Iron-based nano composite material as well as preparation method and application thereof

Abstract

The invention discloses an iron-based nano composite material and a preparation method and application thereof. The invention relates to an iron-based nano composite material which is obtained by taking an iron-based metal organic framework material as a carrier, loading a medicine 1, wrapping the surface of the iron-based metal organic framework material with polydopamine and combining with a medicine 2 through a borate bond, the iron-based metal organic framework material is MIL-88, the medicine 1 is beta-lapamoquinone, and the medicine 2 is chlorambucil. The preparation method comprises the following steps: S1, preparing MIL-88; s2, preparing MIL-88-beta-lap (MIL-88-beta-lap) S3, MIL-88-beta-lap (PDA) is prepared, and MIL-88-beta- S4, cb-BA is prepared; s5, MIL-88-beta-lap (PDA)-cb is prepared, and the PDA-cb is prepared; the iron-based nano composite material is used as a drug carrier to be applied to tumor treatment. According to the invention, combined treatment of ferroptosis, photo-thermal treatment and chemotherapy can be realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to an iron-based nanocomposite material, a preparation method thereof, and an application thereof. Background Art

[0002] The harm of cancer has been intensifying globally and has become the number one "killer" threatening human health. At present, the most commonly used treatment methods in clinical practice are surgery, chemotherapy, and radiotherapy. Chemotherapy can cause a series of acute and chronic organ toxicities. Since many chemotherapeutic drugs and their metabolites are excreted through renal tubular epithelial cells, the kidneys are easily damaged by chemotherapeutic drugs. Therefore, the use of chemotherapeutic drugs is sometimes limited by their nephrotoxic side effects. Chemotherapy-related kidney damage usually leads to insufficient cancer treatment because renal dysfunction requires clinicians to reduce the chemotherapy dose to avoid further kidney damage. Kidney damage can also cause other adverse complications, such as water and nitrogenous waste retention, electrolyte disorders, decreased immunity, etc. Developing effective drug carriers to reduce the inherent adverse reactions of chemotherapeutic drugs and improve the treatment effect has become one of the key issues in cancer treatment. At present, in order to enhance the enhanced permeability and retention effect, drug nanocarriers have been developed to improve the accumulation of drugs in the tumor region, and the nanocarriers include polymer vesicles, micelles, polymer nanoparticles, inorganic nanoparticles, and hybrid porous solids. Among them, nano-metal-organic frameworks (MOFs) have attracted attention as drug nanocarriers with high drug loading capacity due to their high pore volume, large surface area, and adjustable pore size within the framework.

[0003] As an emerging treatment strategy, CDT usually utilizes the Fenton reaction mediated by Fe(II) ions to convert endogenous hydrogen peroxide (H2O2) into highly reactive and toxic hydroxyl radicals (·OH) with strong oxidizing ability. CDT usually functions under acidic conditions and does not require external energy and oxygen, which is a safe and effective tumor treatment method. The efficiency of generating ·OH in a typical Fenton catalytic reaction mainly depends on the type of catalyst and the level of endogenous H2O2. Although the intracellular H2O2 level is higher than that of normal cells due to metabolic changes in tumor cells, the insufficient content of endogenous H2O2 is difficult to achieve satisfactory CDT efficacy.

[0004] β-lapachone, chemically named 3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]-pyran-5,6-dione, belongs to the 1,2-naphthoquinone class and is a compound with a wide range of biological activities. Its potential in tumor treatment has gradually attracted attention. β-lapachone can significantly inhibit the proliferation, invasion, and migration abilities of cancer cells. However, it has large toxic side effects and relatively poor treatment effects.

[0005] Photothermal therapy (PTT) is a new non-invasive tumor treatment method that uses a photothermal conversion agent (PTA) to convert light energy into heat energy under the irradiation of external light sources such as near-infrared light (NIR) to kill tumor cells. Due to the limited transmission ability of lasers, photothermal therapy cannot penetrate deep into the deep tissues of organisms, which limits its therapeutic effect on internal tumors. When photothermal therapy materials are used for treatment, their circulation in the human body has not been fully understood, and there has been no long-term experimental observation, so their potential toxicity remains unknown. This may lead to some unforeseen side effects or long-term health problems.

[0006] Chlorambucil is an alkylating agent and belongs to cytotoxic chemotherapy drugs. It mainly binds to DNA, interferes with cell replication and division, affects the DNA function of tumor cells, thereby inhibiting the growth of cancer cells and achieving the purpose of anti-tumor treatment. However, it has large toxic side effects and relatively poor therapeutic effects.

[0007] Based on the above situation, it is necessary to develop a nano-drug-loaded composite that can directly deliver β-lapachone and chlorambucil to cancer cells and achieve the combined treatment of ferroptosis, photothermal therapy and chemotherapy. Summary of the Invention

[0008] Based on this, it is necessary to provide an iron-based nanocomposite material, a preparation method thereof and an application in view of the defects and deficiencies of the prior art.

[0009] An iron-based nanocomposite material is obtained by using an iron-based metal-organic framework material as a carrier, loading drug 1, and coating polydopamine on its surface and binding drug 2 through a borate ester bond. The iron-based metal-organic framework material is MIL-88, the drug 1 is β-lapachone, and the drug 2 is chlorambucil.

[0010] A preparation method of the iron-based nanocomposite material as described above includes the following steps:

[0011] S1. Prepare MIL-88;

[0012] S2. React MIL-88 prepared in S1 with β-lapachone to obtain MIL-88-β-lap;

[0013] S3. React tris(hydroxymethyl)aminomethane, MIL-88-β-lap prepared in S2 and dopamine hydrochloride in an alkaline environment to obtain MIL-88-β-lap@PDA;

[0014] S4. 4-[Bis(2-chloroethyl)amino]phenylbutyric acid nitrogen mustard (cb), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine and 4-hydroxymethylphenylboronic acid react to obtain cb-BA;

[0015] S5. The MIL-88-β-lap@PDA prepared in S3, the cb-BA prepared in S4 and anhydrous magnesium sulfate react to obtain the iron-based nanocomposite MIL-88-β-lap@PDA-cb.

[0016] As a preferred embodiment, the method for preparing MIL-88 in S1 includes the following steps:

[0017] Ferric chloride hexahydrate and amino-terephthalic acid are subjected to a microwave synthesis reaction to obtain MIL-88.

[0018] As a preferred embodiment, the mass ratio of the ferric chloride hexahydrate to the amino-terephthalic acid is 1.316:1.

[0019] As a preferred embodiment, the mass ratio of the MIL-88 to the β-lapachone in S2 is 2:1.

[0020] As a preferred embodiment, the mass ratio of the tris(hydroxymethyl)aminomethane, the MIL-88-β-lap and the dopamine hydrochloride in S3 is 12:5:10.

[0021] As a preferred embodiment, the pH value of the alkaline environment in S3 is 8.0 - 8.5.

[0022] As a preferred embodiment, the mass ratio of the 4-[bis(2-chloroethyl)amino]phenylbutyric acid nitrogen mustard (cb), the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, the 4-dimethylaminopyridine and the 4-hydroxymethylphenylboronic acid in S4 is 152:192:61:99.

[0023] As a preferred embodiment, the mass ratio of the MIL-88-β-lap@PDA, the cb-BA and the anhydrous magnesium sulfate in S5 is 4:1:8.

[0024] An iron-based nanocomposite as described above is used as a drug carrier in tumor treatment.

[0025] Tumor treatment mechanism: Iron ions in the iron-based nanocomposite can react with endogenous hydrogen peroxide through the Fenton reaction to generate highly toxic hydroxyl radicals, leading to cell death; β-lap inhibits glutathione peroxidase 4 (GPX4), triggering ferroptosis; Polydopamine (PDA) has excellent near-infrared light absorption ability, which can efficiently convert light energy into heat energy, resulting in a rapid increase in local temperature, directly killing tumor cells, and at the same time further accelerating the release of chlorambucil. Chlorambucil (cb) can hinder DNA replication and transcription, inducing apoptosis, thus realizing the combined treatment of ferroptosis, photothermal therapy and chemotherapy.

[0026] The beneficial effects of the present invention are as follows:

[0027] Using the iron-based metal-organic framework material MIL-88 as a carrier, loading β-lapachone (β-lap), and forming polydopamine (PDA) on its surface, further binding chlorambucil (cb) through the formation of borate bonds, and finally obtaining the iron-based nanocomposite MIL-88-β-lap@PDA-cb. Iron ions in the iron-based nanocomposite can react with endogenous hydrogen peroxide through the Fenton reaction to generate highly toxic hydroxyl radicals, leading to cell death. β-lap inhibits glutathione peroxidase 4 (GPX4), triggering ferroptosis. Polydopamine (PDA) has excellent near-infrared light absorption ability, which can efficiently convert light energy into heat energy, resulting in a rapid increase in local temperature, directly killing tumor cells, and at the same time further accelerating the release of chlorambucil. Chlorambucil (cb) can hinder DNA replication and transcription, inducing apoptosis, thus realizing the combined treatment of ferroptosis, photothermal therapy and chemotherapy. Description of the Drawings

[0028] Figure 1 XRD spectra of MIL-88, MIL-88-β-lap, MIL-88-β-lap@PDA and MIL-88-β-lap@PDA-cb of the present invention;

[0029] Figure 2 Particle size distribution diagrams of MIL-88, MIL-88-β-lap, MIL-88-β-lap@PDA and MIL-88-β-lap@PDA-cb of the present invention;

[0030] Figure 3 Zeta potential diagrams of MIL-88, MIL-88-β-lap, MIL-88-β-lap@PDA and MIL-88-β-lap@PDA-cb of the present invention;

[0031] Figure 4Scanning electron microscopy (SEM) images of MIL-88(a), MIL-88-β-lap(b), MIL-88-β-lap@PDA(c), and MIL-88-β-lap@PDA-cb(d) of the present invention;

[0032] Figure 5 Transmission electron microscopy (TEM) images of MIL-88(a), MIL-88-β-lap(b), MIL-88-β-lap@PDA(c), and MIL-88-β-lap@PDA-cb(d) of the present invention;

[0033] Figure 6 Nitrogen adsorption-desorption isotherm curves (a) and pore size distribution diagrams (b) of MIL-88, MIL-88-β-lap, MIL-88-β-lap@PDA, and MIL-88-β-lap@PDA-cb of the present invention;

[0034] Figure 7 Ultraviolet-visible absorption spectra of MIL-88, β-lap, MIL-88-β-lap, MIL-88-β-lap@PDA, cb, and MIL-88-β-lap@PDA-cb of the present invention;

[0035] Figure 8 Fourier transform infrared spectroscopy (FT-IR) spectra of β-lap, DA, cb-BA, MIL-88, MIL-88-β-lap, MIL-88-β-lap@PDA, and MIL-88-β-lap@PDA-cb of the present invention;

[0036] Figure 9 Temperature change diagrams of MIL-88-β-lap@PDA solution with different concentrations under 808 nm laser at a power of 1.0 W cm -2 Power;

[0037] Figure 10 Temperature change diagrams of MIL-88-β-lap@PDA solution with different concentrations under 808 nm laser at a power of 1.0 W cm -2 Power;

[0038] Figure 11 Temperature change diagrams of MIL-88-β-lap@PDA solution with a concentration of 200.0 μg / mL under 808 nm laser at different powers;

[0039] Figure 12 Near-infrared thermal imaging diagrams of MIL-88-β-lap@PDA solution with a concentration of 200.0 μg / mL under 808 nm laser at different powers;

[0040] Figure 13 The temperature change curve (a) of the MIL-88-β-lap@PDA solution with a concentration of 200.0 μg / mL of the present invention under 5 laser on / off cycles of 808 nm laser at a power of 2.0 W / cm -2 ; the natural logarithm plots of the temperature rise curve, the temperature drop curve, and the curve obtained with the cooling cycle (b);

[0041] Figure 14 The inhibition result diagrams of MCF-7 cells cultured for 24 h by MIL-88, cb, β-lap, MIL-88-β-lap, MIL-88-β-lap@PDA, MIL-88-β-lap@PDA+Laser, MIL-88-β-lap@PDA-cb, and MIL-88-β-lap@PDA-cb+Laser of the present invention;

[0042] Figure 15 The inhibition result diagrams of MCF-7 cells cultured for 48 h by MIL-88, cb, β-lap, MIL-88-β-lap, MIL-88-β-lap@PDA, MIL-88-β-lap@PDA+Laser, MIL-88-β-lap@PDA-cb, and MIL-88-β-lap@PDA-cb+Laser of the present invention;

[0043] Figure 16 The uptake situation diagram of MCF-7 cells of the present invention for MIL-88 at different time points (scale bar: 50 μm);

[0044] Figure 17 The reactive oxygen species level diagram of MCF-7 cells detected by DCFH-DA of the present invention (scale bar: 50 μm);

[0045] Figure 18 The intracellular cell viability and toxicity diagram of MCF-7 cells detected by Calcein AM / PI of the present invention (scale bar: 100 μm);

[0046] Figure 19 The change result diagram of mitochondrial membrane potential in MCF-7 cells detected by JC-1 probe of the present invention (scale bar: 50 μm);

[0047] Figure 20 The result diagram of the level of lipid peroxides in MCF-7 cells of the present invention (scale bar: 50 μm). Detailed implementation manners

[0048] The endpoints and any values in the ranges disclosed herein are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0049] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only for the purpose of illustrating and explaining the present invention, and are not used to limit the present invention.

[0050] Example 1

[0051] Preparation of iron-based nanocomposite MIL-88-β-lap@PDA-cb

[0052] S1. Take a clean beaker, add 30 mL of DMF solution thereto, then weigh 329 mg of FeCl3·6H2O and add it thereto, stir or ultrasonicate until completely dissolved. After the solution is completely clarified, weigh 250 mg of amino-terephthalic acid (NH2-BDC) and add it thereto, stir or ultrasonicate until completely dissolved. After the above solution is stirred at room temperature for 15 minutes, transfer it into a microwave synthesis reactor, microwave react at 600 W and 120 °C for 30 min. After the instrument completely drops to room temperature, take out the reactor, centrifuge the product at 11000 rpm for 8 min to collect the product, then wash the product twice with DMF and once with ethanol, and place it in an oven at 60 °C for drying to obtain MIL-88;

[0053] S2. Take 10.0 mg of MIL-88 and dissolve it in a mixed solution of 2.0 mL of DMSO and 9.0 mL of deionized water, ultrasonicate for 20 min to obtain solution ①; weigh 5.0 mg of β-lap and dissolve it in 1.0 mL of DMSO to obtain solution ②; add solution ② dropwise to solution ①, and stir vigorously in the dark for 16 hours. After the stirring is completed, centrifuge at 12000 rpm for 15 min to collect the precipitate product, then wash it three times with deionized water, and finally vacuum dry the obtained product for 12 hours to obtain MIL-88-β-lap;

[0054] S3. Weigh 61.0 mg of Tris and ultrasonicate it in 50.0 mL of deionized water until completely dissolved, and adjust the pH of the solution to 8.0 - 8.5. Then weigh 25.0 mg of MIL-88-β-lap and add it thereto, ultrasonicate for 5 minutes to disperse it evenly. Then weigh 50.0 mg of dopamine hydrochloride and add it thereto, stir at room temperature for 18 hours. After the stirring is completed, centrifuge at 12000 rpm for 15 min to collect the precipitate product, then wash it three times with deionized water, and finally vacuum dry the obtained product for 12 hours to obtain MIL-88-β-lap@PDA;

[0055] S4. Weigh 152.0 mg of 4-[bis(2-chloroethyl)amino]phenylbutyric acid nitrogen mustard (cb), 192.0 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCL), and 61.0 mg of 4-dimethylaminopyridine (DMAP) and dissolve them in 3.0 mL of dimethyl sulfoxide (DMSO). React under stirring at room temperature for 30 minutes. Subsequently, weigh 99.0 mg of 4-hydroxymethylphenylboronic acid (BA) and dissolve it in 2.0 mL of DMSO. Dropwise add it to the solution under stirring at room temperature and react for 24 hours. Subsequently, add ethyl acetate solution, wash it with aqueous sodium bicarbonate solution, and obtain cb-BA by column chromatography;

[0056] S5. Under the protection of nitrogen, take 2.5 mg of cb-BA, 10 mg of MIL-88-β-lap@PDA, and 20.0 mg of anhydrous magnesium sulfate and dissolve them in 10.0 mL of DMSO. Stir and react the mixture at room temperature for 24 hours. Subsequently, centrifuge the solution at 12000 rpm for 10 min, collect the precipitate product, wash it once with deionized water and once with DMSO, and finally vacuum dry the obtained product for 12 hours to obtain MIL-88-β-lap@PDA-cb.

[0057] Test Example 1

[0058] X-ray diffraction experiment

[0059] Perform X-ray diffraction tests on MIL-88 prepared in S1 of Example 1, MIL-88-β-lap prepared in S2 of Example 1, MIL-88-β-lap@PDA prepared in S3 of Example 1, and MIL-88-β-lap@PDA-cb prepared in S5 of Example 1 using an X-ray diffractometer. The test results are as Figure 1 shown.

[0060] It can be seen from Figure 1 that the peak positions of MIL-88 and the typical diffraction peaks are in the range of 3° to 50°, which are 2θ = 8.9°, 9.6°, 10.0°, 16.3°, 17.7°, 18.7°, 24.5°, and 27.7° respectively, and the MIL-88 carrier is successfully prepared. The loading of β-lap does not interfere with the integrity of the MIL-88 crystal structure. The formation of polydopamine covers the surface of the crystal, resulting in a decrease in its crystallinity and a weakening of the peak intensity, indicating the successful preparation of the iron-based nanocomposite.

[0061] Test Example 2

[0062] Particle size distribution experiment

[0063] The particle size tests were carried out on MIL-88 prepared by S1 in Example 1, MIL-88-β-lap prepared by S2 in Example 1, MIL-88-β-lap@PDA prepared by S3 in Example 1, and MIL-88-β-lap@PDA-cb prepared by S5 in Example 1 using a particle size analyzer. The experimental results are as Figure 2 shown.

[0064] It can be Figure 2 seen that the average particle size of MIL-88 is about 166.8 nm. Due to the loading of β-lap, the average particle size of MIL-88-β-lap increases to about 217.2 nm. Polydopamine (PDA) is formed on its surface and the particles are partially aggregated. The average particle size of MIL-88-β-lap@PDA is about 238.4 nm. Further combined with chlorambucil (cb), the particle size increases, and the average particle size of MIL-88-β-lap@PDA-cb is about 271.2 nm.

[0065] Test Example 3

[0066] Zeta potential experiment

[0067] The Zeta potential tests were carried out on MIL-88 prepared by S1 in Example 1, MIL-88-β-lap prepared by S2 in Example 1, MIL-88-β-lap@PDA prepared by S3 in Example 1, and MIL-88-β-lap@PDA-cb prepared by S5 in Example 1 using a Zeta potential tester. The experimental results are as Figure 3 shown.

[0068] It can be Figure 3 seen that the potential of MIL-88 is +13.96 mV. Since β-lap is negatively charged, the potential of MIL-88-β-lap is -6.08 mV. After polydopamine is formed on the surface, there are a large number of hydroxyl groups on the surface. Therefore, the negative potential level of MIL-88-β-lap@PDA is further reduced to -28.66 mV. Further combined with chlorambucil (cb) which is positively charged, the potential of MIL-88-β-lap@PDA-cb is -12.5 mV. The change in Zeta potential indicates the successful preparation of the iron-based nanocomposite.

[0069] Test Example 4

[0070] Scanning electron microscope and transmission electron microscope experiments

[0071] The MIL-88 prepared from S1 in Example 1, the MIL-88-β-lap prepared from S2 in Example 1, the MIL-88-β-lap@PDA prepared from S3 in Example 1, and the MIL-88-β-lap@PDA-cb prepared from S5 in Example 1 were tested using a scanning electron microscope and a transmission electron microscope respectively. The experimental results are as Figure 4 and Figure 5 shown.

[0072] It can be seen from Figure 4 and Figure 5 that MIL-88 has a polyhedral structure with uniform size and distinct edges and corners. After β-lapachone (β-lap) is loaded into the pores of MIL-88, the particle size of MIL-88-β-lap slightly increases, the morphology remains unchanged, and it is uniform and stable. Due to the formation of polydopamine on the surface of MIL-88-β-lap@PDA, the morphology is affected and the edges and corners become smooth. Further combination with chlorambucil (cb) to form MIL-88-β-lap@PDA-cb does not result in an obvious change in morphology.

[0073] Test Example 5

[0074] Specific Surface Area Measurement

[0075] The specific surface area and pore size of the MIL-88 prepared from S1 in Example 1, the MIL-88-β-lap prepared from S2 in Example 1, the MIL-88-β-lap@PDA prepared from S3 in Example 1, and the MIL-88-β-lap@PDA-cb prepared from S5 in Example 1 were measured using a specific surface area sorption instrument. The experimental results are as Figure 6 and Table 1 shows.

[0076] Table 1 shows the specific surface area and pore volume of MIL-88, MIL-88-β-lap, MIL-88-β-lap@PDA, and MIL-88-β-lap@PDA-cb

[0077]

[0078] It can be seen from Figure 6 and Table 1 that MIL-88, MIL-88-β-lap, MIL-88-β-lap@PDA, and MIL-88-β-lap@PDA-cb all exhibit a Type I isotherm curve due to the presence of micropores. The specific surface area and pore volume results are shown in Table 1. The specific surface area of MIL-88 is 625.7 m 2 / g, and the pore volume is 0.18 cm 3 / g. β-lap is loaded in MIL-88, occupying most of the pores, and the specific surface area is reduced to 325.8 m 2 / g, and the pore volume is 0.10 cm 3 / g, proving the effective loading of β-lap. When polydopamine (PDA) is formed on the surface, the pores are almost blocked, and both the specific surface area and pore size are reduced (12.1 m 2 / g, 0.003 cm 3 / g), indicating the successful encapsulation of dopamine hydrochloride (DA). Further combined with chlorambucil (cb), the specific surface area of MIL-88-β-lap@PDA-cb is only 5.0 m 2 / g.

[0079] Test Example 6

[0080] Ultraviolet-visible absorption spectroscopy experiment

[0081] The MIL-88 prepared from S1 in Example 1, commercially available β-lap, MIL-88-β-lap prepared from S2 in Example 1, MIL-88-β-lap@PDA prepared from S3 in Example 1, commercially available cb, and MIL-88-β-lap@PDA-cb prepared from S5 in Example 1 were tested using an ultraviolet absorption spectrometer, and the experimental results are as Figure 7 shown.

[0082] As can be Figure 7 seen, β-lap shows a characteristic absorption peak at 259 nm, and MIL-88-β-lap exhibits the characteristic absorption peak of β-lap at 259 nm, proving the successful loading of β-lap. Cb has a characteristic absorption at 262 nm, and MIL-88-β-lap@PDA-cb shows the characteristic absorption peak of cb at 262 nm. It proves the successful preparation of the iron-based nanocomposite MIL-88-β-lap@PDA-cb.

[0083] Test Example 7

[0084] Fourier transform infrared spectroscopy determination

[0085] The commercially available β-lap, commercially available DA, cb-BA prepared from S4 in Example 1, MIL-88 prepared from S1 in Example 1, MIL-88-β-lap prepared from S2 in Example 1, MIL-88-β-lap@PDA prepared from S3 in Example 1, and MIL-88-β-lap@PDA-cb prepared from S5 in Example 1 were tested using a Fourier transform infrared spectrometer, and the experimental results are as Figure 8 shown.

[0086] As can beFigure 8 It can be seen that at 1386 cm -1 and 1658 cm -1 are the characteristic peaks of the stretching vibration of C═C in the benzene ring and the stretching vibration of C═O in the carboxyl group of the terephthalic acid ligand of MIL-88, indicating that the carboxyl group coordinates with metal ions to form a metal-carboxyl bond. At 770 cm -1 is the vibration absorption peak of the metal-oxygen (M-O) bond, indicating the successful preparation of MIL-88. β-Lapachone (β-lap) will show a characteristic stretching vibration peak of the carbonyl group (C═O) at around 1700 cm -1 . The loading of β-lap retains the basic skeleton of MIL-88. The presence of an amino absorption peak in the region of 1479 cm -1 indicates the formation of polydopamine. The characteristic absorption peak position of the borate ester at 1021 cm -1 indicates the successful preparation of the MIL-88-β-lap@PDA-cb iron-based nanocomposite.

[0087] Test Example 8

[0088] Photothermal effect of MIL-88-β-lap@PDA at different concentrations

[0089] Accurately weigh an appropriate amount of MIL-88-β-lap@PDA-cb prepared by S5 in Example 1 using an analytical balance, dissolve it in a certain amount of DMSO solution to prepare a solution with a concentration of 1 mg / mL, and then dilute it into test solutions with concentrations of 50.0, 100.0, 150.0, and 200.0 μg / mL respectively. Use deionized water as a blank control. Then irradiate the test solutions under the laser irradiation conditions of a wavelength of 808 nm and a power of 1.0 W cm -2 for 0, 100, 200, and 300 s respectively. Then use a thermometer to measure the temperature of the test solution, record the results, and plot a graph with the temperature as the ordinate and the irradiation time as the abscissa. The experimental results are as Figure 9 shown; at the same time, use an infrared thermal imager to detect the corresponding test solution, and the experimental results are as Figure 10 shown.

[0090] It can be seen from Figure 9 and Figure 10 that as the concentration increases, the temperature of MIL-88-β-lap@PDA increases, indicating a concentration dependence. At 200.0 μg / mL, it can reach up to 42.0 °C at most, showing a good photothermal effect.

[0091] Test Example 9

[0092] Photothermal effect of MIL-88-β-lap@PDA at different power densities

[0093] Weigh an appropriate amount of MIL-88-β-lap@PDA-cb prepared in S5 of Example 1 accurately using an analytical balance, dissolve it in a certain amount of DMSO solution to prepare a test solution with a concentration of 200 μg / mL. Then, irradiate the test solution under laser irradiation conditions with a wavelength of 808 nm and powers of 0.5, 1.0, 1.5, and 2.0 W / cm -2 for 0, 100, 200, and 300 s respectively. Then, measure the temperature of the test solution with a thermometer, record the results, plot the temperature on the vertical axis and the irradiation time on the horizontal axis. The experimental results are as shown in Figure 11 ; at the same time, detect the corresponding test solution with an infrared thermal imager, and the experimental results are as shown in Figure 12 .

[0094] It can be seen from Figure 11 and Figure 12 that as the power increases, the temperature of the MIL-88-β-lap@PDA solution increases accordingly, indicating a dependence on the power density. The MIL-88-β-lap@PDA solution with a concentration of 200 μg / mL can reach a maximum temperature of 55.0 °C at a power density of 2.0 W / cm -2 , indicating that the MIL-88-β-lap@PDA solution has good photothermal effects, can effectively convert 808 nm laser into heat energy, and can be effectively used for photothermal therapy.

[0095] Test Example 10

[0096] Photothermal stability of MIL-88-β-lap@PDA

[0097] Weigh an appropriate amount of MIL-88-β-lap@PDA-cb prepared in S5 of Example 1 accurately using an analytical balance, dissolve it in a certain amount of DMSO solution to prepare a test solution with a concentration of 200 μg / mL. Then, irradiate the test solution under laser irradiation conditions with a wavelength of 808 nm and a power of 2.0 W / cm -2 for 5 min, then turn off the laser and let it cool naturally for 5 min. Continuously repeat the laser on / off process 5 times, measure the temperature of the test solution with a thermometer, record the results, plot the temperature on the vertical axis and the irradiation time on the horizontal axis, and finally calculate the photothermal conversion efficiency. The results are as shown in Figure 13 .

[0098] Calculate the photothermal conversion efficiency of the aqueous solution of MIL-88-β-lap@PDA. According to the time constant and the highest temperature, its photothermal conversion efficiency is shown by Equation 1:

[0099]

[0100] Among them, h is the heat transfer coefficient, s is the surface area of the container, and T max is the maximum temperature reached after the aqueous solution of MIL-88-β-lap@PDA is irradiated with laser, and T sur is the ambient temperature, Q Dis is the heat loss, I is the effective power of the aqueous solution receiving the laser, and A 808 is the ultraviolet absorption value of the 200.0 μg / mL aqueous solution of MIL-88-β-lap@PDA at 808 nm.

[0101]

[0102] Among them, m D is the mass of the solution, C D is the specific heat capacity of the solution, and τ s is the system time constant.

[0103]

[0104] Among them, t is the cooling time, T is the temperature at different time points during cooling, and T sur is the ambient temperature, and T max is the maximum temperature reached after the aqueous solution of MIL-88-β-lap@PDA is irradiated with laser.

[0105] Finally, the photothermal conversion efficiency (η) is calculated using Equation 4:

[0106]

[0107] From Figure 13 a, it can be seen that after continuously repeating the laser on / off 5 times, the maximum temperature does not decrease significantly, proving that the photothermal performance of MIL-88-β-lap@PDA is good during the cycling process; from Figure 13 b, it can be seen that based on the time constant and the maximum temperature, the photothermal conversion efficiency of MIL-88-β-lap@PDA is 31.0%, that is, under the condition of 808 nm laser irradiation, 31.0% of the absorbed light energy can be converted into heat energy.

[0108] Test Example 11

[0109] 1) Toxicity experiment on MCF-7 cells

[0110] The cytotoxicity of MIL-88, β-lap, cb, MIL-88-β-lap, MIL-88-β-lap@PDA, MIL-88-β-lap@PDA-cb, MIL-88-β-lap@PDA+Laser, MIL-88-β-lap@PDA-cb+Laser against MCF-7 cells was detected by the MTT method. MCF-7 cells were seeded in 96-well plates at a density of 5000 - 8000 cells, and then incubated in a cell culture incubator for 24 h. After incubation, various nanomaterials with certain concentrations were prepared and diluted to the required concentration with DMEM complete medium. Then, the various nanomaterials were added to the 96-well plates and co-incubated with the cells for 24 h and 48 h. For the laser irradiation group (+Laser), the cells were irradiated with 808 nm laser (2 W cm -2 ) for 5 min and then continued to be incubated. After the incubation time ended, 100.0 μL of MTT solution (5.0 mg / mL) was added to each well and incubated for 4 h. After the incubation time ended, the old culture medium was discarded, 140.0 μL of DMSO solution was added to each well, and the plate was shaken on a shaker at room temperature for 15 min. The absorbance value was measured at a wavelength of 490 nm using a multifunctional microplate reader. During the experiment, a certain number of blank wells, control wells, and experimental wells were set, and 5 replicates were set for each group. The cell survival rate was calculated by formula 5:

[0111]

[0112] The above data were sorted out and charts were drawn. The results are shown as Figure 14 follows;

[0113] 2) Cytotoxicity experiment on L929 cells

[0114] The cytotoxicity of MIL-88, β-lap, cb, MIL-88-β-lap, MIL-88-β-lap@PDA, MIL-88-β-lap@PDA-cb, MIL-88-β-lap@PDA+Laser, MIL-88-β-lap@PDA-cb+Laser against L929 cells was detected by the MTT method. L929 cells were seeded in 96-well plates at a density of 5000 - 8000 cells, and then incubated in a cell culture incubator for 24 h. After incubation, various nanomaterials with certain concentrations were prepared and diluted to the required concentration with DMEM. Then, the various nanomaterials were added to the 96-well plates and co-incubated with the cells for 24 h and 48 h. For the laser irradiation group (+Laser), the cells were irradiated with 808 nm laser (2 W cm -2) Incubate for 5 min and continue incubation. After the incubation time ends, add 100.0 μL of MTT solution (5.0 mg / mL) to each well and incubate for 4 h. After the incubation time ends, discard the old medium, add 140.0 μL of DMSO solution to each well, shake on a shaker at room temperature for 15 min, and measure the absorbance value at a wavelength of 490 nm using a multifunctional microplate reader. During the experiment, set a certain number of blank wells, control wells, and experimental wells, and set 5 replicates for each group. The cell survival rate is calculated using formula 6:

[0115]

[0116] Organize the above data and draw charts. The results are as Figure 15 shown;

[0117] All data are expressed as mean ± standard deviation (x ± s). The differences between groups are compared using one-way ANOVA. After ANOVA, LSD-t test is continued. GraphPad Prism 8.0 software is used for analysis. The test level is α = 0.05. When P < 0.05, the difference is statistically significant. When P > 0.05, the difference is not statistically significant.

[0118] From Figure 14 and Figure 15 it can be seen that when the concentration of β-lap increases from 1.0 μg / mL to 12.0 μg / mL and the concentration of cb increases from 8 μg / mL to 48 μg / mL, the inhibition rates of all samples on MCF-7 cells gradually increase, showing concentration dependence. The higher the concentration, the stronger the inhibitory effect. In addition, it can be clearly seen that the inhibitory effects of MIL-88, MIL-88-β-lap, and MIL-88-β-lap@PDA-cb on cells gradually increase. And compared with MIL-88-β-lap@PDA-cb, MIL-88-β-lap@PDA-cb shows the strongest inhibitory effect on MCF-7 cells after irradiation with 808 nm laser, with an inhibition rate as high as 79.0%. When administered for 48 hours, the inhibitory effect of MIL-88-β-lap@PDA-cb + Laser on MCF-7 cells further increases, with an inhibition rate of 88.0%. This indicates that the combined treatment of ferroptosis, photothermal therapy, and chemotherapy has a stronger inhibitory effect on tumor cells.

[0119] Test Example 12

[0120] MCF-7 Cell Uptake Experiment

[0121] Rhodamine B (RhB) is a commonly used cell fluorescent staining agent in the laboratory, which has good fluorescence characteristics. Its characteristics are utilized to detect fluorescence signals to determine the uptake of materials by MCF-7 cells. Rhodamine B (RhB) was loaded into MIL-88 to obtain MIL-88-RhB. MCF-7 cells were evenly seeded in a 12-well plate at a cell density of 40,000 per well and incubated for 24 h. A certain concentration of MIL-88-RhB was prepared and diluted to 60.0 mg / mL with DMEM complete medium. To explore the uptake of materials by MCF-7 cells at different times, the incubation times were set to 0, 1, 3, 6, 9, and 12 h during the experiment. That is, according to the time points, 1.0 mL of MIL-88-RhB solution was added to the 12-well plate. After incubation in the cell culture incubator for the corresponding time, the old medium was discarded, and the cells were washed twice with PBS. Subsequently, the uptake of materials by MCF-7 cells was observed and photographed under an inverted fluorescence microscope. The results are as Figure 16 shown.

[0122] As Figure 16 can be seen, MCF-7 cells were co-incubated with MIL-88-RhB for 0, 1, 3, 6, 9, and 12 h. With the increase of the administration time, the intensity of the red fluorescence presented gradually increased, indicating that the uptake amount of materials by MCF-7 cells increased. At 12 h of administration, the fluorescence intensity presented was the strongest, reflecting that the uptake of materials by MCF-7 cells was time-dependent.

[0123] Test Example 13

[0124] Experiment on the generation of reactive oxygen species (ROS) in MCF-7 cells

[0125] An appropriate amount of MIL-88 prepared in S1 of Example 1, MIL-88-β-lap@PDA prepared in S3 of Example 1, and MIL-88-β-lap@PDA-cb prepared in S5 of Example 1 were respectively added with DCFH-DA probe, and sample solutions with a concentration of 1 mg / mL were prepared with DMEM medium and diluted to a concentration of 100 μg / mL with DMEM medium. The PBS solution was used as the blank control group.

[0126] Take MCF-7 cells in good condition and seed them in a 12-well cell plate at a cell density of 60,000 per well. Shake evenly and place them in the cell culture incubator for 24 hours. After the cells are completely adherent, discard the original medium, wash once with PBS, and add 1.0 mL of each material to co-incubate with MCF-7 cells for 4 h. For the laser irradiation group (+Laser), irradiate with 808 nm laser (2 Wcm -2) Incubate for 5 min and continue incubation. After the incubation is completed, discard the old medium, wash twice with PBS, then add 1.0 mL of DCFH-DA (serum-free DMEM:DCFH-DA = 1:1000) probe, place it in a cell culture incubator and incubate at 37 °C for 30 min. After the incubation is completed, wash twice with PBS, observe the fluorescence under an inverted fluorescence microscope and take pictures. The results are as Figure 17 shown.

[0127] As Figure 17 can be seen, after treatment with MIL-88, weak green fluorescence appears in MCF-7 cells because the iron ions in MIL-88 react with hydrogen peroxide to generate a small amount of reactive oxygen species. After treatment with MIL-88-β-lap, the intensity of green fluorescence in MCF-7 cells increases slightly. β-lap generates hydrogen peroxide through the reduction reaction mediated by quinone oxidoreductase 1 (NQO1), thereby increasing the generation of reactive oxygen species. After treatment of MIL-88-β-lap@PDA with near-infrared laser, the level of reactive oxygen species in MCF-7 cells is significantly enhanced, indicating that the photothermal conditions induce the rupture of the polydopamine shell, accelerate the release of β-lap, and thus enhance the generation of reactive oxygen species. After treatment with MIL-88-β-lap@PDA-cb+Laser, the green fluorescence in MCF-7 cells is enhanced, generating a large amount of reactive oxygen species, thereby killing tumor cells.

[0128] Test Example 14

[0129] Calcein AM / PI staining experiment

[0130] Seed MCF-7 cells in a 12-well plate at a cell density of 100,000 cells per well, shake well, and place in a cell culture incubator for 24 h. The experiment is divided into 10 groups, namely: PBS, MIL-88, β-lap, cb, MIL-88-β-lap, MIL-88-β-lap@PDA, MIL-88-β-lap@PDA-cb, PBS+Laser, MIL-88-β-lap@PDA+Laser, MIL-88-β-lap@PDA-cb+Laser. Prepare certain concentrations of each nanomaterial and dilute them to the required concentration with DMEM, and add 1.0 mL of each material to co-incubate with MCF-7 cells for 4 h. For the laser irradiation group (+Laser), irradiate with an 808 nm laser (2 W cm -2 ) for 10 min and continue incubation. After the incubation is completed, discard the old medium, wash twice with PBS, then add 1 mL of Calcein AM / PI probe, place it in a cell culture incubator and incubate at 37 °C for 30 min. After the incubation time is completed, wash twice with PBS, observe the fluorescence under an inverted fluorescence microscope and take pictures. The experimental results are as Figure 18 shown.

[0131] As can be seen from Figure 18 , almost no red fluorescence was observed in MCF-7 cells after PBS and PBS+Laser treatment, indicating that neither caused damage to MCF-7 cells. Only a small amount of red fluorescence was seen in MCF-7 cells after β-lap and MIL-88 treatment, indicating that they were basically non-toxic to MCF-7 cells. The red fluorescence in MCF-7 cells increased after MIL-88-β-lap treatment, indicating that β-lap triggered the ferroptosis pathway and ultimately induced the death of MCF-7 cells. The red fluorescence in MCF-7 cells increased after MIL-88-β-lap@PDA+Laser treatment. Due to the photothermal effect caused by near-infrared laser irradiation, the polydopamine shell ruptured, increasing the release amount of β-lap. At the same time, the photothermal conditions were conducive to the generation of reactive oxygen species, indicating that there was a synergistic effect between ferroptosis and photothermal therapy, promoting the cell death process. The red fluorescence in MCF-7 cells increased after MIL-88-β-lap@PDA-cb+Laser treatment, and most cells were in a state of death, fully reflecting that the combined treatment of ferroptosis, photothermal therapy and chemotherapy had a strong inhibitory effect on MCF-7 cells.

[0132] Test Example 15

[0133] Experiment on detecting the change of mitochondrial membrane potential in MCF-7 cells with JC-1 probe

[0134] When detecting the mitochondrial membrane potential, the transformation of the JC-1 probe from the aggregate state to the monomer state, which is accompanied by a significant change in fluorescence color from red to green, can be used as a detection index for early apoptosis of cells. MCF-7 cells were inoculated into confocal dishes at a cell density of 30,000 per dish, shaken evenly, and placed in a cell culture incubator for 24 h. The experiment was divided into 10 groups, namely: PBS, MIL-88, β-lap, cb, MIL-88-β-lap, MIL-88-β-lap@PDA, MIL-88-β-lap@PDA-cb, PBS+Laser, MIL-88-β-lap@PDA+Laser, MIL-88-β-lap@PDA-cb+Laser. Each nanomaterial was prepared at a certain concentration and diluted to the required concentration with DMEM, and 1.0 mL of each material was added to co-incubate with MCF-7 cells for 6 h. For the laser irradiation group (+Laser), it was irradiated with 808 nm laser (2 W cm -2 ) for 5 min. After the incubation, the old culture medium was discarded, washed twice with PBS, then 1.0 mL of JC-1 probe was added, and placed in a cell culture incubator at 37 °C for 30 min. After the incubation, 1.0 mL of JC-1 staining buffer was added and washed twice, and the fluorescence was observed and photographed under a laser confocal microscope. The experimental results are asFigure 19 as shown

[0135] As can be seen from Figure 19 the above, the red fluorescence in MCF-7 cells treated with PBS, PBS+Laser, and MIL-88 increased, while the green fluorescence decreased, indicating that there was no significant damage to the mitochondria in the cells. The green fluorescence in MCF-7 cells treated with β-lap increased. After β-lap was catalytically reduced by quinone oxidoreductase-1 (NQO1), it reacted with oxygen to produce a large amount of reactive oxygen species (ROS). Excessive ROS attacked the mitochondrial membrane lipids, ultimately leading to a decrease in mitochondrial membrane potential. The green fluorescence in MCF-7 cells treated with MIL-88-β-lap increased slightly, and the mitochondrial membrane potential decreased. For MIL-88-β-lap@PDA+Laser and MIL-88-β-lap@PDA-cb+Laser, the green fluorescence in MCF-7 cells increased after near-infrared laser irradiation. Through the photothermal effect, more ROS could be generated in the cells, and the mitochondrial function was damaged, reducing the mitochondrial membrane potential. The introduction of the chemotherapeutic drug chlorambucil further decreased the mitochondrial membrane potential. This indicates that the combined treatment of ferroptosis, photothermal therapy, and chemotherapy can lead to mitochondrial dysfunction.

[0136] Test Example 16

[0137] Experiment for detecting lipid peroxidation (LPO) in MCF-7 cells

[0138] MCF-7 cells were seeded in confocal dishes at a cell density of 60,000 per dish, shaken evenly, and incubated in a cell culture incubator for 24 h. The experiment was divided into 10 groups, namely: PBS, MIL-88, β-lap, cb, MIL-88-β-lap, MIL-88-β-lap@PDA, MIL-88-β-lap@PDA-cb, PBS+Laser, MIL-88-β-lap@PDA+Laser, MIL-88-β-lap@PDA-cb+Laser. Nanomaterials with certain concentrations were prepared and diluted to the required concentrations with DMEM, and 1.0 mL of each material was added to co-incubate with MCF-7 cells for 4 h. For the laser irradiation group (+Laser), it was irradiated with 808 nm laser (2 W cm -2 ) for 5 min, and then continued to be incubated. After the incubation, the old culture medium was discarded, and the cells were washed twice with PBS. C11-BODIPY was pre-diluted to 2 μM, then 1.0 mL of C11-BODIPY was added, and the cells were incubated in a cell culture incubator at 37 °C for 30 min. After the incubation, the cells were washed twice with PBS, then an appropriate amount of DAPI nuclear staining solution was added, and the cells were incubated in a cell culture incubator for 15 min. After the incubation, the fluorescence was observed and photographed under a laser confocal microscope. The experimental results are as Figure 20 shown

[0139] It can be seen from Figure 20 that after treatment with PBS, PBS+Laser, and MIL-88, the green fluorescence in MCF-7 cells is weak, indicating a low level of lipid peroxidation in the cells. After treatment with β-lap, the green fluorescence in MCF-7 cells is enhanced. β-lap can induce a certain degree of lipid peroxidation, leading to ferroptosis of the cells. After treatment with MIL-88-β-lap@PDA+Laser and MIL-88-β-lap@PDA-cb+Laser due to near-infrared laser irradiation, the green fluorescence in MCF-7 cells is enhanced, indicating that the outer shell of polydopamine formed by the photothermal effect is broken, and the increase in temperature increases the release of β-lap, improving the level of lipid peroxides. The introduction of the chemotherapeutic drug chlorambucil results in more accumulation of lipid peroxides on the cell membrane, leading to the death of tumor cells.

[0140] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0141] The above-described embodiments merely represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. A kind of iron-based nanocomposite material, characterized in that, It is obtained by using an iron-based metal-organic framework material as a carrier, loading drug 1, and coating polydopamine on its surface, and binding drug 2 through a borate ester bond. The iron-based metal-organic framework material is MIL-88, drug 1 is β-lapachone, and drug 2 is chlorambucil.

2. A method for preparing the iron-based nanocomposite material as described in claim 1, characterized in that, It includes the following steps: S1. Prepare MIL-88; S2. React MIL-88 prepared in S1 with β-lapachone to obtain MIL-88-β-lap; S3. React tris(hydroxymethyl)aminomethane, MIL-88-β-lap prepared in S2, and dopamine hydrochloride in an alkaline environment to obtain MIL-88-β-lap@PDA; S4. React 4-[bis(2-chloroethyl)amino]phenylbutyric acid nitrogen mustard (cb), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, and 4-hydroxymethylphenylboric acid to obtain cb-BA; S5. React MIL-88-β-lap@PDA prepared in S3, cb-BA prepared in S4, and anhydrous magnesium sulfate to obtain the iron-based nanocomposite MIL-88-β-lap@PDA-cb.

3. The preparation method of the iron-based nanocomposite material according to claim 2, characterized in that, The method for preparing MIL-88 in S1 includes the following steps: React ferric chloride hexahydrate with amino-terephthalic acid through microwave synthesis reaction to obtain MIL-88.

4. The preparation method of the iron-based nanocomposite material according to claim 3, characterized in that, The mass ratio of the ferric chloride hexahydrate to the amino-terephthalic acid is 1.316:

1.

5. The preparation method of the iron-based nanocomposite according to claim 2, wherein In S2, the mass ratio of the MIL-88 to the β-lapachone is 2:

1.

6. The preparation method of the iron-based nanocomposite material according to claim 2, characterized in that, In S3, the mass ratio of the tris(hydroxymethyl)aminomethane, the MIL-88-β-lap, and the dopamine hydrochloride is 12:5:

10.

7. The preparation method of the iron-based nanocomposite according to claim 2, wherein In S3, the pH value of the alkaline environment is 8.0 - 8.

5.

8. The preparation method of the iron-based nanocomposite material according to claim 2, characterized in that, In S4, the mass ratio of the 4-[bis(2-chloroethyl)amino]phenylbutyric acid nitrogen mustard (cb), the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, the 4-dimethylaminopyridine, and the 4-hydroxymethylphenylboric acid is 152:192:61:

99.

9. The preparation method of the iron-based nanocomposite material according to claim 2, wherein, In S5, the mass ratio of the MIL-88-β-lap@PDA, the cb-BA, and the anhydrous magnesium sulfate is 4:1:

8.

10. An iron-based nanocomposite as described in claim 1 is used as a drug carrier in tumor treatment.

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