Magnetic layered double hydroxide, tumor-targeting drug-loaded nano-preparation, preparation method and application of tumor-targeting drug-loaded nano-preparation

Abstract

The application belongs to the technical field of medicine preparation, and particularly relates to a magnetic layered double hydroxide, a targeted drug-loaded nano preparation, a preparation method and application of a tumor-targeted drug-loaded nano preparation. The preparation method of the magnetic layered double hydroxide provided by the application can prepare Fe3O4 nanoparticles with small particle size, and further prepare the magnetic layered double hydroxide which can be used as a water-soluble drug carrier. The magnetic layered double hydroxide is used for preparing the targeted drug-loaded nano preparation and the tumor-targeted drug-loaded nano preparation, and can be used for delivering water-soluble drugs such as 5-fluorouracil. The tumor-targeted drug-loaded nano preparation has good dispersity and can be actively targeted to HepG2 cells with high FR expression, has a good inhibitory effect on liver cancer cells, and is expected to provide a potential new treatment system for magnetic chemotherapy.

Description

Technical Field

[0001] This invention belongs to the field of drug preparation technology, specifically relating to a method for preparing and applying a magnetic layered double hydroxide, a targeted drug-loaded nanoparticle, and a tumor-targeted drug-loaded nanoparticle. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a major form of primary liver cancer, with complex causes and mechanisms. Many treatment options exist for HCC, including surgical resection, liver transplantation, radiotherapy, and chemotherapy. However, these traditional therapies each have their limitations, such as high recurrence rates after surgical resection, immune irritation after transplantation, and the development of drug resistance with chemotherapy. The emergence of nanotechnology has provided new directions for cancer treatment, not only effectively treating cancer but also potentially improving cancer diagnosis and imaging. Among these, the use of magnetic nanocomposites for controlled drug release and targeted drug delivery holds great potential in exploring new methods of cancer chemotherapy.

[0003] Magnetic nanoparticles (MNPs) hold great promise in the field of nanomaterials for diagnosis and treatment due to their unique metallic properties. Fe3O4 nanoparticles are a commonly used magnetic material. They can achieve targeted delivery under static magnetic fields and are widely used in targeted therapy for various diseases; they can also generate heat under external alternating magnetic fields for magnetothermal therapy to kill tumors, and are used in magnetic resonance imaging (MRI) for diagnosis and monitoring. Their medical applications and biological distribution are related to particle size. Small-sized Fe3O4 nanoparticles may yield more ideal nanocomposites capable of loading water-soluble drugs, but these are not easily obtained.

[0004] Nanocomposite carriers are among the most promising tools in nanomedicine because they can exhibit the mixed properties of different substances within a single system, thus endowing them with superior performance in therapeutic and diagnostic applications. Layered double hydroxides (LDHs) have a layered structure, consisting of positively charged laminations and orderly stacking of interlayer anions. LDHs are often used as biocompatible and pH-responsive nanocarriers to regulate drug release in vivo, primarily due to their excellent properties, including high anion exchange capacity, large surface area, low toxicity, and pH-controlled release. Magnetic nanocomposites prepared using Fe3O4 nanoparticles as magnetically responsive carriers and LDH to load drugs exhibit high anticancer activity and good magnetic responsiveness. However, a limitation of LDHs is their tendency to aggregate in PBS buffer. Summary of the Invention

[0005] To address the above problems, this invention provides a method for preparing magnetic layered double hydroxides, targeted drug-loaded nanoparticles, and tumor-targeted drug-loaded nanoparticles, as well as their applications. The method for preparing magnetic layered double hydroxides provided by this invention can produce layered double hydroxide nanocomposite carriers internally loaded with small-particle-size Fe3O4. These nanocomposite carriers can be used to prepare targeted drug-loaded nanoparticles and tumor-targeted drug-loaded nanoparticles. The method for preparing targeted drug-loaded nanoparticles provided by this invention can achieve loading of water-soluble drugs; the method for preparing tumor-targeted drug-loaded nanoparticles not only achieves loading of water-soluble drugs but also exhibits good dispersibility, overcoming the limitation of LDH's tendency to aggregate in PBS buffer.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing a magnetic layered double hydroxide, comprising the following steps:

[0008] Step A: Dissolve ferric chloride in diethylene glycol (DEG), add sodium citrate (Na3Cit) and stir to dissolve, then add sodium acetate (NaAc) and continue stirring until completely dissolved. Heat the resulting solution at 100-200℃ for 10-20 hours, collect the product with a magnet, wash with ethanol and dry to obtain Fe3O4 nanoparticles.

[0009] Step B: The Fe3O4 nanoparticles are uniformly dispersed in deionized water, magnesium chloride and aluminum chloride are added and dissolved, and then an alkaline solution is added while stirring under an inert atmosphere. After the reaction is completed, the solid and liquid phases are separated, the solid phase is collected and washed with water to obtain the magnetic layered double hydroxide.

[0010] By employing the above-described preparation process, the Fe3O4 nanoparticles obtained in step A have a small particle size of only 1-4 nm, enabling them to be loaded between LDH layers without significantly affecting the interlayer spacing of the LDH. In step B, adding an alkaline solution under an inert atmosphere prevents carbon dioxide from entering the reaction solution during stirring and generating carbonate ions, which could interfere with the formation of LDH anions. Continued stirring after adding the alkaline solution accelerates the removal of gas from the reaction solution.

[0011] The final magnetic layered double hydroxide obtained by this preparation method is a layered double hydroxide nanocomposite carrier (Fe3O4@LDH) internally loaded with Fe3O4, which can be used as a carrier for the preparation of water-soluble targeted drug formulations. This nanocomposite carrier exhibits the ability to mix different substances within a single system, thus endowing it with superior performance in therapeutic and diagnostic applications, including high anion exchange capacity, large surface area, low toxicity, and pH-controlled release, thus showing promising application prospects.

[0012] In conjunction with the first aspect, it is preferable to stir and dissolve sodium citrate at 60–80°C after its addition.

[0013] In conjunction with the first aspect, it is preferable to heat the obtained liquid at 200°C for 10 hours in step A.

[0014] In conjunction with the first aspect, the solid-liquid separation described in step B can be performed by centrifugation at a speed of 3000–5000 rpm.

[0015] In conjunction with the first aspect, the molar ratio of magnesium chloride to aluminum sulfate in step B is 3:1.

[0016] In conjunction with the first aspect, the magnesium chloride mentioned in step B can be magnesium chloride hexahydrate, the aluminum chloride can be aluminum chloride hexahydrate, and the total mass of the magnesium chloride and the aluminum chloride is 20 to 30 times that of the Fe3O4 nanoparticles.

[0017] In conjunction with the first aspect, the alkaline solution mentioned in step B is preferably an aqueous sodium hydroxide solution with a concentration of 0.1–0.2 mol / L, preferably 0.15 mol / L.

[0018] In conjunction with the first aspect, in step B, after adding the alkaline solution, stirring at a speed of 500-700 rpm for 30-60 minutes allows air to be quickly expelled from the reaction vessel and enables the reactants to quickly form a precipitate in an alkaline environment.

[0019] In conjunction with the first aspect, the deionized water is deionized water that has been boiled to remove CO2.

[0020] Secondly, the present invention provides a magnetic layered double hydroxide, which is prepared according to the above-described method for preparing magnetic layered double hydroxides.

[0021] Thirdly, the present invention provides the application of the above-mentioned magnetic layered double hydroxide in the preparation of targeted drug-loaded nanoparticles: the carrier of the targeted drug-loaded nanoparticles is the above-mentioned magnetic layered double hydroxide, and the active ingredient is a water-soluble drug.

[0022] Fourthly, the present invention also provides a method for preparing targeted drug-loaded nanoparticles, specifically including the following operations:

[0023] The water-soluble drug is dissolved in an alkaline solution to prepare a drug solution; the above-mentioned magnetic layered double hydroxide is dispersed in the drug solution and stirred for at least 2 hours; solid-liquid separation is performed, the solid phase is collected and washed with an alkaline solution, and then dispersed in deionized water for hydrothermal treatment. After the treatment is completed, the solvent is removed to obtain the final product.

[0024] The targeted drug-loaded nanoformulation prepared by the above method uses magnetic layered double hydroxides as carriers to encapsulate water-soluble drugs, which can insert water-soluble drugs into the LDH layers, thereby achieving the loading of water-soluble drugs.

[0025] In conjunction with the fourth aspect, the alkaline solution may be an aqueous sodium hydroxide solution with a pH of 8 to 9.

[0026] In conjunction with the fourth aspect, the hydrothermal treatment temperature is 80–120°C, and the time is 6–12 hours.

[0027] In conjunction with the fourth aspect, the deionized water is deionized water that has been boiled to remove CO2.

[0028] Fifthly, the present invention provides the application of the above-mentioned magnetic layered double hydroxide in the preparation of tumor-targeting drug-loaded nanoparticles: the carrier of the tumor-targeting drug-loaded nanoparticles is the above-mentioned magnetic layered double hydroxide, the active ingredient is a water-soluble anti-tumor drug, and the surface of the tumor-targeting drug-loaded nanoparticles is modified with a folic acid (FA)-bovine serum albumin (BSA) complex (FA-BSA).

[0029] Fatty acid (FA) is a water-soluble B vitamin, an essential element for cell growth, and participates in the synthesis of purines and thymine during DNA replication. It has a small molecular weight, is non-toxic, and has no immunogenicity. Bsamin (BSA) is a major protein in blood plasma, with hydrophobic and hydrophilic groups on its surface, making it easy to modify. As an endogenous substance, it is non-toxic and non-immunogenic, often used as a stabilizer and reducing agent in drug delivery systems. Its long half-life prevents rapid clearance of drug carriers. Abundant functional groups on its surface allow FA to be covalently bound to BSA, thus endowing it with active targeting capabilities. The folic acid receptor (FR) is typically overexpressed on the surface of some tumor cells (ovarian cancer, liver cancer, breast cancer, etc.), but not expressed or expressed sparingly in normal cells. FA can specifically target FR and then be transported into the cell via receptor-mediated endocytosis, thereby achieving active targeting. Therefore, FA-BSA modification is beneficial for the enrichment of this nanomaterial at tumor sites, increasing its bioavailability, while also improving the dispersibility and surface activity of the Fe3O4@LDH nanocarrier.

[0030] In conjunction with the fifth aspect, the water-soluble antitumor drug is 5-fluorouracil (5-Fluorouracil, 5-Fu).

[0031] In conjunction with the fifth aspect, the tumor-targeting drug-loaded nanoparticle is a targeted drug-loaded nanoparticle for liver cancer. Experiments have shown that this tumor-targeting drug-loaded nanoparticle can actively target HepG2 cells with high FR expression and has a good inhibitory effect on liver cancer cells.

[0032] Sixthly, the present invention provides a method for preparing tumor-targeting drug-loaded nanoparticles, specifically including the following steps:

[0033] Step a: Dissolve the water-soluble antitumor drug in an alkaline solution to prepare a drug solution; disperse the above-mentioned magnetic layered double hydroxide in the drug solution and stir for at least 2 hours; separate the solid and liquid phases, collect the solid phase and wash it with an alkaline solution, then disperse it in water for hydrothermal treatment, remove the solvent after treatment, and obtain the initial product.

[0034] Step b: Dissolve folic acid in an organic solvent, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS), and vortex in the dark for at least 3 hours; dissolve bovine serum albumin in a sodium carbonate-sodium bicarbonate buffer solution at pH 8-9, mix with the organic solution of folic acid in the dark for at least 10 hours, dialyze the resulting product with deionized water for at least 40 hours, and dry to obtain the folic acid-bovine serum albumin complex;

[0035] Step c: Add the suspension of the initial product obtained in step a to the solution of the folic acid-bovine serum albumin complex obtained in step b under ultrasonic conditions, vortex for at least 25 minutes, and then remove the solvent to obtain the final product.

[0036] The tumor-targeting drug-loaded nanoparticles prepared by the above method encapsulate water-soluble antitumor drugs using magnetic layered double hydroxides as carriers. The nanocarriers are modified with FA coupled with BSA to seal the pores on the surface of the nanoparticles, without significantly affecting the crystal structure of Fe3O4@LDH.

[0037] Experiments have demonstrated that this tumor-targeting drug-loaded nanoformulation can deliver water-soluble anti-tumor drugs, making it easier for cells to take up the water-soluble anti-tumor drugs in the formulation, thereby achieving better therapeutic effects. It has good stability, pH responsiveness, magnetic properties, and active targeting ability, and can produce good controlled release under acidic conditions. It has good dispersibility, overcoming the limitation of LDH's easy aggregation in PBS buffer. It has good blood compatibility and is relatively safe for injection.

[0038] In step b, the carboxyl group of FA and the amino group of BSA are covalently bonded via an amide reaction. The reaction mechanism is as follows:

[0039]

[0040] In step c, the initial product obtained in step a should be added slowly to the solution of the folic acid-bovine serum albumin complex obtained in step b.

[0041] In conjunction with the sixth aspect, the water-soluble antitumor drug is 5-fluorouracil.

[0042] In conjunction with the sixth aspect, the tumor-targeting drug-loaded nano-formulation is a targeted drug-loaded nano-formulation for liver cancer.

[0043] In conjunction with the sixth aspect, the alkaline solution mentioned in step a can be an aqueous sodium hydroxide solution with a pH of 8 to 9.

[0044] In conjunction with the sixth aspect, the mass ratio of the water-soluble antitumor drug to the alkaline solution is 1.5 to 2:1.

[0045] In conjunction with the sixth aspect, the hydrothermal treatment in step a is performed at a temperature of 80–120°C for a duration of 6–12 hours.

[0046] In conjunction with the sixth aspect, the organic solvent mentioned in step b is preferably DMSO.

[0047] In conjunction with the sixth aspect, the mass ratio of folic acid to bovine serum albumin in step b is 1:2 to 3.

[0048] In conjunction with the sixth aspect, the mass ratio of folic acid to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in step b is 1:0.6 to 0.7, and the mass ratio of folic acid to N-hydroxysuccinimide is 1:0.35 to 0.4.

[0049] In conjunction with the sixth aspect, the mass ratio of the initial product obtained in step c to the folic acid-bovine serum albumin complex is 1:0.8 to 1.2, preferably 1:1.

[0050] In conjunction with the sixth aspect, the solvent removal method in step c can be freeze drying.

[0051] In conjunction with the sixth aspect, the deionized water is deionized water that has been boiled to remove CO2.

[0052] Compared with the prior art, the beneficial effects of the present invention are:

[0053] This invention obtains small-sized Fe3O4 nanoparticles through the above-described method for preparing magnetic layered double hydroxides, and loads these Fe3O4 nanoparticles onto the interlayer of LDH, thereby obtaining magnetic layered double hydroxides that can serve as water-soluble drug carriers. The method for preparing tumor-targeting drug-loaded nanoparticles provided by this invention utilizes FA-BSA to modify the drug-loaded magnetic layered double hydroxides, further endowing them with active targeting properties. This preparation method can be used for loading the water-soluble drug 5-Fu, and exhibits good 5-Fu loading rates. Furthermore, the folic acid-conjugated bovine serum albumin-modified magnetic layered double hydroxide nanocomplex constructed by this method possesses good stability, pH responsiveness, magnetic properties, and active targeting ability, and can produce good controlled-release effects under acidic conditions. Experiments have demonstrated that the magnetic nanocomplex prepared by this method has a good inhibitory effect on liver cancer cells and good dispersibility, overcoming the limitation of LDH's tendency to aggregate in PBS buffer, and holds promise for providing a potentially improved nanoplatform for magnetic chemotherapy of tumors. Attached Figure Description

[0054] Figure 1 Transmission electron microscope (TEM) images of Fe3O4@LDH nanocarrier prepared in Example 1 of the present invention (Figure a) and FA-BSA / Fe3O4@LDH prepared in the test example (Figure b);

[0055] Figure 2 Particle size diagrams of Fe3O4 and Fe3O4@LDH prepared in Example 1, Fe3O4@LDH / 5-Fu prepared in Example 2, FA-BSA / Fe3O4@LDH / 5-Fu prepared in Example 3, and LDH and FA-BSA / Fe3O4@LDH prepared in the test examples.

[0056] Figure 3 The XRD spectra of Fe3O4 and Fe3O4@LDH prepared in Example 1 of this invention, and LDH and FA-BSA / Fe3O4@LDH prepared in the test example;

[0057] Figure 4 XPS spectra of Fe3O4 prepared in Example 1 of this invention and LDH and FA-BSA / Fe3O4@LDH prepared in the test example;

[0058] Figure 5 Fourier transform infrared (FTIR) characterization spectra of 5-Fu, Fe3O4 and Fe3O4@LDH prepared in Example 1 of this invention, FA-BSA / Fe3O4@LDH / 5-Fu prepared in Example 3, and LDH and FA-BSA / Fe3O4@LDH prepared in the test examples.

[0059] Figure 6TGA images of Fe3O4@LDH prepared in Example 1 of the present invention, and LDH and FA-BSA / Fe3O4@LDH prepared in the test example;

[0060] Figure 7 VSM diagrams of Fe3O4 and Fe3O4@LDH prepared in Example 1 and FA-BSA / Fe3O4@LDH / 5-Fu prepared in Example 3 of this invention;

[0061] Figure 8 Release curves of FA-BSA / Fe3O4@LDH / 5-Fu prepared in Example 3 of this invention in release media at pH 7.4 and pH 5.0;

[0062] Figure 9 The hemolysis test results are shown in the figure for FA-BSA / Fe3O4@LDH / 5-Fu prepared in Example 3 and FA-BSA / Fe3O4@LDH prepared in the test example.

[0063] Figure 10 CCK-8 assay results for 5-Fu, BSA / Fe3O4@LDH / 5-Fu prepared in the test examples of this invention, and FA-BSA / Fe3O4@LDH / 5-Fu prepared in Example 3;

[0064] Figure 11 The image shows the cellular uptake results of BSA / Fe3O4@LDH and FA-BSA / Fe3O4@LDH prepared in the test examples of this invention. Detailed Implementation

[0065] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0066] The use of magnetic nanocomposites for controlled drug release and targeted drug delivery holds great potential in cancer chemotherapy. Fe3O4 nanoparticles are commonly used magnetic materials, and smaller-sized Fe3O4 nanoparticles may be able to yield more ideal nanocomposites capable of loading water-soluble drugs, but these are not easily obtained.

[0067] LDH is often used as a biocompatible and pH-responsive nanocarrier. Magnetic nanocomposites prepared by using Fe3O4 nanoparticles as a magnetically responsive carrier and LDH to load drugs exhibit high anticancer activity and good magnetic responsiveness. However, LDH tends to aggregate in PBS buffer, which limits the application of this magnetic nanocarrier.

[0068] To address the above problems, embodiments of the present invention provide a method for preparing magnetic layered double hydroxides, comprising the following steps:

[0069] Step A: Dissolve ferric chloride in diethylene glycol (DEG), add sodium citrate (Na3Cit) and stir to dissolve, then add sodium acetate (NaAc) and continue stirring until completely dissolved. Heat the resulting solution at 100-200℃ for 10-20 hours, collect the product with a magnet, wash with ethanol and dry to obtain Fe3O4 nanoparticles.

[0070] Step B: The Fe3O4 nanoparticles are uniformly dispersed in deionized water, magnesium chloride and aluminum chloride are added and dissolved, and then an alkaline solution is added while stirring under an inert atmosphere. After the reaction is completed, the solid and liquid phases are separated, the solid phase is collected and washed with water to obtain the magnetic layered double hydroxide.

[0071] The Fe3O4 nanoparticles obtained in step A of this preparation method have a particle size of only 1-4 nm. They can be loaded between the layers of layered double hydroxide (LDH) without significantly affecting the interlayer spacing of LDH. They can be used as a carrier for the preparation of water-soluble drug targeted formulations.

[0072] The embodiments of the present invention also provide the application of magnetic layered double hydroxides in the preparation of targeted drug-loaded nanoformulations and in the preparation of tumor-targeted drug-loaded nanoformulations, and provide methods for preparing targeted drug-loaded nanoformulations and tumor-targeted drug-loaded nanoformulations.

[0073] The preparation method of the targeted drug-loaded nano-formulation provided in this embodiment of the invention specifically includes the following operations: dissolving a water-soluble drug in an alkaline solution to prepare a drug solution; dispersing the above-mentioned magnetic layered double hydroxide in the drug solution and stirring for at least 2 hours; separating the solid and liquid phases, collecting the solid phase and washing it with an alkaline solution, then dispersing it in deionized water for hydrothermal treatment, and removing the solvent after treatment to obtain the final product.

[0074] The method for preparing tumor-targeting drug-loaded nanoparticles provided in this embodiment of the invention specifically includes the following steps:

[0075] Step a: Dissolve the water-soluble antitumor drug in an alkaline solution to prepare a drug solution; disperse the above-mentioned magnetic layered double hydroxide in the drug solution and stir for at least 2 hours; separate the solid and liquid phases, collect the solid phase and wash it with an alkaline solution, then disperse it in water for hydrothermal treatment, remove the solvent after treatment, and obtain the initial product.

[0076] Step b: Dissolve folic acid in an organic solvent, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS), and vortex in the dark for at least 3 hours; dissolve bovine serum albumin in a sodium carbonate-sodium bicarbonate buffer solution at pH 8-9, mix with the organic solution of folic acid in the dark for at least 10 hours, dialyze the resulting product with deionized water for at least 40 hours, and dry to obtain folic acid-bovine serum albumin complex (FA-BSA);

[0077] Step c: Add the suspension of the initial product obtained in step a to the solution of the folic acid-bovine serum albumin complex obtained in step b under ultrasonic conditions, vortex for at least 25 minutes, remove the solvent, and the product is obtained.

[0078] The present invention will be described below through specific embodiments.

[0079] Example 1

[0080] This embodiment provides a magnetic layered double hydroxide and its preparation method. All deionized water mentioned in this embodiment is boiled deionized water with CO2 removed.

[0081] The preparation method of this magnetic layered double hydroxide is as follows:

[0082] (1) Dissolve 1081 mg FeCl3·6H2O in 40 mL DEG, add 471 mg Na3Cit·2H2O and stir at 70 °C to dissolve. Then, add 1312 mg NaAc and continue stirring until completely dissolved. Place the mixed solution in a polytetrafluoroethylene liner and heat in a reaction vessel at 200 °C for 10 h. Finally, collect the prepared product with a magnet, purify it three times with ethanol, and obtain Fe3O4 nanoparticles by vacuum drying for further use;

[0083] (2) 30 mg Fe3O4 was uniformly dispersed in 10 mL of deionized water, and 610 mg MgCl2·6H2O and 241 mg AlCl3·6H2O were dissolved in the solution. Then, after stirring under N2 conditions for 45 min, 40 mL of NaOH (0.15 mol / L) was quickly added and the mixture was stirred vigorously at 600 rpm for 45 min. The prepared preproduct was centrifuged at 4000 rpm for 5 min, the supernatant was removed, and the product was washed twice with deionized water (4000 rpm, 5 min) by centrifugation. The product was then freeze-dried to obtain magnetic layered double hydroxide Fe3O4@LDH.

[0084] Example 2

[0085] This embodiment provides the application of magnetic layered double hydroxides in the preparation of targeted drug-loaded nanoparticles. The magnetic layered double hydroxide in this embodiment is Fe3O4@LDH obtained in Example 1; all deionized water mentioned in this embodiment is boiled deionized water with CO2 removed.

[0086] The preparation method of this targeted drug-loaded nanoformulation is as follows:

[0087] 30 mg of 5-Fu (purity: 99%) was dissolved in 20 mL of an aqueous solution adjusted to pH 8.5 with NaOH solution. Fe3O4@LDH was suspended in the 5-Fu solution and stirred with a mechanical stirrer for 2.5 h. The reaction mixture was obtained by centrifugation at 5000 rpm for 10 min and washed with NaOH aqueous solution (pH = 8-9) to remove free 5-Fu. The mixture was then redispersed in deionized water for further hydrothermal treatment at 100 °C for 6 h to obtain the Fe3O4@LDH / 5-Fu solution, which was then lyophilized.

[0088] A portion of Fe3O4@LDH / 5-Fu was pretreated with HCl solution to disrupt the drug-loaded complex. After adjusting the volume, the concentration of 5-Fu was determined by monitoring the absorbance of 5-Fu at 265 nm. The drug loading rate of Fe3O4@LDH / 5-Fu was determined by UV-Vis spectroscopy according to a standard curve. The final drug loading of Fe3O4@LDH / 5-Fu was calculated to be 11.68%.

[0089] Example 3

[0090] This embodiment provides the application of magnetic layered double hydroxides in the preparation of tumor-targeted drug-loaded nanoparticles. The Fe3O4@LDH / 5-Fu in this embodiment was prepared in Example 2; all deionized water mentioned in this embodiment was boiled and CO2-removed.

[0091] The preparation method of this tumor-targeting drug-loaded nanoparticle formulation is as follows:

[0092] (1) The carboxyl group of FA and the amino group of BSA were covalently bonded via an amide reaction. First, 20 mg of FA was dissolved in DMSO, and then 12.94 mg of EDC·HCl and 7.77 mg of NHS were added, and the mixture was continuously vortexed in the dark for 4 h. Next, 50 mg of BSA was dissolved in Na2CO3 / NaHCO3 buffer solution (pH 8.5), and the FA solution was slowly added to the BSA solution, while stirring continuously in the dark for 12 h. Finally, the product was dialyzed against deionized water for two days. FA-BSA was obtained after freeze-drying.

[0093] (2) 1 mL of 2 mg / mL Fe3O4@LDH / 5-Fu aqueous suspension was added dropwise to 1 mL of 2 mg / mL FA-BSA aqueous solution under ultrasonic bath conditions in an ultrasonic cleaner, and vortexed for 30 min. After the reaction was completed, FA-BSA / Fe3O4@LDH / 5-Fu was obtained by freeze drying.

[0094] Test Example

[0095] Preparation of LDH: 610 mg MgCl2·6H2O and 241 mg AlCl3·6H2O were dissolved in deionized water. Then, the mixture was stirred under N2 conditions for 45 min, followed by rapid addition of 40 mL NaOH (0.15 mol / L) and vigorous stirring at 600 rpm for 45 min. The prepared preproduct was centrifuged at 4000 rpm for 5 min, the supernatant was removed, and the product was washed twice with deionized water (4000 rpm, 5 min) by centrifugation. Finally, it was freeze-dried to obtain LDH.

[0096] Preparation of FA-BSA / Fe3O4@LDH: Fe3O4@LDH was prepared according to the method in Example 1, and FA-BSA was prepared according to the method in Example 3. 1 mL of an aqueous suspension of 2 mg / mL Fe3O4@LDH was added dropwise to 1 mL of an aqueous solution of 2 mg / mL FA-BSA under ultrasonic bath conditions in an ultrasonic cleaner, and vortexing was continued for 30 min. After the reaction was complete, FA-BSA / Fe3O4@LDH was obtained by freeze-drying.

[0097] Preparation of BSA / Fe3O4@LDH / 5-Fu: Fe3O4@LDH / 5-Fu was prepared according to the method in Example 2. 1 mL of an aqueous suspension of 2 mg / mL Fe3O4@LDH / 5-Fu was added dropwise to 1 mL of an aqueous solution of 2 mg / mL BSA under ultrasonic bath conditions in an ultrasonic cleaner, and vortexing was continued for 30 min. After the reaction was complete, BSA / Fe3O4@LDH / 5-Fu was obtained by freeze-drying.

[0098] The Fe3O4, Fe3O4@LDH, Fe3O4@LDH / 5-Fu, and FA-BSA / Fe3O4@LDH / 5-Fu prepared in Examples 1-3 were tested, and the results are as follows: Figures 1-11 As shown.

[0099] Figure 1The images show transmission electron microscopy (TEM) images of Fe3O4@LDH prepared in Example 1 (Figure a) and FA-BSA / Fe3O4@LDH prepared by the above method (Figure b). It can be seen from the figures that the Fe3O4 particles are very small, with a particle size of 1-4 nm. It can be clearly observed that Fe3O4 is successfully bound to the LDH nanolayer. The structure of Fe3O4@LDH modified by FA-BSA does not undergo significant changes.

[0100] Figure 2 The particle size changes of the materials prepared in Examples 1-3, as well as LDH and FA-BSA / Fe3O4@LDH prepared according to the above method, were measured by DLS during the preparation process, as shown in the figure. After Fe3O4 was combined with LDH, the particle size of Fe3O4@LDH increased. After further modification with FA-BSA, the particle size of the final support FA-BSA / Fe3O4@LDH increased to about 180 nm. After loading 5-Fu onto Fe3O4@LDH / 5-Fu, the particle size increased to 170 nm, which is about 35 nm larger than the blank Fe3O4@LDH support. After modification with FA-BSA, the particle size of FA-BSA / Fe3O4@LDH / 5-Fu increased to 195 nm. The hydrodynamic particle size of Fe3O4 is approximately 80–90 nm. In the DLS characterization method, when light shines on colloidal particles, the light is scattered, and the intensity depends on the particle size. The detected signal is the result of the superposition of multiple scattered photons. Due to the high surface energy and anisotropic effects of Fe3O4, they tend to aggregate, thus the measured particle size is larger than the TEM particle size. The relatively large PDI is also due to the inherent magnetism of Fe3O4 nanoparticles, which inevitably leads to some aggregation in aqueous solution.

[0101] Figure 3The XRD patterns of Fe3O4, Fe3O4@LDH, and LDH and FA-BSA / Fe3O4@LDH prepared in Example 1 are shown, displaying their typical crystal plane diffraction peaks. The results show that the diffraction peaks of Fe3O4@LDH at 30.10°, 35.54°, 43.30°, 53.38°, 57.28°, and 62.72° correspond to the characteristic diffraction peaks of Fe3O4 at (220), (311), (400), (422), (511), and (440), respectively. Furthermore, the diffraction peaks of Fe3O4@LDH at 2θ angles of 11.5°, 23.38°, and 60.8° correspond to the characteristic diffraction peaks of LDH at (003), (006), and (110), respectively. The above results confirm the presence of typical diffraction peaks of LDH and Fe3O4 in Fe3O4@LDH, indicating that Fe3O4 and LDH are successfully combined. Furthermore, the positions of the characteristic diffraction peaks of LDH remain largely unchanged, suggesting that the introduction of Fe3O4 has minimal impact on the interlayer spacing of LDH. In addition, the XRD pattern of FA-BSA / Fe3O4@LDH shows no significant changes compared to Fe3O4@LDH, although the peak intensity of Fe3O4 is very weak, possibly due to the introduction of FA-BSA further reducing the content of magnetic components in the support. Other diffraction peak shapes do not change significantly, demonstrating that FA-BSA has minimal impact on the crystal structure of the support, and the crystal structure of Fe3O4@LDH can still be maintained after modification with FA-BSA.

[0102] Figure 4 The figure shows the changes in surface elements of Fe3O4 obtained in Example 1, LDH prepared by the above method, and FA-BSA / Fe3O4@LDH, as analyzed by X-ray photoelectron spectroscopy. Analysis of the XPS full-scan spectra of Fe3O4, LDH, and FA-BSA / Fe3O4@LDH revealed that FA-BSA / Fe3O4@LDH contains Al, Mg, Fe, O, C, and N elements. The peak value of Fe in FA-BSA / Fe3O4@LDH decreased significantly. This is likely due to the presence of FA-BSA coupling on the Fe3O4@LDH surface after FA-BSA modification, which, influenced by the XPS detection depth, led to a decrease in the absorption peak intensity of each element. Furthermore, the newly added characteristic peak of N in FA-BSA / Fe3O4@LDH is mainly due to the presence of N element in FA-BSA, confirming the successful introduction of FA-BSA.

[0103] Figure 5The functional groups of different samples from Examples 1 and 3 were analyzed by Fourier transform infrared spectroscopy. The FT-IR spectra of 5-Fu, Fe3O4 and Fe3O4@LDH obtained in Example 1, FA-BSA / Fe3O4@LDH / 5-Fu obtained in Example 3, and LDH and FA-BSA / Fe3O4@LDH prepared according to the above method are shown below. Figure 5 As shown in Figure a: Fe-O can be observed at 580 cm⁻¹ in the FT-IR spectrum of Fe₃O₄. -1 The tensile vibrations at 781, 679, and 450 cm⁻¹ are observed in the FT-IR spectrum of LDH. -1 MO and OMO vibrations are observed at 3460 cm. -1 The broad and strong absorption peak of -OH is mainly due to the water molecules carried by the layers and interlayers; the Fe3O4@LDH exhibits a strong absorption peak at 580-680 cm⁻¹ due to the overlapping vibrations of MO and Fe-O. -1 A strong and broad absorption band is observed at 1651 and 1540 cm⁻¹, indicating that Fe₃O₄ has successfully combined with LDH. Due to slight changes in the surrounding electron cloud distribution and steric hindrance after combination, the absorption peaks differ; in the FA-BSA / Fe₃O₄@LDH spectrum, the peaks are located at 1651 and 1540 cm⁻¹. -1 The image shows the stretching vibration of the amide bond, indicating that FA-BSA was successfully modified onto Fe3O4@LDH. Figure 5 Figure b in the image shows the FT-IR spectra of 5-Fu and FA-BSA / Fe3O4@LDH / 5-Fu: The 1720 and 1660 cm⁻¹ spectra of 5-Fu are shown. -1 The peaks at 1240 and 1220 cm⁻¹ are related to the imine C=O stretching vibration and the amide C=O stretching vibration, respectively. -1 The peaks at these locations are related to the tensile vibrations of CN and the stretching vibrations of CF, respectively; compared with 5-Fu, FA-BSA / Fe3O4@LDH / 5-Fu observed a peak at 1720 cm⁻¹. -1 The disappearance of the peak at the point and the presence of the CN stretching vibration peak attributed to 5-Fu prove that 5-Fu was successfully loaded.

[0104] Figure 6The thermal stability of Fe3O4@LDH prepared in Example 1, as well as LDH and FA-BSA / Fe3O4@LDH prepared by the above method, was tested by TGA, specifically the weight change of different samples heated from 0°C to 800°C. The weight loss of LDH was mainly divided into two stages: the initial weight loss of approximately 23% (0-200°C) was due to the evaporation of water between the LDH sheets, followed by approximately 51% (200-800°C) attributable to the destruction of the layered structure. Compared to LDH, Fe3O4@LDH and FA-BSA / Fe3O4@LDH lost weight more slowly, which is attributed to the presence of highly stable Fe3O4 in the support, reducing the specific gravity of LDH. Furthermore, the weight loss of Fe3O4@LDH was similar to that of FA-BSA / Fe3O4@LDH in the 0-200°C range, with the main weight loss also being the evaporation of adsorbed water molecules in the sample. However, within the temperature range of 200-800℃, as the temperature continues to rise, the weight loss becomes more and more obvious, and the difference in weight loss between the two gradually increases. The final weight loss of Fe3O4@LDH is about 42%, and the final weight loss of FA-BSA / Fe3O4@LDH is about 63%. This is mainly due to the gradual decomposition of FA-BSA modified on the Fe3O4@LDH surface in FA-BSA / Fe3O4@LDH at high temperatures. This also confirms that FA-BSA was successfully modified on the Fe3O4@LDH composite support.

[0105] Figure 7 The saturation magnetization and coercivity of Fe3O4 and Fe3O4@LDH prepared in Example 1, and FA-BSA / Fe3O4@LDH / 5-Fu prepared in Example 3, were measured at room temperature using a vibrating sample magnetometer. Figure 7 As shown in Figure a, the saturation magnetization of the Fe3O4 nanoparticles was determined to be 37.6 emu / g based on the magnetization curve, and the coercivity was almost zero, indicating that the prepared Fe3O4 exhibits superparamagnetism. Similarly, as... Figure 7 As shown in Figure b, the coercivity of Fe3O4@LDH and FA-BSA / Fe3O4@LDH / 5-Fu is almost zero, indicating paramagnetism. The saturation magnetization of Fe3O4@LDH is 3.4 emu / g, meaning that Fe3O4 and LDH have successfully combined, giving the composite nanocarrier magnetism. With the modification of FA-BSA and the loading of 5-Fu, the saturation magnetization of FA-BSA / Fe3O4@LDH / 5-Fu decreases to 1 emu / g, which is due to the reduction in effective magnetic content.

[0106] Figure 8The release behavior of 5-Fu from the nanocomposite prepared in Example 3 was investigated in PBS release media at pH 7.4 and pH 5. The drug loading rate of FA-BSA / Fe3O4@LDH / 5-Fu was determined by UV-Vis spectroscopy, and the calculated final drug loading was 5.84%. Since LDH laminae are easily degraded under acidic conditions, and the pH of the tumor cell environment is acidic, this experiment simulated in vivo physiological and tumor environments to examine the in vitro release behavior of the nanocomposite constructed in Example 3, thereby evaluating its controlled-release performance. Figure 8 It is evident that the release of 5-Fu at different pH values ​​initially proceeds rapidly, followed by a slow release until reaching equilibrium. At pH 7.4, due to the presence of high Cl- content in the PBS buffer... - With PO4 3- The 5-Fu released from the nanocomposite in the dialysis bag readily exchanges with these anions, and the degradation of a small amount of LDH nanocarrier leads to a 46% release of 5-Fu loaded in FA-BSA / Fe3O4@LDH within 6 hours. The release rate then slows down and gradually stabilizes, reaching a total release of 55% within 24 hours. When the pH of the PBS buffer decreases from 7.4 to 5.0, the release of 5-Fu from the FA-BSA / Fe3O4@LDH nanocomposite increases to 60% within 6 hours, 14% higher than the release at pH 7.4. This is mainly because the LDH layer gradually degrades under acidic conditions, causing the release of 5-Fu from the interlayer. After 12 hours, the release of 5-Fu reaches 78%, after which the drug release rate gradually slows down and reaches equilibrium, ultimately reaching a 5-Fu release rate as high as 84%. In vitro release curves demonstrated that the release amount and release time of FA-BSA / Fe3O4@LDH / 5-Fu under acidic conditions were significantly higher than those under neutral conditions, indicating that FA-BSA / Fe3O4@LDH / 5-Fu can achieve a good controlled release effect and selectively and continuously release the drug in the acidic environment of the tumor.

[0107] Figure 9 The hemolysis results of FA-BSA / Fe3O4@LDH / 5-Fu prepared in Example 3 and FA-BSA / Fe3O4@LDH prepared by the above method were all less than 5%, which proves that FA-BSA / Fe3O4@LDH and FA-BSA / Fe3O4@LDH / 5-Fu of different concentrations have good blood compatibility and good safety, and can be used for intravenous injection.

[0108] Figure 10The antitumor activities of 5-Fu, BSA / Fe3O4@LDH / 5-Fu prepared according to the above method, and FA-BSA / Fe3O4@LDH / 5-Fu prepared in Example 3 were detected by the CCK-8 assay kit. As shown in the figure, different samples all showed dose-dependent cytotoxic effects when treating HepG2 cells; the higher the sample concentration, the greater the effect on cell viability. At the same 5-Fu concentration, FA-BSA / Fe3O4@LDH / 5-Fu had a stronger inhibitory effect on HepG2 cells than BSA / Fe3O4@LDH / 5-Fu. This is because FA has targeting properties, targeting the overexpressed FR on the surface of cancer cells, and its targeting specificity makes FA-BSA / Fe3O4@LDH / 5-Fu more easily taken up by HepG2 cells. The IC50 values ​​of 5-Fu and FA-BSA / Fe3O4@LDH / 5-Fu are shown in the figure. 50 The calculated values ​​were 9.3 μg / mL and 60 μg / mL (equivalent to 3.6 μg / mL of 5-Fu), respectively. This indicates that the cell inhibition rate of 5-Fu delivered by FA-BSA / Fe3O4@LDH / 5-Fu is increased, demonstrating the effective delivery of the nanocomposite. The presence of FA-BSA makes it easier for cells to take up the nanocomposite, thereby achieving better therapeutic effects.

[0109] Figure 11 BSA / Fe3O4@LDH and FA-BSA / Fe3O4@LDH, prepared according to the above method and labeled with the fluorescent agent R6G, were used to study the cellular uptake ability of HepG2 cells and the targeting ability of FA. After treating HepG2 cells with the modified or unmodified FA nanocomposites for 0.5, 1, 2, and 4 h, both BSA / Fe3O4@LDH and FA-BSA / Fe3O4@LDH showed different degrees of fluorescence, indicating that the constructed nanocomposite carriers could be successfully internalized by HepG2 cells. When cells were...

[0110] After co-incubation with BSA / Fe3O4@LDH / R6G for a period of time, only weak red fluorescence was observed, while stronger fluorescence was observed when co-incubated with FA-BSA / Fe3O4@LDH / R6G. Furthermore, the average fluorescence intensity of the FA-BSA / Fe3O4@LDH / R6G group was significantly higher than that of the BSA / Fe3O4@LDH / R6G group (117.42±4.23 vs 191.30±6.42, P<0.001). These results indicate that HepG2 cells uptake FA-BSA / Fe3O4@LDH / R6G more than BSA / Fe3O4@LDH / R6G, suggesting that the FA-targeting-FR pathway can enhance the uptake of the nanocomposite material by cells. Moreover, the fluorescence intensity of both groups showed an increasing trend over time, indicating that the nanocomposite material exhibits a time-dependent effect in treating HepG2 cells.

[0111] Example 4

[0112] This embodiment provides a magnetic layered double hydroxide and its preparation method. All deionized water mentioned in this embodiment is boiled deionized water with CO2 removed.

[0113] The preparation method of this magnetic layered double hydroxide is as follows:

[0114] (1) 1081 mg FeCl3·6H2O was dissolved in 40 mL DEG, and 471 mg Na3Cit·2H2O was added and stirred at 60 °C to dissolve. Subsequently, 1312 mg NaAc was added and stirring was continued until completely dissolved. The mixed solution was placed in a polytetrafluoroethylene liner and heated in a reaction vessel at 100 °C for 20 h. Finally, the prepared product was collected with a magnet, purified three times with ethanol, and Fe3O4 nanoparticles were obtained by vacuum drying for further use.

[0115] (2) 30 mg Fe3O4 was uniformly dispersed in 10 mL of deionized water, and 430 mg MgCl2·6H2O and 170 mg AlCl3·6H2O were dissolved in the solution. Then, after stirring under N2 conditions for 30 min, 40 mL NaOH (0.2 mol / L) was quickly added and the mixture was stirred vigorously at 700 rpm for 30 min. The prepared preproduct was centrifuged at 3000 rpm for 6 min, the supernatant was removed, and the product was washed twice with deionized water (3000 rpm, 6 min), and then freeze-dried to obtain magnetic layered double hydroxide Fe3O4@LDH.

[0116] Example 5

[0117] This embodiment provides a magnetic layered double hydroxide and its preparation method. All deionized water mentioned in this embodiment is boiled deionized water with CO2 removed.

[0118] The preparation method of this magnetic layered double hydroxide is as follows:

[0119] (1) Dissolve 1081 mg FeCl3·6H2O in 40 mL DEG, add 471 mg Na3Cit·2H2O and stir at 80 °C to dissolve. Then, add 1312 mg NaAc and continue stirring until completely dissolved. Place the mixed solution in a polytetrafluoroethylene liner and heat in a reaction vessel at 200 °C for 10 h. Finally, collect the prepared product with a magnet, purify it three times with ethanol, and obtain Fe3O4 nanoparticles by vacuum drying for further use;

[0120] (2) 30 mg Fe3O4 was uniformly dispersed in 10 mL of deionized water, and 645 mg MgCl2·6H2O and 255 mg AlCl3·6H2O were dissolved in the solution. Then, after stirring under N2 conditions for 60 min, 40 mL of NaOH (0.1 mol / L) was quickly added and the mixture was stirred vigorously at 500 rpm for 60 min. The prepared preproduct was centrifuged at 5000 rpm for 4 min, the supernatant was removed, and the product was washed twice with deionized water (5000 rpm, 4 min) by centrifugation. The product was then freeze-dried to obtain magnetic layered double hydroxide Fe3O4@LDH.

[0121] Example 6

[0122] This embodiment provides the application of magnetic layered double hydroxides in the preparation of targeted drug-loaded nanoparticles. The magnetic layered double hydroxide in this embodiment is Fe3O4@LDH obtained in Example 4; all deionized water mentioned in this embodiment is boiled deionized water with CO2 removed.

[0123] The preparation method of this targeted drug-loaded nanoformulation is as follows:

[0124] 30 mg of 5-Fu (purity: 99%) was dissolved in 20 mL of an aqueous solution adjusted to pH 8 with NaOH solution. Fe3O4@LDH was suspended in the 5-Fu solution and stirred with a mechanical stirrer for 2.5 h. The reaction mixture was obtained by centrifugation and washed with NaOH aqueous solution (pH = 8) to remove free 5-Fu. Then, it was redispersed in deionized water for further hydrothermal treatment at 80 °C for 12 h to obtain the Fe3O4@LDH / 5-Fu solution, which was then lyophilized.

[0125] Example 7

[0126] This embodiment provides the application of magnetic layered double hydroxides in the preparation of targeted drug-loaded nanoparticles. The magnetic layered double hydroxide in this embodiment is Fe3O4@LDH obtained in Example 5; all deionized water mentioned in this embodiment is boiled deionized water with CO2 removed.

[0127] The preparation method of this targeted drug-loaded nanoformulation is as follows:

[0128] 40 mg of 5-Fu (purity: 99%) was dissolved in 20 mL of an aqueous solution adjusted to pH 9 with NaOH solution. Fe3O4@LDH was suspended in the 5-Fu solution and stirred with a mechanical stirrer for 2.5 h. The reaction mixture was obtained by centrifugation and washed with NaOH aqueous solution (pH = 9) to remove free 5-Fu. It was then redispersed in deionized water for further hydrothermal treatment at 120 °C for 6 h to obtain the Fe3O4@LDH / 5-Fu solution, which was then lyophilized.

[0129] Example 8

[0130] This embodiment provides the application of magnetic layered double hydroxides in the preparation of tumor-targeted drug-loaded nanoparticles. The Fe3O4@LDH / 5-Fu in this embodiment was prepared in Example 6; all deionized water mentioned in this embodiment was boiled and CO2-removed.

[0131] The preparation method of this tumor-targeting drug-loaded nanoparticle formulation is as follows:

[0132] (1) The carboxyl group of FA and the amino group of BSA were covalently bonded via an amide reaction. First, 20 mg of FA was dissolved in DMSO, and then 12.0 mg of EDC·HCl and 7.0 mg of NHS were added, and the mixture was continuously vortexed in the dark for 4 h. Next, 40 mg of BSA was dissolved in Na2CO3 / NaHCO3 buffer solution (pH 8), and the FA solution was slowly added to the BSA solution, while stirring continuously in the dark for 12 h. Finally, the product was dialyzed against deionized water for two days. FA-BSA was obtained after freeze-drying.

[0133] (2) 1 mL of 2 mg / mL Fe3O4@LDH / 5-Fu aqueous suspension was added dropwise to 1.2 mL of 2 mg / mL FA-BSA aqueous solution under ultrasonic bath conditions in an ultrasonic cleaner, and vortexed for 30 min. After the reaction was completed, FA-BSA / Fe3O4@LDH / 5-Fu was obtained by freeze drying.

[0134] Example 9

[0135] This embodiment provides the application of magnetic layered double hydroxides in the preparation of tumor-targeted drug-loaded nanoparticles. The Fe3O4@LDH / 5-Fu in this embodiment was prepared in Example 7; all deionized water mentioned in this embodiment was boiled and CO2-removed.

[0136] The preparation method of this tumor-targeting drug-loaded nanoparticle formulation is as follows:

[0137] (1) The carboxyl group of FA and the amino group of BSA were covalently bonded via an amide reaction. First, 20 mg of FA was dissolved in DMSO, and then 14.0 mg of EDC·HCl and 8.0 mg of NHS were added, and the mixture was continuously vortexed in the dark for 4 h. Next, 60 mg of BSA was dissolved in Na2CO3 / NaHCO3 buffer solution (pH 9), and the FA solution was slowly added to the BSA solution, while stirring continuously in the dark for 12 h. Finally, the product was dialyzed against deionized water for two days. FA-BSA was obtained after freeze-drying.

[0138] (2) 1 mL of 2 mg / mL Fe3O4@LDH / 5-Fu aqueous suspension was added dropwise to 0.8 mL of 2 mg / mL FA-BSA aqueous solution under ultrasonic bath conditions in an ultrasonic cleaner, and vortexed for 30 min. After the reaction was completed, FA-BSA / Fe3O4@LDH / 5-Fu was obtained by freeze drying.

[0139] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a tumor-targeting drug-loaded nanoformulation, characterized in that, The carrier of the tumor-targeting drug-loaded nanoparticle is a magnetic layered double hydroxide, and the active ingredient is a water-soluble antitumor drug. The surface of the tumor-targeting drug-loaded nanoparticle is modified with a folic acid-bovine serum albumin complex. The preparation method of the tumor-targeting drug-loaded nanoparticle specifically includes the following steps: Step a: Prepare the initial product according to the preparation method of targeted drug-loaded nano-formulation: Dissolve the water-soluble antitumor drug in an alkaline solution to prepare a drug solution; disperse the magnetic layered double hydroxide in the drug solution and stir for at least 2 hours; separate the solid and liquid phases, collect the solid phase and wash it with an alkaline solution, then disperse it in water for hydrothermal treatment, remove the solvent after treatment, and obtain the initial product; Step b: Dissolve folic acid in an organic solvent, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and vortex in the dark for at least 3 hours; dissolve bovine serum albumin in a sodium carbonate-sodium bicarbonate buffer solution at pH 8-9, mix with the organic solution of folic acid in the dark for at least 10 hours, dialyze the resulting product with deionized water for at least 40 hours, and dry to obtain the folic acid-bovine serum albumin complex; Step c: Add the suspension of the initial product obtained in step a to the solution of the folic acid-bovine serum albumin complex obtained in step b under ultrasonic conditions, vortex for at least 25 minutes, and then remove the solvent to obtain the final product. The preparation method of the magnetic layered double hydroxide in step a includes the following steps: Step A: Dissolve ferric chloride in diethylene glycol, add sodium citrate and stir to dissolve, then add sodium acetate and continue stirring until completely dissolved. Heat the resulting liquid at 100~200℃ for 10~20h, collect the product with a magnet, wash with ethanol and dry to obtain Fe3O4 nanoparticles. Step B: The Fe3O4 nanoparticles are uniformly dispersed in deionized water, magnesium chloride and aluminum chloride are added and dissolved, and then an alkaline solution is added while stirring under an inert atmosphere. After the reaction is completed, the solid and liquid phases are separated, the solid phase is collected and washed with water to obtain the magnetic layered double hydroxide.

2. The method for preparing tumor-targeting drug-loaded nanoparticles according to claim 1, characterized in that, After adding sodium citrate, stir to dissolve at 60-80℃; and / or In step A, the obtained liquid is heated at 200°C for 10 hours; and / or The solid-liquid separation in step B is performed by centrifugation at a speed of 3000-5000 rpm; and / or In step B, the molar ratio of magnesium chloride to aluminum chloride is 3:1; and / or The magnesium chloride mentioned in step B is magnesium chloride hexahydrate, the aluminum chloride is aluminum chloride hexahydrate, and the total mass of the magnesium chloride and the aluminum chloride is 20 to 30 times that of the Fe3O4 nanoparticles; and / or The alkaline solution mentioned in step B is a 0.1~0.2 mol / L sodium hydroxide aqueous solution; and / or In step B, after adding the alkaline solution, stir at 500-700 rpm for 30-60 minutes; and / or The deionized water is deionized water that has been boiled to remove CO2.

3. The method for preparing tumor-targeting drug-loaded nanoparticles according to claim 1, characterized in that, The water-soluble antitumor drug is 5-fluorouracil; and / or The tumor-targeting drug-loaded nanoformulation is a targeted drug-loaded nanoformulation for liver cancer; and / or The alkaline solution mentioned in step a is an aqueous sodium hydroxide solution with a pH of 8-9; and / or The mass ratio of the water-soluble antitumor drug to the alkaline solution in step a is 1.5~2:1; and / or The hydrothermal treatment in step a is performed at a temperature of 80-120°C for a time of 6-12 hours; and / or The organic solvent used in step b is DMSO; and / or The mass ratio of folic acid to bovine serum albumin in step b is 1:2~3; and / or In step b, the mass ratio of folic acid to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:0.6~0.7, and the mass ratio of folic acid to N-hydroxysuccinimide is 1:0.35~0.4; and / or The mass ratio of the initial product obtained in step c to the folic acid-bovine serum albumin complex is 1:0.8~1.2; and / or The solvent removal method in step c is freeze drying; and / or The deionized water is deionized water that has been boiled to remove CO2.

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