Biocompatible tumor-inducing ferroptosis magnetic resonance nanoprobe and its preparation and application

Abstract

The invention discloses a biocompatible tumor-inducing ferroptosis magnetic resonance nanoprobe, the nanoprobe comprises a core-shell structure with ferroferric oxide nanoparticles as a core and apple pectin as a reducing agent as a shell, and ferroptosis inducing agents salazine and RGD tumor targeting peptide are loaded on the surface of the core-shell structure, and the core-shell structure composite nanoprobe finally formed is a magnetic resonance nanoprobe, which is spherical and has an average particle size of 20 to 30 nm. The magnetic resonance nanoprobe of the invention has good biocompatibility, MR T2 imaging capability and ferroptosis inducing activity, and has no toxic side effects. Through specific enrichment at the tumor site, ferroptosis treatment of the tumor and MR visualization monitoring are achieved. The magnetic resonance nanoprobe is used as a drug carrier, and an amide reaction method is used to load doxorubicin on its surface for combined chemotherapy drugs. It has the characteristics of magnetic resonance T2 weighted imaging, inducing ferroptosis and apoptosis of cancer cells, and can achieve the purpose of tumor inhibition.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomedicine, and in particular relates to a preparation method and application of a biocompatible magnetic resonance visualization nanoprobe that can be used to induce tumor ferroptosis. Background Art

[0002] With the aging of the population and changes in diet and lifestyle, the incidence of tumors is not optimistic. At present, most conventional chemotherapy drugs and targeted drugs inhibit tumor progression by inducing apoptosis. However, in many cases, the results of inducing apoptosis are far from satisfactory. Overexpression of apoptosis protein inhibitors in malignant tumor cells leads to their poor response to apoptosis therapy. Multidrug resistance (MDR) effects often occur in malignant cells as a means of self-defense against apoptosis caused by chemotherapy drugs. In addition, due to endogenous apoptosis inhibition in RAS mutations, apoptosis therapy cannot handle cancer cells with RAS (a protein family belonging to small GTPases), which account for about 30% of all cancers. These inevitable obstacles have triggered an urgent need for non-apoptotic cancer treatments. Therefore, the study of new non-apoptotic-related death methods provides new ideas for cancer treatment.

[0003] Ferroptosis is a new type of programmed cell death discovered in recent years. It is a regulated cell death driven by iron-dependent lipid peroxidation, which is different from other cell death modes in morphology and mechanism. Phospholipids containing polyunsaturated fatty acids in cell membranes are prone to peroxidation under conditions rich in iron and reactive oxygen species. The accumulation of such lipid peroxides in cell membranes eventually destroys the integrity of the membrane, leading to ferroptosis. Recent studies have shown that ferroptosis plays an important role in tumor suppression and the treatment of certain cancers. Targeting ferroptosis has become a promising strategy for cancer treatment. Given the important role that ferroptosis plays in the occurrence, development and metastasis of tumors, the use of ferroptosis for anti-tumor treatment has great potential advantages. In order to further improve the anti-tumor effect of ferroptosis, it is of great significance to study the mechanism of ferroptosis and combine it with other treatment methods to develop multifunctional and intelligent nanosystems. Summary of the invention

[0004] In view of the requirements of the prior art, the object of the present invention is to provide a method for preparing a biocompatible magnetic resonance visualization nanoprobe that can be used to induce tumor ferroptosis. The nanoprobe has good biocompatibility and MRI 2 It has imaging capabilities and inducing ferroptosis activity, and has no toxic side effects. It can achieve ferroptosis treatment of tumors through specific enrichment at the tumor site, as well as MR visualization monitoring of treatment effects. In addition, the nanoprobe can also be used as a drug carrier and combined with chemotherapy drugs to achieve the purpose of tumor inhibition.

[0005] In order to solve the above technical problems, the present invention proposes a biocompatible tumor-inducing ferroptosis magnetic resonance nanoprobe, which comprises ferroferric oxide (Fe 3 O 4 ) nanoparticles as the core and apple pectin (AP) as the shell, and the surface of the core-shell structure is loaded with ferroptosis inducer and RGD tumor targeting peptide. The core-shell structure composite nanoprobe finally formed is the magnetic resonance nanoprobe. The magnetic resonance nanoprobe is spherical, with a particle size range of 15 to 50 nm and an average particle size of 20 to 30 nm. The magnetic resonance nanoprobe has a magnetic resonance T 2 Weighted imaging capability (MR T 2 ) and the property of inducing ferroptosis.

[0006] Ferroptosis inducers are hydrophobic drugs that have the effect of inducing ferroptosis, and sulfasalazine (SAS) is selected; RGD tumor targeting peptide is a five-membered cyclic peptide composed of arginine, glycine, aspartic acid, D-phenylalanine, and lysine, referred to as c(RGDfK). c(RGDfK) can bind to the αvβ3 receptor and is a highly effective and selective inhibitor of the αvβ3 integrin receptor. In an appropriately labeled form, these peptides can be used as useful tools for diagnostic imaging and peptide-targeted therapy of certain types of cancer.

[0007] The present invention proposes a method for preparing the above-mentioned magnetic resonance nanoprobe, which mainly comprises: modifying the reducing agent apple pectin on the surface of ferroferric oxide nanoparticles through a one-step hydrothermal synthesis to obtain the core-shell structure, referred to as FA nanoparticles; modifying the ferroptosis inducer pyridine sulfoxide on the surface of FA nanoparticles through electrostatic action, and the synthesized product is referred to as FAS nanoparticles; then modifying the RGD tumor targeting peptide on the surface of the FA nanoparticles through an amide reaction method, and the resulting product is the magnetic resonance nanoprobe, referred to as FASR nanoprobe. The specific steps are as follows

[0008] Step 1) Preparation of FA nanoparticles: FeCl 3 6H 2 O and FeSO 4 7H 2 O was mixed in water under vigorous stirring to obtain solution A. In solution A, FeCl 3 6H 2 O and water mass volume ratio 54mg / ml, FeSO 4 7H 2O and water have a mass volume ratio of 27.8 mg / ml; heated to 80°C in a three-necked flask under nitrogen protection for 5 minutes; quickly drop 10% ammonia water into the above reaction solution, and continue to react at 80°C for 30 minutes to obtain solution B, wherein the volume ratio of 10% ammonia water to water is 0.35:1; prepare apple pectin aqueous solution at a mass volume concentration of 100 mg / ml, wherein the molecular weight of the apple pectin is 50000-300000, which can accelerate the conversion of Fe(III) / Fe(II), regenerate Fe(II) with high peroxidase activity, and enhance the ferroptosis effect. According to the volume ratio of 12:27, add the above apple pectin aqueous solution to the above solution B, heat to 95°C, stir for 90 minutes; cool naturally to room temperature, separate the product from the reaction mixture with a magnet, then wash it several times with deionized water, collect the precipitate, and the synthetic product is Fe 3 O 4 Apple pectin nanoparticles, denoted as Fe 3 O 4 @AP, referred to as FA nanoparticles; stored at 4°C until use;

[0009] Step 2) Preparation of FAS nanoparticles: dissolve ferroptosis inducer pyralidone in dimethyl sulfoxide at a mass volume ratio of 50 mg / mL to obtain solution C; dissolve FA nanoparticles obtained in step 1) in water at a mass volume ratio of 2.55 mg / mL to obtain solution D; slowly add solution C to solution D at a volume ratio of 1:49, and react at 37°C with vigorous stirring for 24 hours; wash with a water-dimethyl sulfoxide mixed solution after centrifugation, collect the dark brown precipitate, and the synthetic product is Fe 3 O 4 Apple pectin-flavone pyridoxine nanoparticles, denoted as Fe 3 O 4 @AP-SAS, referred to as FAS nanoparticles; stored at 4°C until use;

[0010] Step 3) Preparation of FASR nanoprobe: Disperse the FAS nanoparticles obtained in step 2) in deionized water at a mass volume ratio of 6 mg / mL, and then add a condensing agent thereto to activate the carboxyl group, wherein the mass ratio of the condensing agent to the FAS nanoparticles is 11:60; stir in a 37°C water bath for 1 hour to obtain solution E; add RGD tumor targeting peptide to solution E at a mass volume ratio of 0.2 mg / mL, stir in a 37°C water bath in the dark for 10 to 12 hours, so that the carboxyl group on the surface of the FAS nanoparticles is covalently bonded to the amino group of the RGD tumor targeting peptide; separate the product from the reaction mixture with a magnet, wash it several times with deionized water, collect the precipitate, and the obtained product is Fe 3 O 4Apple pectin-salazine / RGD tumor targeting peptide nanoparticles, denoted as Fe 3 O 4 @AP-SAS / RGD, referred to as FASR nanoprobe, is a biocompatible magnetic resonance visualization nanoprobe that can be used to induce tumor ferroptosis.

[0011] Furthermore, in the above preparation method: the stirring speed is 500-1000 rpm. The speed of adding ammonia water dropwise in step 1) is 0.35 mL / s.

[0012] The magnetic resonance nanoprobe prepared by the present invention is used as a carrier for preparing chemotherapy drugs. Doxorubicin is an anti-tumor antibiotic that can inhibit the synthesis of RNA and DNA. It has the strongest inhibitory effect on RNA and a broad anti-tumor spectrum. It is effective against a variety of tumors. It is a cycle non-specific drug and has a killing effect on tumor cells in various growth cycles. In the present invention, doxorubicin (Dox) is loaded on the surface of the FASR nanoprobe by an amide reaction method, and the process is: the FASR nanoprobe is dispersed in deionized water, and a condensing agent is added thereto to activate the carboxyl group, wherein the mass ratio of the condensing agent to the FASR nanoprobe is 11:30, and the solution F is obtained by stirring in a 37°C water bath for 1 hour; doxorubicin is added to the solution F according to the mass ratio of doxorubicin to the FASR nanoprobe being 1:6, and the solution is stirred in a 37°C water bath in the dark for 10 to 12 hours to make the carboxyl group on the surface of the FASR nanoparticles covalently bonded with the amino group of doxorubicin; the product is separated from the reaction mixture by a magnet, washed with deionized water several times, and the precipitate is collected. The final product is a FASR nanoprobe loaded with doxorubicin, which is recorded as Fe 3 O 4 @AP-SAS / RGD / Dox, referred to as FASDR nanoprobe, is a nanoprobe that combines ferroptosis and chemotherapy to achieve tumor inhibition. 2 Weighted imaging, characteristics of induced ferroptosis and apoptosis in cancer cells.

[0013] In the present invention, the condensing agent is 4-(-4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride or is composed of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide in a mass ratio of 6:5.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) The present invention aims to solve the problem of lack of highly active ferrous ions in the treatment of tumor ferroptosis. Based on the reducing properties of apple pectin in an acidic environment, the present invention constructs a FASR nanoprobe that can accelerate the conversion of Fe(III) / Fe(II) using apple pectin (AP) and regenerate Fe(II) with highly active peroxidase activity. The present invention is the first to use AP as a reducing agent in promoting the efficacy of tumor ferroptosis, which expands the research and development ideas of iron-based magnetic nanoparticles in the application of tumor ferroptosis.

[0016] (2) The present invention is based on the high performance T of iron 2 Imaging characteristics, from T 2 The mapping technology has the ability to non-invasively and accurately detect iron deposition in mouse tissues, organs and tumors, making full use of the potential application value of FASR nanoprobes in tumor assessment.

[0017] (3) The present invention starts from the perspective of enhancing and real-time evaluation of tumor ferroptosis, and uses self-reducing Fe(II) ultra-efficient nanomaterials as dual drug carriers, combined with ferroptosis and chemotherapy to inhibit tumor growth. It closely combines MRI, ferroptosis, chemotherapy and nanomaterial construction, and strives to promote the solution and transformation of clinical problems through the intersection of medical and engineering disciplines. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the structure of the FASR nanoprobe of the present invention;

[0019] Figure 2 is a characterization diagram of the FASR nanoprobe prepared in Example 1;

[0020] Figure 3 is the X-ray photoelectron spectroscopy (XPS) of the FASR nanoprobe;

[0021] Figure 4 is the in vitro MR imaging and relaxivity of the FASR nanoprobe;

[0022] Figure 5 This is the statistical graph of PC3 cell survival rate and GPX4 protein expression of FASR nanoprobe;

[0023] Figure 6 The MR T of mice in each group were injected intravenously with different concentrations of FASR nanoprobes. 2 Mapping of tumor T 2 Values ​​and correlation analysis with GPX4 expression in tumors after treatment;

[0024] Figure 7This is a graph showing the therapeutic effects of each group of mice after three injections of FASDR nanoprobes and other drugs into the tail vein. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the following examples are by no means intended to limit the present invention in any way. All raw materials of the present invention are not particularly limited in their sources and can be purchased on the market or prepared according to conventional methods well known to those skilled in the art.

[0026] Example 1: A biocompatible magnetic resonance nanoprobe for tumor ferroptosis, such as Figure 1 As shown, the nanoprobe includes a core-shell structure with ferroferric oxide nanoparticles as the core and apple pectin as the shell, and the surface of the core-shell structure is loaded with ferroptosis inducers and RGD tumor targeting peptides. The core-shell structure composite nanoprobe finally formed is the biocompatible tumor-inducing ferroptosis magnetic resonance nanoprobe. The magnetic resonance nanoprobe is spherical, with a particle size range of 15 to 50 nm and an average particle size of 20 to 30 nm. It has a magnetic resonance T 2 Weighted imaging capabilities and properties of ferroptosis induction.

[0027] In this embodiment, the molecular weight of the reducing agent apple pectin is 50000-300000; the ferroptosis inducer is salazopyridine, but is not limited to hydrophobic drugs with the effect of inducing ferroptosis; the RGD tumor targeting peptide is a five-membered cyclic peptide composed of arginine, glycine, aspartic acid, D-phenylalanine, and lysine, referred to as RGD tumor targeting peptide, which can bind to the αvβ3 receptor and is a highly efficient and selective inhibitor of the αvβ3 integrin receptor.

[0028] The preparation of the magnetic resonance nanoprobe (FASR) is mainly as follows: the reducing agent apple pectin is modified on the surface of ferroferric oxide nanoparticles by a one-step hydrothermal method to obtain the core-shell structure, referred to as FA nanoparticles; the ferroptosis inducer pyridine is modified on the surface of FA nanoparticles by electrostatic action, and the synthetic product is referred to as FAS nanoparticles; then the RGD tumor targeting peptide is modified on the surface of the FA nanoparticles by an amide reaction method, and the resulting product is the magnetic resonance nanoprobe, referred to as FASR nanoprobe. The specific steps are as follows:

[0029] Step 1): Preparation of FA nanoparticles:

[0030] 540 mg FeCl 3 6H 2 O and 278mg FeSO 4 7H 2 O was dissolved in 10 mL of water to obtain a mixed solution;

[0031] The mixed solution was added into a three-necked flask under nitrogen protection, heated to 80°C, and reacted for 5 minutes under stirring at 1000 rpm; then, 3.5 mL of 10% ammonia water was rapidly added dropwise into the three-necked flask at a rate of 0.35 mL / s, and reacted for 30 minutes to obtain a reaction solution.

[0032] 600 mg AP (molecular weight 50000-300000) was weighed and dissolved in 6 ml deionized water at 80°C to prepare an apple pectin aqueous solution. Then, the apple pectin aqueous solution was slowly added to the above reaction solution at a rate of 50 ul / s. The temperature was raised to 95°C and stirred for 90 minutes. The solution was then cooled naturally to room temperature, and the product was separated from the reaction mixture with a magnet and washed 3 times with deionized water. The collected precipitate was the synthetic product of Fe 3 O 4 Apple pectin nanoparticles, denoted as Fe 3 O 4 @AP, referred to as FA nanoparticles, was stored at 4°C until use.

[0033] Step 2): Preparation of FAS nanoparticles:

[0034] 50 mg of salazine was weighed and dissolved in 1 mL of dimethyl sulfoxide, which was slowly added dropwise to 49 mL of an aqueous solution containing 125 mg of FA nanoparticles, and reacted at 37°C with stirring at 1000 rpm for 24 h. Subsequently, the mixture was centrifuged at 12000 rpm for 10 min, washed three times with deionized water / DMSO at a volume ratio of 49:1, each time for 10 min, and the dark brown precipitate was collected. The synthetic product of this step was Fe 3 O 4 Apple pectin-flavone pyridoxine nanoparticles, denoted as Fe 3 O 4 @AP-SAS, referred to as FAS nanoparticles; stored at 4°C until use.

[0035] Step 3): Preparation of FASR nanoparticles:

[0036] 60 mg of FAS nanoparticles prepared in step 2) were dispersed in 10 ml of deionized water, and then 6 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 5 mg of N-hydroxysuccinimide (NHS) were added thereto for carboxyl activation treatment, and stirred in a 37°C water bath for 1 hour. Then 2 mg of RGD tumor targeting peptide was added to the above solution, and stirred in a 37°C water bath in the dark for 12 hours to covalently bind the carboxyl groups on the surface of apple pectin to the amino groups of RGD. After the reaction was completed, the product was separated from the reaction mixture with a magnet, washed 3 times with deionized water, and the collected precipitate was finally obtained as Fe 3 O 4 Apple pectin-salazine / RGD tumor targeting peptide nanoparticles, denoted as Fe 3 O 4 @AP-SAS / RGD, referred to as FASR nanoprobe.

[0037] like Figure 2 As shown, A is the transmission electron microscope (TEM) image of the FASR nanoprobe prepared in Example 1. It can be seen that the FASR nanoprobe has a spherical structure; B is a dynamic light scattering hydration particle size image, and the average particle size of the FASR nanoprobe is 20 to 30 nm.

[0038] Example 2: Verification of FASR nanoprobes using AP to accelerate Fe(III) / Fe(II) conversion in an acidic environment in vitro and regenerate Fe(II) with high peroxidase activity:

[0039] Step 1) 100 mg of FASR prepared in Example 1 was divided into two equal parts and placed in each 5 ml centrifuge tube, and 3 ml of phosphate-balanced saline (PBS) with a pH of 7.4 and 5.0 were added respectively. After treatment for 3 days, the two groups of samples were frozen in a -80°C refrigerator;

[0040] Step 2) After the two groups of samples in step 1) are frozen and solid, take them out, seal the mouth of the centrifuge tube with a perforated plastic wrap, and place them in a freeze dryer for 24 hours. After freeze drying, take out the two groups of samples for XPS detection.

[0041] like Figure 3As shown, Figure A is the XPS peak fitting diagram of FASR freeze-dried powder placed in PBS buffer at pH 7.4, and Figure B is the XPS peak fitting diagram of FASR freeze-dried powder placed in PBS buffer at pH 5.0. Peaks corresponding to Fe(II) (712.7eV) and Fe(III) (710.8eV) were observed. The results show that after FASR freeze-dried powder was treated in a neutral environment at pH 7.4, the peak area ratio of Fe(III) / Fe(II) was about 1.4:1; after being treated in an acidic environment at pH 5.0, the peak area ratio of Fe(III) / Fe(II) was about 0.3:1. The results showed that after treatment, Fe in FASR existed in a mixed valence state, consisting of Fe(III) and Fe(II). After treatment in a neutral environment of pH 7.4, Fe(III) was the main component, while after treatment in an acidic environment of pH 5.0, Fe(II) was the main component. This verified the ability of FASR nanoprobes to accelerate Fe(III) / Fe(II) conversion using AP in an acidic environment in vitro and regenerate Fe(II) with high peroxidase activity.

[0042] Example 3: In vitro MR imaging experiment of FASR nanoprobe:

[0043] Step 1) Take 10 mL of deionized water and the FASR nanoprobe prepared in Example 1 to form a FASR stock solution, dilute the FASR stock solution by 150, 300, 500, 750, and 1000 times, respectively, and take 1.5 ml of each and place them in a 1.5 mL centrifuge tube to obtain 5 groups of FASR nanoprobe samples, and mix the FASR nanoprobe samples of each group evenly at room temperature; and take 1.5 ml of deionized water and place it in a 1.5 mL centrifuge tube as a group of control samples.

[0044] Step 2) Fix the 6 groups of samples prepared in step 1) in a plastic tube rack and place them in a 3.0T MR scanner. Adjust the MR scanning parameters and scan the T 2 Weighted imaging, the acquired data are transferred to the post-processing workstation.

[0045] like Figure 4 As shown in the figure, A is the T of the five groups of FASR nanoprobe samples and the control sample water. 2 Weighted imaging diagram, B is the relaxation rate curve of FASR nanoprobe. Figure 4 As can be seen from the figure, with the increase of FASR nanoprobe concentration, T 2 The signal of weighted imaging gradually decreases; the T of FASR nanoprobe 2 Relaxation rate: 124.52mM -1 s -1, proving that the FASR nanoprobe prepared in the present invention has excellent magnetic resonance T 2 Weighted imaging performance.

[0046] Example 4: FASR nanoprobes can promote Fe(III) / Fe(II) conversion through AP, regenerate Fe(II) with high peroxidase activity, generate a large number of hydroxyl radicals, and cooperate with the ferroptosis inducer sulfasalazine (SAS) to explore its effect on ferroptosis in tumor cells.

[0047] First, prostate cancer cells PC3 were seeded in a 96-well plate (about 3000 cells per well) and incubated in an incubator overnight. The culture medium was then replaced with fresh culture medium containing different concentrations (0, 50, 100, 150, 200 μg / mL) of the FASR nanoprobes prepared in Example 1 and incubated together for 48 hours. Cell viability was detected by standard MTT assay, as shown in Figure 2. Figure 5 As shown in A.

[0048] Then, PC3 were seeded in 6-well plates (about 2×10 5 Cells were added and incubated overnight in an incubator. The culture medium was then replaced with fresh culture medium containing different concentrations (0, 100, 200, 300, 400, 500 μg / mL) of FASR prepared in Example 1 and incubated for 48 hours. Cell protein was extracted and the expression of ferroptosis marker GPX4 was detected by Western Blot. Figure 5 As shown in B.

[0049] Figure 5 The data were derived from three independent samples and expressed as mean ± standard deviation. Figure 5 In A, it can be observed that as the concentration of FASR nanoprobe increases, the cell survival rate gradually decreases. When the concentration of FASR nanoprobe reaches 200 μg / mL, the cell survival rate drops to about 54%. Figure 5 In B, it can be observed that as the concentration of FASR nanoprobe increases, GPX4, one of the iron death indicators in tumor cells, gradually decreases, proving that the nanoprobe achieves the effect of reducing cancer cells by causing iron death of tumor cells; the above experimental results show that the FASR nanoprobe prepared by the present invention can cause iron death of tumor cells and produce a killing effect.

[0050] Example 5: Application of FASR nanoprobe in PC3 tumor-bearing nude mouse model to induce tumor ferroptosis and visualize the effect.

[0051] A PC3 tumor-bearing nude mouse model was selected, and the FASR nanoprobe prepared in Example 1 was injected into the mouse tail vein. The nude mice were imaged in real time using MR. The MR T values ​​of the nude mice were calculated based on the time when the tumor of the nude mice accumulated the nanoprobes to the highest level.2 Mapping visualizes the ferroptosis effect on tumors. The specific implementation steps are as follows:

[0052] Step 1) taking logarithmically growing PC3 cells, digesting with trypsin, resuspending in culture medium, collecting the cells, and adjusting the cell density with sterile phosphate buffered saline (PBS) for later use;

[0053] Step 2) Nude mice were selected to establish a PC3 tumor nude mouse model. Each nude mouse was subcutaneously injected with 5x10 6 / L PC3 cell suspension 0.1ml;

[0054] Step 3) Wait until the subcutaneous tumor of the tumor-bearing mouse model established in step 2) reaches 50 mm 3 After that, the mice were randomly divided into 6 groups and injected with PBS, 10 mg / kg / mouse FASR nanoprobe prepared in Example 1, 20 mg / kg / mouse FASR nanoprobe, 30 mg / kg / mouse FASR nanoprobe, 40 mg / kg / mouse FASR nanoprobe, and 50 mg / kg / mouse FASR nanoprobe respectively through the tail vein. After injection, real-time T 2 WI and T 2 Mapping imaging was used to detect the highest accumulation of FASR in tumors of tumor-bearing mice. 2 Mapping determination of T 2 The values ​​were analyzed for correlation with the therapeutic effects on mouse tumors.

[0055] Step 4) The corresponding amount of nanoprobes was intravenously injected into the tumor-bearing mice in step 3) on days 3 and 6. The tumor size was measured every 2 days, and the mice were weighed. On day 14, the mice were euthanized, and the tumors were taken to prepare pathological sections for immunohistochemical staining of GPX4, one of the ferroptosis indicators.

[0056] like Figure 6 As shown, Figure 6 A is the T value when FASR accumulation in the tumor was the highest (6 h) after tumor-bearing mice were divided into groups and injected with PBS, 10 mg / kg / mouse FASR nanoprobe, 20 mg / kg / mouse FASR nanoprobe, 30 mg / kg / mouse FASR nanoprobe, 40 mg / kg / mouse FASR nanoprobe, and 50 mg / kg / mouse FASR nanoprobe through the tail vein. 2 T obtained by mapping 2 Value quantification; Figure 6 B is the GPX4 and Figure 6 A in T 2 Correlation analysis of the values. Figure 6As can be seen from Figure A, with the increase in the injection concentration of the FASR nanoprobe prepared in Example 1, the tumor T 2 The value gradually decreased, indicating that the aggregation of nanomaterials in the tumor increased with the increase of FASR nanoprobe injection concentration; Figure B shows that after the tumor-bearing mice in each group were injected with FASR nanoprobes, T 2 Mapping imaging T 2 The value was significantly positively correlated with the final tumor GPX4 expression, and the r value was 0.9494. The above experimental results verified the ability of the FASR nanoprobe prepared by the present invention to induce tumor ferroptosis and visualize the effect in the PC3 tumor-bearing nude mouse model.

[0057] Example 6: Using FASR nanoprobes as carriers to load the chemotherapy drug doxorubicin (Dox) to obtain Fe 3 O 4 @AP-SAS / RGD / Dox nanoparticles, combined with ferroptosis and chemotherapy, achieve tumor inhibitory effects.

[0058] In this example, Dox was loaded onto the FASR nanoparticles prepared in Example 1 through an amide reaction, and then a PC3 tumor-bearing nude mouse model was selected, and the FASDR NPs of the present invention were injected through the tail vein of the mouse to explore its tumor inhibition effect of combined ferroptosis and chemotherapy. The specific implementation steps are as follows:

[0059] Step 1) 30 mg of the FASR nanoparticles prepared in Example 1 were dispersed in 10 ml of deionized water, and then 6 mg of EDC and 5 mg of NHS were added thereto for carboxyl activation treatment, and the mixture was stirred in a 37°C water bath for 1 hour; then 5 mg of the chemotherapy drug Dox was added to the solution, and the mixture was stirred in a 37°C water bath in the dark for 12 hours to allow the carboxyl groups on the surface of the apple pectin of the FASR nanoparticles to covalently bind to the amino groups of Dox, and then the product was separated from the reaction mixture by a magnet, washed several times with deionized water, and the precipitate was collected. The final product obtained in this step was a FASR nanoprobe loaded with doxorubicin, denoted as Fe 3 O 4 @AP-SAS / RGD / Dox, referred to as FASDR nanoprobe.

[0060] Step 2) taking logarithmically growing PC3 cells, digesting with trypsin, resuspending in culture medium, collecting the cells, and adjusting the cell density with sterile phosphate buffered saline (PBS) for later use;

[0061] Step 3) Nude mice were selected to establish a PC3 tumor nude mouse model. Each nude mouse was subcutaneously injected with 5x10 6 / L PC3 cell suspension 0.1ml;

[0062] Step 4) Wait until the subcutaneous tumor of the tumor-bearing mouse model established in step 3) reaches 50 mm 3 Afterwards, the mice were randomly divided into 5 groups, namely group 1, group 2, group 3, group 4 and group 5. Group 1 was injected with PBS in the tail vein on days 0, 3, 6 and 9 (this group was the control group), and group 2 was injected with Fe in the tail vein on days 0, 3, 6 and 9. 3 O 4 Nanoparticles were injected into the tail vein of mice in group 3 on days 0, 3, 6, and 9. FASR nanoprobes were injected into the tail vein of mice in group 4 on days 0, 3, 6, and 9. FASDR nanoprobes prepared in this example were injected into the tail vein of mice in group 5 on days 0, 3, 6, and 9. Meanwhile, the tumor size was measured every 2 days, and the mice were weighed once. On the 14th day, the mice were euthanized. 3 O 4 The nanoparticles, FAS nanoparticles and FASR nanoprobes are all obtained according to the preparation method of Example 1 of the present invention.

[0063] like Figure 7 As shown in Figure A, tumor-bearing mice were injected with PBS, Fe 3 O 4 Nanoparticles, FAS nanoparticles, FASR nanoprobes, and FASDR nanoprobes were used to evaluate the changes in tumor volume within 14 days. Figure B shows the changes in tumor volume within 14 days after the tumor-bearing mice were injected with PBS, Fe 3 O 4 Nanoparticles, FAS nanoparticles, FASR nanoprobes, and FASDR nanoprobes. Photos and weights of tumors in each group after 14 days. Figure 7 As can be seen from A and B, the tumors of the tumor-bearing mice in the control group injected with PBS grew rapidly within 14 days, while those injected with Fe 3 O 4 The two groups of tumor-bearing mice after nanoparticles were obtained. 3 O 4 Nanoparticles have only a small inhibitory effect on tumor growth; after the injection of FAS nanoparticles, the tumor growth inhibition of the 3 groups of tumor-bearing mice, that is, after the addition of AP and SAS, was increased compared with the 2 groups; and after the injection of FASR nanoprobes, the tumor growth inhibition of the 4 groups of tumor-bearing mice, that is, after the addition of tumor targeting peptide RGD, was further inhibited; after the injection of FASDR nanoprobes, the tumor growth inhibition of the 5 groups of tumor-bearing mice, that is, after the addition of chemotherapy drug Dox, was significantly inhibited. The above experimental results show that the FASR nanoprobes prepared by the present invention can be used to load chemotherapy drugs, combined with ferroptosis and chemotherapy to achieve tumor inhibition effects.

[0064] Although the present invention has been described above in conjunction with the accompanying drawings, the present invention is not limited to the above-mentioned specific embodiments, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can make many changes without departing from the purpose of the present invention, all of which are within the protection of the present invention.

Claims

1. A biocompatible tumor-inducing ferroptosis magnetic resonance nanoprobe, characterized in that: The nanoprobe includes a core-shell structure with ferroferric oxide nanoparticles as the core and apple pectin as the shell. The iron death inducer pyralidone and RGD tumor targeting peptide are loaded on the surface of the core-shell structure. The core-shell structure composite nanoprobe finally formed is the magnetic resonance nanoprobe. The magnetic resonance nanoprobe is spherical, with a particle size range of 15-50 nm and an average particle size of 20-30 nm.

2. A method for preparing a magnetic resonance nanoprobe according to claim 1, characterized in that: The reducing agent apple pectin is modified on the surface of ferroferric oxide nanoparticles by a one-step hydrothermal method to obtain the core-shell structure, referred to as FA nanoparticles; the ferroptosis inducer pyridine is modified on the surface of FA nanoparticles by electrostatic action, and the synthetic product is referred to as FAS nanoparticles; then the RGD tumor targeting peptide is modified on the surface of the FA nanoparticles by an amide reaction method, and the resulting product is the magnetic resonance nanoprobe, referred to as FASR nanoprobe.

3. The preparation method according to claim 2, characterized in that: The following steps are involved: Step 1) Preparation of FA nanoparticles: Mix FeCl3·6H2O and FeSO4·7H2O in water under vigorous stirring to obtain solution A, in which the mass volume ratio of FeCl3·6H2O to water is 54 mg / ml, and the mass volume ratio of FeSO4·7H2O to water is 27.8 mg / ml; heat to 80°C in a three-necked flask under nitrogen protection for 5 minutes; Rapidly drop ammonia water with a mass concentration of 10% into the reaction solution, and continue to react at 80° C. for 30 minutes to obtain solution B, wherein the volume ratio of 10% ammonia water to water in solution A is 0.35:1; Prepare an apple pectin aqueous solution at a mass volume concentration of 100 mg / ml, wherein the molecular weight of the apple pectin is 50000-300000; add the apple pectin aqueous solution to the solution B at a volume ratio of 12:27, heat to 95°C, and stir for 90 minutes; cool naturally to room temperature, separate the product from the reaction mixture with a magnet, and then wash with deionized water several times, collect the precipitate, which is FA nanoparticles; store at 4°C for later use; Step 2) Preparation of FAS nanoparticles: The ferroptosis inducer pyralidone was dissolved in dimethyl sulfoxide at a mass volume ratio of 50 mg / mL to obtain solution C; Dissolve the FA nanoparticles obtained in step 1) in water at a mass volume ratio of 2.55 mg / mL to obtain solution D; Solution C was slowly added dropwise to solution D at a volume ratio of 1:49, and the mixture was stirred vigorously at 37°C for 24 h; after centrifugation, the mixture was washed with a water-dimethyl sulfoxide mixed solution, and the dark brown precipitate was collected, which was the FAS nanoparticles; the mixture was stored at 4°C for later use; Step 3) Preparation of FASR nanoprobes: The FAS nanoparticles obtained in step 2) are dispersed in deionized water at a mass volume ratio of 6 mg / mL, and a condensing agent is added thereto to activate the carboxyl group, wherein the mass ratio of the condensing agent to the FAS nanoparticles is 11:60; the mixture is stirred in a water bath at 37°C for 1 hour to obtain a solution E; RGD tumor targeting peptide was added to solution E at a mass volume ratio of 0.2 mg / mL, and stirred in a light-proof water bath at 37°C for 10-12 h to allow the carboxyl groups on the surface of the FAS nanoparticles to covalently bind to the amino groups of the RGD tumor targeting peptide. The product was separated from the reaction mixture using a magnet, washed several times with deionized water, and the precipitate was collected to obtain the FASR nanoprobe.

4. The preparation method according to claim 3, characterized in that: In step 1), the rate of adding ammonia water is 0.35 mL / s.

5. The preparation method according to claim 3, characterized in that: The condensation agent in step 3) is 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride or is composed of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide in a mass ratio of 6:

5.

6. Use of a magnetic resonance nanoprobe prepared by the preparation method according to any one of claims 3 to 5 in preparing a carrier of a chemotherapeutic drug, wherein doxorubicin is loaded on the surface of the FASR nanoprobe by an amide reaction method, and the process is: The FASR nanoprobe is dispersed in deionized water, and a condensing agent is added thereto to activate the carboxyl group, wherein: The mass ratio of the condensing agent to the FASR nanoprobe was 11:30, and the mixture was stirred in a water bath at 37°C for 1 h to obtain solution F; Doxorubicin was added to solution F at a mass ratio of 1:6 between doxorubicin and FASR nanoprobes, and stirred in a water bath at 37°C in the dark for 10-12 h to allow the carboxyl groups on the surface of FASR nanoparticles to covalently bind to the amino groups of doxorubicin; The product was separated from the reaction mixture by a magnet, washed several times with deionized water, and the precipitate was collected, which was the FASR nanoprobe loaded with doxorubicin, referred to as FASDR nanoprobe.

7. Use of the magnetic resonance nanoprobe according to claim 6 in preparing a carrier of a chemotherapeutic drug, characterized in that: The condensing agent is 4 (-4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride or is composed of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide in a mass ratio of 6:5.

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