphenol drugs and iron salts synergistically enhance the ferroptosis effect after coordination self-assembly.

[0020] (3) According to an embodiment of the present invention, the nanoparticles formed by the coordination self-assembly of polyphenol drugs and iron salts have a photothermal effect within the wavelength range of 730-808nm, and the wavelength of 730-808nm has good tissue penetration ability, so that after the nanomedicine reaches the tumor cells, the efficiency of the nanomedicine in inducing ferroptosis of tumor cells is enhanced by converting light into heat. In addition, iron ions can also consume glutathione in cells, reducing the inhibitory effect of overexpressed glutathione in tumor cells on lipid peroxidation, thereby improving the efficiency of inducing ferroptosis of tumor cells.

[0021] (4) According to the embodiments of the present invention, the nanomedicine used to induce ferroptosis does not require other carriers, and its components are clinically approved materials, which reduces the possibility of immune rejection by the body and has high biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a transmission electron microscopy image of the nanomedicine used to induce ferroptosis in Example 1 of the present invention;

[0023] Figure 2 This is a hydrated particle size distribution diagram of the nano drug used to induce ferroptosis in Example 1 of the present invention;

[0024] Figure 3 This is an X-ray photoelectron spectrum of the nanomedicine used to induce ferroptosis in Example 1 of the present invention;

[0025] Figure 4 This is a UV-visible absorption spectrum of the nanomedicine used to induce ferroptosis in Example 1 of the present invention;

[0026] Figure 5 This is a test graph of absorbance of the nanomedicine used to induce ferroptosis in Example 2 of the present invention catalyzing hydrogen peroxide to oxidize 3,3',5,5'-tetramethylbenzidine under different pH conditions;

[0027] Figure 6 This is an electron paramagnetic resonance analysis diagram of the nanomedicine used to induce ferroptosis in Example 2 of the present invention catalyzing hydrogen peroxide to produce hydroxyl radicals;

[0028] Figure 7is a curve diagram of glutathione consumption by the nanomedicine used to induce ferroptosis in Example 3 of the present invention;

[0029] Figure 8 This is a temperature rise line graph of the nanomedicine used to induce ferroptosis under near-infrared light irradiation in Example 4 of the present invention;

[0030] Fig. 9 This is a diagram showing the catalytic effect of the nanomedicine used to induce ferroptosis under near-infrared light irradiation in Example 4 of the present invention;

[0031] Fig.10 This is a graph showing the results of inductively coupled plasma mass spectrometry measurement of iron ion uptake by tumor cells in Example 5 of the present invention;

[0032] Fig.11 This is a fluorescence spectrum of the iron ion level in tumor cells in Example 5 of the present invention;

[0033] Fig.12 This is a fluorescence spectrum of the reactive oxygen level in tumor cells in Example 5 of the present invention;

[0034] Fig.13 is a bar graph of the survival rate of U87 tumor cells under different conditions in Example 6 of the present invention;

[0035] Fig.14 It is a bar graph of the survival rate of U87 tumor cells under light conditions of Fe@MT in Example 6 of the present invention;

[0036] Fig.15 This is a fluorescence image of lipid peroxidation levels in U87 tumor cells in Example 7 of the present invention;

[0037] Fig.16 This is a fluorescence image of the expression level of SLC47A1 in U87 tumor cells after different treatments in Example 7 of the present invention;

[0038] Fig.17 This is a fluorescence image of the expression level of GPX4 protein in U87 tumor cells after different treatments in Example 7 of the present invention;

[0039] Fig.18 This is a UV-visible absorption spectrum of hemoglobin detected in the hemolysis test in Example 8 of the present invention;

[0040] Fig.19 is a hemolysis ratio diagram of the hemolysis test in Example 8 of the present invention;

[0041] Fig. 20 This is a thermal imaging comparison of the tumor sites of mice in the control group and the experimental group in Example 9 of the present invention;

[0042] Fig.21This is a graph showing the change of the tumor growth curve over time in mice after treatment in Example 9 of the present invention. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be noted in advance that, unless otherwise defined, the technical terms used in the following embodiments have the same meanings as those generally understood by those skilled in the art to which the present invention belongs.

[0044] Ferroptosis mainly occurs through the Fenton reaction of intracellular iron ions to produce reactive oxygen species that oxidize polyunsaturated fatty acids, inducing lipid peroxidation in cells, leading to cell membrane destruction and organelle damage, and ultimately cell death. However, the weak catalytic performance of iron ions in tumors, the low concentration of free iron ions and unsaturated fatty acids in tumor cells greatly limit the therapeutic efficacy of ferroptosis. In addition, the antioxidant system in tumor cells further limits the development and use of ferroptosis.

[0045] In the process of realizing the present invention, it was found that the particle size of inorganic nanomaterials, their metabolic distribution in the body, and their immune stimulation are the key issues that researchers are concerned about. Therefore, it is necessary to develop a nanomedicine that has good biocompatibility, is suitable for the tumor microenvironment, and can efficiently induce catalytic overexpression of hydrogen peroxide, consume glutathione, and upregulate intracellular polyunsaturated fatty acids, so as to improve the therapeutic effect of tumor cell ferroptosis.

[0046] According to an embodiment of the first aspect of the present invention, there is provided a nanomedicine for inducing ferroptosis, which is a nanoparticle formed by coordination self-assembly of an iron salt and a polyphenol drug.

[0047] According to an embodiment of the present invention, polyphenol drugs have multiple phenolic hydroxyl groups, which are weakly acidic and can form oxygen anions, and then coordinate self-assembly with free iron ions in iron salts, and chelate to form nano-scale particles. Polyphenol drugs can increase the content of polyunsaturated fatty acids in cells, iron ions can consume glutathione in cells, and nanoparticles formed by iron ions and polyphenol drugs can catalyze the overexpression of hydrogen peroxide to generate hydroxyl radicals in the slightly acidic environment of tumors, which can effectively oxidize polyunsaturated fatty acids, promote lipid peroxidation in cells, and increase the level of active oxygen in cells. Since nanoparticles have good photothermal conversion performance, this effect can be further enhanced under laser irradiation, and finally efficiently induce ferroptosis in tumor cells.

[0048] According to an embodiment of the present invention, the iron salt includes any one of the following: ferric chloride, ferric sulfate, ferric nitrate; the polyphenol drugs include any one of the following: rhein, dihydromyricetin, baicalein, mitoxantrone dihydrochloride.

[0049] According to an embodiment of the present invention, the iron salt has free iron ions, and the polyphenol drugs have multiple phenolic hydroxyl groups. The empty electron pairs on the phenolic hydroxyl groups can chelate with the empty orbitals of the iron ions to form nano-scale particles by coordination self-assembly. Among them, the endogenous metal iron ions used in the present invention and the polyphenol drugs approved for clinical use are coordinated and self-assembled, and the formed nanoparticles have good stability and good biocompatibility, which is convenient for clinical transformation.

[0050] According to an embodiment of the present invention, in the coordination self-assembly, the concentration of the iron salt is 1 to 10 mM, for example, 1 mM, 3 mM, 5 mM, 7 mM, 9 mM, etc.; the concentration of the polyphenols is 4 to 40 mM. For example, it can be 5 mM, 10 mM, 15 mM, 25 mM, 45 mM, etc. However, it is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable. The ratio of iron salt to polyphenols can be adjusted according to the above concentration according to actual conditions. If the iron salt concentration is too high, the iron ion concentration will increase, which will induce the polyphenols to form aggregates and form larger particles, which is not conducive to diffusion in cells; if the polyphenol concentration is too high, due to the limited coordination of iron ions, the utilization rate of the polyphenols will decrease, affecting the effect.

[0051] According to an embodiment of the present invention, the coordination self-assembly is carried out in an aqueous solution environment with a pH of 7 to 9. Iron ions and polyphenol drugs undergo coordination self-assembly to form nanoparticles under a neutral pH of 7 to 9. For example, the pH may be 7, 7.4, 7.9, 8.2, 8.6, etc., and may be appropriately adjusted within this range. When the pH is too low, the coordination self-assembly effect may be affected. When the pH is too high, the iron salt may form iron hydroxide, inhibiting the formation of nanomedicines.

[0052] According to an embodiment of the present invention, the average particle size of the nano drug is 1 to 5 nm, for example, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, etc., but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable. The particle size of the nano drug is small, which is convenient for enrichment in the tumor area, thereby inducing ferroptosis of tumor cells.

[0053] According to an embodiment of the second aspect of the present invention, a method for preparing the above-mentioned nanomedicine is provided, comprising: dropping an organic solution containing iron salts and polyphenol drugs into a liquid culture medium or a phosphate buffer solution to obtain a mixed solution; stirring the mixed solution to allow the iron salts and polyphenol drugs to self-assemble to obtain the nanomedicine.

[0054] According to an embodiment of the present invention, the free iron ions of the iron salt in the organic solution chelate with the phenolic hydroxyl groups of the polyphenol drugs to form a complex, and the formed complex is slowly added dropwise to a liquid culture medium or a phosphate buffer solution in the form of an aqueous solution, and since the iron ion end is hydrophilic and the polyphenol drug end is hydrophobic, the coordination self-assembly is performed to obtain a nano drug. The nano drug obtained by the coordination self-assembly of the iron salt and the polyphenol drugs has a uniform particle size, good dispersibility, stable catalytic performance, and good biocompatibility.

[0055] According to an embodiment of the present invention, the pH of the liquid culture medium or the phosphate buffer solution is 7 to 9. For example, it can be a liquid culture medium with a pH of 7, a phosphate buffer solution with a pH of 7.5, a liquid culture medium with a pH of 8, a phosphate buffer solution with a pH of 8.5, etc., and its pH can be appropriately adjusted within this range, but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0056] According to an embodiment of the present invention, the concentration of the iron salt in the mixed solution is 1 to 10 mM, for example, 1 mM, 3 mM, 5 mM, 7 mM, 9 mM, etc., but is not limited to the listed values, and other values ​​not listed in the numerical range are also applicable. The concentration of the iron salt within this range can avoid excessive iron ions, which will cause hydrolysis and change the pH of the solution. If the iron ion concentration is too low, it will lead to incomplete coordination with the polyphenol drug, resulting in a low amount of nano-drug production, affecting the therapeutic effect.

[0057] According to an embodiment of the present invention, the concentration of the polyphenol drugs in the mixed solution is 4 to 40 mM, for example, 5 mM, 10 mM, 15 mM, 25 mM, 45 mM, etc. However, it is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable. Within the concentration range, efficient coordination self-assembly of iron ions and polyphenol drugs can be achieved, and at the same time, too much free drug will not be wasted due to excessive content of polyphenol drugs.

[0058] According to an embodiment of the present invention, the solvent of the organic solution includes any one of the following: dimethyl sulfoxide, N,N-dimethylformamide. Polyphenol drugs are difficult to dissolve in water, but can be dissolved in organic solvents. At the same time, iron salts can also be dissolved in the above organic solvents, which is conducive to the chelation of iron ions with polyphenol drugs to form a complex. The complex also has good solubility in organic solvents, which can effectively avoid agglomeration and cause the particle size to be too large, thereby affecting the enrichment of drugs in the tumor area.

[0059] According to an embodiment of the third aspect of the present invention, there is provided an application of the above-mentioned nanomedicine in the preparation of a drug for inducing ferroptosis of tumor cells, especially in the preparation of a drug for photothermal enhancement to induce ferroptosis of tumor cells. According to an embodiment of the present invention, the metabolic level of tumor cells inside the tumor is high, which will lead to acidification of the tumor microenvironment and a significant increase in the levels of hydrogen peroxide and the reducing substance glutathione in the tumor microenvironment. The nanomedicine provided by the present invention has the function of catalyzing the production of reactive oxygen and consuming glutathione in the tumor microenvironment, which can increase the level of polyunsaturated fatty acids in the cell, and the reactive oxygen produced, the consumed glutathione and the increased level of polyunsaturated fatty acids have the effect of promoting the process of lipid peroxidation and inducing ferroptosis of tumor cells. And after the above-mentioned nanomedicine is enriched in the tumor area, light can be converted into heat by laser irradiation, thereby further enhancing the catalytic therapeutic effect.

[0060] According to an embodiment of the present invention, the above-mentioned photothermal enhancement-induced tumor ferroptosis uses laser irradiation with a wavelength of 730 to 808 nm, which has good tissue penetration within this range and can effectively promote tumor cell ferroptosis.

[0061] The following are several specific embodiments to illustrate the technical solution of the present invention in detail. It should be noted that the following specific embodiments are only used as examples, and the protection scope of the present disclosure is not limited thereto.

[0062] Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial sources. Unless otherwise specified, the materials, reagents, instruments, etc. used in the examples are all conventional methods and can be carried out according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0063] Example 1

[0064] 16.2 mg of ferric chloride was weighed and dissolved in 1 mL (100 mM) of dimethyl sulfoxide to obtain a ferric chloride solution; 51.7 mg of mitoxantrone dihydrochloride (MT) was weighed and dissolved in 1 mL (100 mM) of dimethyl sulfoxide to obtain a mitoxantrone dihydrochloride solution. 10 μL of ferric chloride solution and 40 μL of mitoxantrone dihydrochloride solution were respectively added to 950 mL of dimethyl sulfoxide, ultrasonicated for 10 minutes, slowly added dropwise to the liquid culture medium, and stirred for 2 hours. Ultrafiltration was performed 3 times using an ultrafiltration tube with a molecular weight cutoff of MWCO = 10 kDa to remove free ferric chloride and mitoxantrone dihydrochloride, and the product was finally concentrated and collected to obtain a nanomedicine for inducing ferroptosis, labeled as Fe@MT.

[0065] Figure 1 is a transmission electron micrograph of the nanomedicine used to induce ferroptosis in Example 1 of the present invention, Figure 1It can be seen that the particle size of the nano drug Fe@MT prepared by the method provided by the present invention is about 2 nm, and the metal trivalent iron ions and the polyphenol structure of mitoxantrone dihydrochloride coordinate self-assemble to form carrier-free nanoparticles, indicating that the nano drug was successfully prepared by the coordination self-assembly of iron ions and polyphenol drugs.

[0066] The hydrated particle size of Fe@MT was tested and the results are as follows Figure 2 As shown, Figure 2 This is the hydrated particle size distribution diagram of the nanomedicine used to induce ferroptosis in Example 1 of the present invention. It can be seen that Fe@MT has a narrow size distribution, and the coordination self-assembly effect of iron salts and polyphenol drugs effectively improves the water solubility of polyphenol drugs. Compared with free polyphenol drugs, Fe@MT is more easily enriched in tumor tissues.

[0067] The valence state of iron in Fe@MT was analyzed by X-ray photoelectron spectroscopy. Figure 3 is the X-ray photoelectron spectrum of the nanomedicine used to induce ferroptosis in Example 1 of the present invention, such as Figure 3 As shown, there are two characteristic peaks in the figure, namely Fe 3+ 2p1 / 2 (724.5 eV) and 2p2 / 3 (711.0 eV), indicating that Fe 3+ Successfully carried out coordination self-assembly with polyphenol drugs.

[0068] Figure 4 is the UV-visible absorption spectrum of the nano drug used to induce ferroptosis in Example 1 of the present invention, Figure 4 It can be seen that compared with mitoxantrone dihydrochloride (MT), the ultraviolet absorption peak of Fe@MT has undergone a red shift, further proving that iron ions and mitoxantrone dihydrochloride have completed coordination self-assembly. In addition, it can be seen that Fe@MT has strong ultraviolet absorption at about 808nm, which provides Fe@MT with photothermal conversion performance.

[0069] Example 2

[0070] The performance of Fe@MT prepared in Example 1 of the present invention in catalyzing the decomposition of hydrogen peroxide was studied:

[0071] Under the condition of adding 100μM hydrogen peroxide, 100μg / mL Fe@MT was dispersed in buffer solutions containing 3,3',5,5'-tetramethylbenzidine at different pH values ​​(7.4, 6.0 and 5.0) to simulate the physiological environment of normal tissue and tumor tissue respectively, and the effect of Fe@MT under neutral (simulating the physiological environment of normal tissue) and slightly acidic (simulating the physiological environment of tumor tissue) conditions was analyzed. The ability of Fe@MT to catalyze hydrogen peroxide to produce hydroxyl radicals was analyzed by measuring the absorbance at 652nm using a UV-visible spectrophotometer. Figure 5 is the absorbance of the nanomedicine for inducing ferroptosis in Example 2 of the present invention catalyzing hydrogen peroxide to oxidize 3,3',5,5'-tetramethylbenzidine under different pH conditions, such as Figure 5 As shown, Fe@MT is more efficient in catalyzing the decomposition of hydrogen peroxide under acidic conditions. It can be seen that when hydrogen peroxide is present under acidic conditions, Fe@MT can catalyze the decomposition of hydrogen peroxide to produce hydroxyl radicals, which can further oxidize polyunsaturated fatty acids.

[0072] At the same time, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was used as a spin trapping agent, and the efficiency of Fe@MT prepared in Example 1 and hydrogen peroxide in generating hydroxyl radicals was determined by electron spin resonance (ESR) spectroscopy. The blank control group was set to be treated with hydrogen peroxide and trapping agent (DMPO) alone, and the hydroxyl radicals generated by the reaction of Fe@MT and hydrogen peroxide were further analyzed by electron paramagnetic resonance.

[0073] Figure 6 This is an electron paramagnetic resonance analysis diagram of the nanomedicine used to induce ferroptosis in Example 2 of the present invention catalyzing hydrogen peroxide to produce hydroxyl radicals, such as Figure 6 As shown, the characteristic absorption peaks of hydroxyl radicals (1:2:2:1) were detected using electron paramagnetic resonance. The results showed that Fe@MT can catalyze the decomposition of hydrogen peroxide in the slightly acidic environment of the tumor to produce highly active hydroxyl radicals to deoxidize polyunsaturated fatty acids, promote lipid peroxidation, and ultimately induce tumor ferroptosis.

[0074] Example 3

[0075] The glutathione consumption performance of Fe@MT prepared in Example 1 of the present invention was studied:

[0076] The antioxidant system in the tumor (overexpressed glutathione) inhibits the lipid peroxidation process and significantly reduces the effect of ferroptosis treatment. Therefore, based on the principle that 5,5-dithio-bis-(2-nitrobenzoic acid) can react with glutathione to generate a yellow product with a characteristic absorption peak at 412nm, 1mM glutathione and 100μg / mL Fe@MT were added to phosphate buffer solution and incubated at 37°C. 5,5-dithio-bis-(2-nitrobenzoic acid) was used as a probe to test the ability of Fe@MT to consume glutathione. The group treated with glutathione alone and 5,5-dithio-bis-(2-nitrobenzoic acid) was set as the blank control group.

[0077] The absorbance of the product at 412 nm at different times was measured using a UV-visible spectrophotometer to analyze the reaction rate of Fe@MT consuming glutathione. Figure 7 is a curve diagram of glutathione consumption by the nanomedicine used to induce ferroptosis in Example 3 of the present invention, Figure 7 It can be seen that after Fe@MT treatment, the absorption at 412 nm dropped rapidly within 2 h, proving that Fe@MT can effectively consume glutathione.

[0078] Example 4

[0079] The photothermal conversion performance and catalytic decomposition performance of hydrogen peroxide of Fe@MT prepared in Example 1 of the present invention under light conditions were studied:

[0080] It can be seen from Example 1 that Fe@MT has strong absorption in the near-infrared region. Therefore, the photothermal conversion performance of Fe@MT was analyzed using an infrared thermal imaging instrument. The results are as follows: Figure 8 As shown, Figure 8 This is a temperature rise line graph of the nanomedicine used to induce ferroptosis in Example 4 of the present invention under near-infrared light irradiation. It can be seen that the temperature of the Fe@MT solution with different concentrations (10μg / mL, 20μg / mL, 40μg / mL, 80μg / mL) can rise rapidly under 808nm laser irradiation, proving that Fe@MT has good photothermal conversion performance and has the potential to enhance catalytic performance.

[0081] Furthermore, the effect of laser irradiation on the catalytic performance of Fe@MT was determined by UV-visible spectrophotometer. 100 μg / mL Fe@MT was dispersed in a buffer solution containing 3,3',5,5'-tetramethylbenzidine and 100 μM hydrogen peroxide at pH 5.0 with or without 808 nm laser irradiation to simulate the physiological environment of tumor tissue and analyze the catalytic effect of Fe@MT under slightly acidic conditions. The ability of Fe@MT to catalyze the decomposition of hydrogen peroxide to produce hydroxyl radicals was analyzed by measuring the absorbance at 652 nm using a UV-visible spectrophotometer.

[0082] Fig. 9 This is a diagram showing the catalytic effect of the nanomedicine used to induce ferroptosis under near-infrared light irradiation in Example 4 of the present invention, as shown in FIG. Fig. 9 As shown, compared with the group without laser irradiation, the absorption of Fe@MT at 652nm under laser irradiation was significantly increased, proving that the catalytic activity of Fe@MT was significantly improved under laser irradiation.

[0083] Example 5

[0084] The efficiency of tumor cells taking up Fe@MT prepared in Example 1 of the present invention and the level of intracellular reactive oxygen species were studied:

[0085] U87 tumor cells were cultured in confocal culture dishes and incubated overnight. The original DMEM medium was removed and 5 μg / mL Fe 3+ The medium and the medium containing 40 μg / mL Fe@MT were added. After incubation for 4 hours, the medium was removed and the cells were washed three times with phosphate buffer solution (PBS). The cells were collected and broken, and centrifuged at 12000 rpm for 10 min. The supernatant was digested with aqua regia, and the iron ion concentration in the cell supernatant was measured by inductively coupled plasma mass spectrometry. Cells not treated with drugs were used as blank control group. Fig.10 This is a graph showing the results of inductively coupled plasma mass spectrometry measuring iron ion uptake by tumor cells in Example 5 of the present invention. Fig.10 As shown, compared with cells not treated with drugs, cells treated with Fe 3+ The iron ion concentration in the experimental group with and Fe@MT was higher, proving that Fe@MT can be effectively taken up by tumor cells and enriched in tumor cells.

[0086] In addition, U87 tumor cells were treated with different methods, and 5 μg / mL Fe 3+ , 40 μg / mL mitoxantrone dihydrochloride (MT), 45 μg / mL Fe@MT, and a Fe@MT followed by laser irradiation group (Fe@MT+L) was set up, and cells only irradiated with laser were used as the blank control group.

[0087] Calcein was used to analyze the changes in iron ion concentration in tumor cells after different treatments. After calcein interacted with iron ions, the green fluorescence was significantly reduced. Fig.11 As shown, Fig.11 The fluorescence spectrum of the iron ion level in tumor cells in Example 5 of the present invention shows that compared with the blank control group, Fe 3+ After treatment with Fe@MT, the green fluorescence in the cells was significantly reduced, further proving that the iron ion concentration in tumor cells was increased.

[0088] In addition, the reactive oxygen species probe 2,7-dichlorofluororesin diacetate was used to detect the changes in the reactive oxygen species levels in tumor cells after the above-mentioned different treatments. The probe can react with reactive oxygen species to display green fluorescence. Fig.12 is a fluorescence spectrum of the reactive oxygen level in tumor cells in Example 5 of the present invention, such as Fig.12 As shown, with Fe 3+ Compared with MT alone, Fe@MT contains not only Fe 3+ Fe@MT catalyzes the decomposition of hydrogen peroxide in cells and consumes glutathione, and mitoxantrone dihydrochloride (MT) increases the content of polyunsaturated fatty acids in cells. Fe@MT shows brighter green fluorescence. After laser irradiation, the green fluorescence is further enhanced, indicating that Fe@MT converts light into heat, enhances catalytic performance, and greatly increases the level of reactive oxygen in cells.

[0089] Example 6

[0090] The effect of Fe@MT prepared in Example 1 of the present invention on the growth of tumor cells before and after illumination was studied:

[0091] The cell counting kit-8 (CCK-8) kit was used to test the effect of drugs on cell proliferation rate and Fe 3+ , MT and Fe@MT on the growth of U87 tumor cells before and after illumination, and evaluate the therapeutic effect of Fe@MT. U87 tumor cells were seeded in 96-well plates at 6000 cells per well, and incubated with DMEM medium containing 10% fetal bovine serum (FBS) for 24 hours. The original medium was removed and different concentrations of Fe@MT were added. 3+ , MT and Fe@MT culture medium, continue incubation for 24 hours, rinse with PBS, then add 100 μL of culture medium containing CCK-8, incubate for 1-2 hours, and use an enzyme reader to detect the absorbance value at 450 nm to analyze the survival rate of tumor cells.

[0092] Among them, the Fe@MT culture medium was set with another set of illumination conditions (Fe@MT+L). After adding the nanodrug, the illumination continued for 4 hours, and then the culture medium was incubated for another 20 hours before rinsing with PBS for the above-mentioned detection.

[0093] Fig.13 is a bar graph of the survival rate of U87 tumor cells under different conditions in Example 6 of the present invention, Fig.14 The bar graph of the survival rate of U87 tumor cells under light conditions of Fe@MT in Example 6 of the present invention. Fig.13 It can be seen that Fe 3+There was almost no effect on the growth of U87 tumor cells. The survival rate of tumor cells after free MT treatment was reduced to 32.5%, while the survival rate of tumor cells after Fe@MT treatment was reduced to 15.1%, which proved that Fe@MT was more effective in inhibiting tumor growth. Fig.14 It can be seen that under laser irradiation, the survival rate of Fe@MT-treated tumor cells is only 4.1%. Fe@MT can convert light into heat, further enhancing the catalytic performance to increase the level of reactive oxygen in cells. Therefore, Fe@MT can efficiently consume the reducing substance glutathione in cells and catalyze the decomposition of hydrogen peroxide in tumors to produce highly toxic reactive oxygen. At the same time, it can also upregulate the content of polyunsaturated fatty acids in cells. The reactive oxygen produced oxidizes polyunsaturated fatty acids and promotes the lipid peroxidation process, thereby improving the therapeutic effect of ferroptosis.

[0094] Example 7

[0095] The ferroptosis mechanism induced by Fe@MT prepared in Example 1 of the present invention was studied, and specific indicators of ferroptosis were analyzed, including lipid peroxidation levels, GPX4 and SCL47A1 protein expression levels:

[0096] Using C11-BODIPY 581 / 591 The probe was used to analyze the changes in intracellular lipid peroxidation levels after different treatments. Specifically, 50,000 U87 tumor cells were seeded in a confocal dish per well and incubated for 24 hours with DMEM medium containing 10% FBS. The original medium was removed and Fe 3+ , mitoxantrone dihydrochloride (MT) and Fe@MT medium, and continue to incubate for 24 hours; and set up the Fe@MT medium illumination group (Fe@MT+L), start illuminating for 5 minutes 4 hours after adding the drug, and then continue to incubate for 20 hours. After removing the medium, rinse with PBS and add the nuclear dye Hoechst. Using C11-BODIPY 581 / 591 The probe analyzes the changes in the level of intracellular lipid peroxidation after different treatments. When lipid peroxidation does not occur in the cells, the probe shows red fluorescence; when lipid peroxidation occurs in the cells, the probe shows green fluorescence.

[0097] Fig.15 This is a fluorescence image of lipid peroxidation levels in U87 tumor cells in Example 7 of the present invention. Cells irradiated only with laser light are set as a blank control group. Fig.15 As shown, Fe 3+Compared with MT alone, cells treated with Fe@MT showed brighter green fluorescence, because Fe@MT can not only catalyze the decomposition of hydrogen peroxide and consume glutathione in cells, but also increase the content of polyunsaturated fatty acids in cells. The reactive oxygen species produced can efficiently oxidize unsaturated fatty acids and induce lipid peroxidation. After laser irradiation, the green fluorescence is further enhanced, because Fe@MT converts light into heat, enhances catalytic performance, and greatly promotes lipid peroxidation.

[0098] In addition, immunofluorescence experiments were used to analyze the expression levels of phospholipid transporter solute carrier family 47 member 1 (SLC47A1) and glutathione peroxidase 4 (GPX4 protein), important indicators of ferroptosis. Specifically, U87 tumor cells were seeded in confocal dishes at 80,000 cells per well and incubated for 24 hours with DMEM medium containing 10% FBS. The original culture medium was removed and Fe 3+ , mitoxantrone dihydrochloride (MT) and Fe@MT culture medium as different experimental groups, and continue to incubate for 24 hours; and set up the Fe@MT culture medium illumination group (Fe@MT+L), start illuminating for 5 minutes 4 hours after adding the drug, and then continue to incubate for 20 hours. Then remove the culture medium and rinse with PBS 3 times, add paraformaldehyde to fix at room temperature for 15 minutes, and then wash three times with PBS; add Triton X-100 to permeabilize at room temperature for 15 minutes, wash three times with PBS; add goat serum to block for 45 minutes; add diluted primary antibody, incubate at 37℃ for 1 hour, wash three times with PBS; add fluorescent secondary antibody, incubate at 37℃ in the dark for 45 minutes, wash three times with PBS, and finally use confocal microscope to capture fluorescence.

[0099] Fig.16 This is a fluorescence graph of the expression level of SLC47A1 in U87 tumor cells after different treatments in Example 7 of the present invention. Fig.17 : is a fluorescence graph of the expression level of GPX4 protein in U87 tumor cells after different treatments in Example 7 of the present invention, such as Fig.16 and Fig.17 As shown, compared with the cell group irradiated with laser only (blank control group), the red fluorescence in tumor cells after treatment with MT and Fe@MT decreased significantly, indicating that MT and Fe@MT can effectively downregulate the expression levels of SLC47A1 and GPX4 proteins. The results show that Fe@MT can effectively consume overexpressed glutathione in tumor cells and upregulate the content of polyunsaturated fatty acids, ultimately inducing ferroptosis of tumor cells.

[0100] Example 8

[0101] The biological safety of Fe@MT prepared in Example 1 of the present invention was studied:

[0102] The hemolysis test was used to analyze the hemolysis ratio of red blood cells after treatment with different concentrations of Fe@MT. Specifically, 200μL of blood was collected from the eye socket of a mouse, 10μL of 1mg / mL heparin sodium was added for anticoagulation, 20mL of pre-cooled saline was added to dilute the blood, and then the red blood cells were collected by centrifugation at 4 degrees and 3000 rpm. Repeat three times until the supernatant has no obvious color, and finally dispersed in 1mL PBS. Take 40μL of red blood cells respectively, add 960μL ultrapure water and different concentrations of PBS dissolved Fe@MT solution (0, 100, 200, 400, 600, 800, 1000μg / mL), incubate at room temperature for 2 hours, then take pictures with a camera and use a UV-visible spectrophotometer to test the absorbance value. Among them, when red blood cells are hemolyzed, hemoglobin will be released, which will cause the UV-visible absorption spectrum to have a double absorption peak at 500-600nm.

[0103] Fig.18 is a UV-visible absorption spectrum of hemoglobin detected in the hemolysis test in Example 8 of the present invention, Fig.19 : is a hemolysis ratio diagram of the hemolysis test in Example 8 of the present invention, such as Fig.18 and Fig.19 As shown, compared with the hemolysis of red blood cells in the ultrapure water group alone (blank control group), after incubation with different concentrations of Fe@MT solutions for 2 hours, the camera photography results showed that there was no obvious hemolysis of red blood cells. The UV-visible absorption spectrum also further demonstrated that different concentrations of Fe@MT solutions did not cause obvious hemolysis of red blood cells.

[0104] Example 9

[0105] The ferroptosis therapeutic effect of Fe@MT was verified in a mouse model loaded with tumor cells. All in vivo animal experiments were conducted according to the protocol approved by the Ethics Committee of Fujian Medical University. The specific protocol of the experiment is as follows:

[0106] The dorsal flank of Balb / c nude mice was subcutaneously injected with 100 μL of U87 cells (1×10 7 The U87 glioma model was constructed by 30 cells per well. When the tumor volume reached about 70 mm 3 When the U87 brain glioma mouse model was obtained, it was used for in vivo anti-tumor treatment, where tumor volume = (tumor length) × (tumor width) 2 / 2.

[0107] First, in order to verify the photothermal conversion effect of Fe@MT in mouse tumor sites, we randomly divided U87 glioma mice into two groups and applied different drug treatments, including 100 μL phosphate buffer (PBS) plus light as the control group and 100 μL 0.8 mg mL Fe@MT plus light as the experimental group. The mice in the Fe@MT plus light group were injected through the tail vein for 24 hours and then exposed to laser light conditions (1 W / cm 2 The tumor was irradiated at 808 nm for 5 minutes. During this period, an infrared thermal imager was used to detect the temperature changes of the tumor site in real time.

[0108] Fig. 20 This is a thermal image of the tumor site of the mouse in Example 8 of the present invention. Fig. 20 As shown, the temperature of the tumor of mice in the PBS control group increased by only 5 degrees after five minutes of laser irradiation, which was due to the absorption of 808 nm laser by the skin surface; while the temperature of the tumor of mice in the Fe@MT experimental group increased by 22 degrees after five minutes of laser irradiation, indicating that Fe@MT can effectively accumulate at the tumor site and convert 808 nm laser into heat, thereby effectively improving the catalytic performance of Fe@MT in the tumor, promoting lipid peroxidation, inducing tumor ferroptosis and ultimately inhibiting tumor growth.

[0109] In addition, U87 glioma-bearing mice were randomly divided into six groups (5 mice in each group) and subjected to different drug treatments, including: 100 μL phosphate buffer (PBS) plus light, 100 μL 4 mg / mL FeCl3 plus light, 100 μL 0.8 mg / mL mitoxantrone dihydrochloride (MT), 100 μL 0.8 mg mL MT plus light, 100 μL 0.8 mg mL Fe@MT, and 100 μL 0.8 mg mL Fe@MT plus light. Among them, the mice in the Fe@MT plus light group were injected through the tail vein for 24 hours and then exposed to laser light conditions (1 W / cm 2 The tumor volume was recorded using a vernier caliper under the above conditions.

[0110] Fig.21 Graph showing the change of tumor growth curve over time in mice after treatment in Example 9 of the present invention. Fig.21As shown, the tumor growth rate of mice in the group treated with FeCl3 alone was no different from that in the group treated with phosphate buffer alone; the addition of MT and MT plus illumination inhibited some tumor growth; the tumor growth in mice treated with Fe@MT was significantly inhibited; further, four tumors in the mice in the Fe@MT plus illumination group were completely ablated. Since Fe@MT can not only catalyze the decomposition of hydrogen peroxide and consume glutathione in cells, but also increase the content of polyunsaturated fatty acids in cells, the reactive oxygen species produced can efficiently oxidize unsaturated fatty acids and induce lipid peroxidation; after laser irradiation, Fe@MT converts light into heat, enhances catalytic performance, further promotes lipid peroxidation, and ultimately causes tumor ferroptosis. Therefore, by conducting ferroptosis treatment experiments in U87 glioma mouse models, the experimental results further confirmed that Fe@MT can amplify oxidative stress in tumors to inhibit tumor growth under laser irradiation.

[0111] Through the above examples, it can be found that the nano drug provided by the present invention can affect the lipid peroxidation level and cell survival rate in tumor cells, can effectively catalyze the decomposition of hydrogen peroxide in tumor cells to produce reactive oxygen, consume overexpressed glutathione in cells, and can also increase the content of polyunsaturated fatty acids in cells. The reactive oxygen generated can efficiently oxidize unsaturated fatty acids, induce lipid peroxidation, and finally induce ferroptosis in tumor cells. And the nano drug can convert light into heat, enhance the catalytic performance, greatly promote the lipid peroxidation process, thereby enhancing the therapeutic effect of ferroptosis.

[0112] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A nano drug for inducing ferroptosis, which is a nanoparticle formed by coordination self-assembly of an iron salt and a polyphenol drug, wherein the average particle size of the nano drug is 1 to 5 nm; The preparation method of the nano drug comprises: The organic solution containing iron salt and polyphenol drugs is dripped into a liquid culture medium or a phosphate buffer solution with a pH of 7 to 9 to obtain a mixed solution; Stirring the mixed solution to allow the iron salt and the polyphenol drug to self-assemble to obtain the nano drug; Wherein, the iron salt includes any one of the following: ferric chloride, ferric sulfate, ferric nitrate; the polyphenol drug is mitoxantrone dihydrochloride; and the solvent of the organic solution is dimethyl sulfoxide.

2. The nano drug according to claim 1, wherein In the coordination self-assembly, the concentration of the iron salt is 1-10 mM; the concentration of the polyphenol drug is 4-40 mM.

3. A method for preparing the nano drug according to claim 1 or 2, comprising: The organic solution containing iron salt and polyphenol drugs is dripped into a liquid culture medium or a phosphate buffer solution with a pH of 7 to 9 to obtain a mixed solution; Stirring the mixed solution to allow the iron salt and the polyphenol drug to self-assemble to obtain the nano drug; Wherein, the solvent of the organic solution is dimethyl sulfoxide; and the polyphenol drug is mitoxantrone dihydrochloride.

4. The preparation method according to claim 3, wherein: The concentration of the iron salt in the mixed solution is 1-10 mM; The concentration of the polyphenol drug in the mixed solution is 4-40 mM.

5. Use of the nano drug according to claim 1 or 2 in the preparation of a drug for inducing ferroptosis of tumor cells.

6. Use of the nanomedicine as claimed in claim 1 or 2 in the preparation of a drug for photothermal enhancement-induced ferroptosis of tumor cells.

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