Dual-targeting superparamagnetic nano-drug carrier and preparation method thereof

Abstract

The invention discloses a dual-targeting superparamagnetic nano-drug carrier as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. Superparamagnetic Fe3O4 is used as an inner core, PHis and chemotherapeutic drugs are sequentially wrapped, 2-DG and CB-839 are co-modified on the outermost layer, and efficient treatment is realized through a triple synergistic mechanism: (1) dual metabolism targeted blocking: 2-DG competitively inhibits glycolysis, CB-839 specifically blocks glutamine metabolism, and synergistically cuts off energy supply of tumor cells; (2) pH-responsive accurate drug release: protonizing and swelling the PHis in a tumor microenvironment (pH is 6.5-7.0) to trigger rapid release of the regorafenib; and (3) magnetocaloric synergistic interaction: Fe3O4 generates local high temperature (42-45 DEG C) under an external magnetic field, accelerates drug release and directly induces tumor cell apoptosis. The average hydrated particle size of the prepared nano-drug carrier is 42 + / -5nm, the regorafenib encapsulation efficiency is greater than 85%, the drug loading rate is greater than 11%, in vitro experiments and tumor-bearing mouse models prove that the inhibition rate of the nano-drug carrier on liver cancer cell proliferation is obviously superior to that of the traditional therapy, and an innovative technical scheme is provided for precise targeted therapy of liver cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a targeted nano drug delivery system; more specifically, the present invention relates to a dual-targeted superparamagnetic nano drug carrier and its preparation method and application. Background Art

[0002] Hepatocellular Carcinoma (HCC), the third leading cause of cancer-related deaths worldwide, faces multiple challenges in its treatment, including high heterogeneity, metabolic reprogramming, tumor microenvironment (TME) barriers, and drug resistance. HCC has significant heterogeneity at the genomic, epigenetic, and metabolic levels, which makes single-target strategies prone to failure. In addition, liver cancer cells (such as HCC-LM3) rely on glycolysis (Warburg effect) and glutamine metabolism (Glutaminolysis) for dual energy supply, making it difficult to completely block them with traditional chemotherapy. The immunosuppressive nature of the tumor microenvironment (high expression of molecules such as PD-L1 and TGF-β) and the dense fibrotic matrix (collagen deposition hinders drug penetration) further increase the difficulty of treatment. Targeted drugs (such as sorafenib and regorafenib) are also prone to failure due to mutations in the RAF / MEK / ERK pathway or compensatory metabolic activation.

[0003] Current nanocarriers for liver cancer (e.g., liposomes, polymer nanoparticles, and magnetic nanoparticles) have significant limitations in targeting, metabolic blockade, and drug release control. In terms of targeting, passive targeting (EPR effect) relies on tumor vascular leakage, but HCC vascular distribution is uneven, resulting in an enrichment efficiency of less than 5%. Active targeting strategies (e.g., GalNAc-ASGPR targeting) only target a subset of HCC cells (ASGPR expression heterogeneity), or 2-DG-glycolysis targeting fails to cover HCC subpopulations dependent on glutamine metabolism. In terms of metabolic blockade, existing studies have focused solely on targeting folate receptors, ignoring metabolic intervention; or have only inhibited glycolysis without blocking glutamine metabolism (e.g., the GLS1 enzyme targeted by CB-839), potentially leading to HCC cell escape through metabolic compensation. In terms of drug release control, most carriers rely on passive diffusion release and lack microenvironmental responsiveness. Magnetic nanoparticles are used solely for targeting, without developing magnetothermal release enhancement capabilities.

[0004] The metabolic heterogeneity of HCC cells is characterized by glycolysis-dominant (high expression of GLUT1 and HK2, sensitive to 2-DG) and glutamine-dependent (high expression of GLS1 and ASCT2, dependent on CB-839 inhibition). However, there are currently no patents or research reports on nanocarriers that simultaneously target glycolysis and glutamine metabolism, nor are there any studies combining metabolic blockade with pH-responsive drug release or magnetic hyperthermia.

[0005] Based on the above reasons, this application is filed. Summary of the Invention

[0006] Based on the above reasons, in view of the problems or defects in the prior art, the purpose of the present invention is to provide a dual-targeted superparamagnetic nanoparticle drug carrier and its preparation method and application, so as to solve or at least partially solve the above technical defects in the prior art:

[0007] To address the technical gaps in the above-mentioned existing technologies, the present invention proposes an innovative solution: dual metabolic blockade is achieved by inhibiting glycolysis through 2-DG and glutamine metabolism through CB-839, covering all metabolic subtypes of HCC; the swelling properties of polyhistidine PHis in the HCC microenvironment (pH 6.5-7.0) are utilized to trigger the release of regorafenib, achieving intelligent drug release; and the magnetothermal effect of Fe3O4 under an external magnetic field is used to accelerate drug release and directly kill tumors, thereby verifying its synergistic therapeutic advantages in HCC-LM3 cell models and tumor-bearing mice.

[0008] In order to achieve the above-mentioned first object of the present invention, the technical solution adopted by the present invention is as follows:

[0009] A dual-targeted superparamagnetic nanodrug carrier comprises an inner core, an encapsulating layer, and an outer layer in sequence; the inner core is a superparamagnetic Fe3O4 nanoparticle; the encapsulating layer is a pH-sensitive polymer polyhistidine (PHis), which is arranged on the surface of the inner core and loaded with a chemotherapy drug; the outer layer is composed of 2-deoxyglucose (2-DG) and the glutaminase inhibitor CB-839.

[0010] Furthermore, in the above technical solution, the particle size of the superparamagnetic Fe3O4 nanoparticles is 5 to 10 nm.

[0011] Furthermore, in the above technical solution, the thickness of the wrapping layer is 5 to 20 nm.

[0012] Furthermore, in a preferred embodiment of the present invention, the chemotherapy drug is Regorafenib. That is, when the chemotherapy drug is Regorafenib, the dual-targeted superparamagnetic nanoparticle drug carrier is specifically Fe3O4@PHis@Regorafenib@2-DG / CB-839.

[0013] Furthermore, in the above technical solution, the superparamagnetic Fe3O4 nanoparticles are prepared by a co-precipitation method.

[0014] Furthermore, in the above technical solution, the specific steps of preparing the superparamagnetic Fe3O4 nanoparticles by co-precipitation method are as follows:

[0015] (A) Under normal temperature and inert gas protection, according to the proportion, ferric chloride hexahydrate (FeCl3·6H2O) and ferrous chloride tetrahydrate (FeCl2·4H2O) are sequentially dissolved in deionized water;

[0016] (B) Under the conditions of inert gas protection and stirring, an ammonia water solution is added dropwise into the mixed solution 1 obtained in step (A) according to the proportion; after the dropwise addition is completed, the obtained mixed reaction solution 1 is continuously reacted under stirring for 20-40 min;

[0017] (C) After the reaction is completed, citric acid is added to the obtained product, the obtained mixed reaction solution 2 is heated to 80°C and refluxed for 2 hours; after the reaction is completed, the obtained product is magnetically separated and washed to obtain the superparamagnetic Fe3O4 nanoparticles.

[0018] Specifically, the above technical solution, the normal temperature of the present application refers to the natural room temperature condition in four seasons, without additional cooling or heating treatment, and the general normal temperature is controlled at 10-30°C, preferably 15-25°C.

[0019] Preferably, in the above technical solution, in one embodiment of the present application, the mass ratio of ferric chloride hexahydrate to ferrous chloride tetrahydrate in step (A) is 1:0.3-0.5, preferably 1:0.4.

[0020] Preferably, in the above technical solution, the total concentration of iron salt in the mixed solution 1 in step (B) is 5-25 mmol / L.

[0021] Preferably, in the above technical solution, the mass fraction of the ammonia water solution in step (B) is 15-30%.

[0022] Preferably, in the above technical solution, the dropwise addition speed of the ammonia water solution in step (B) is 0.8-1.2 mL / min.

[0023] Preferably, in the above technical solution, the dosage ratio of ferric chloride hexahydrate to ammonia water solution in step (B) is 1 mole part:(2-5) volume parts; wherein: the mole part and the volume part are based on mmol:mL.

[0024] Preferably, in the above technical solution, the stirring speed in step (B) is 300-1200 rpm, preferably 900 rpm.

[0025] Preferably, in the above technical solution, the molar ratio of citric acid to ferric chloride hexahydrate in step (C) is (0.4-1.0):1.

[0026] The second object of the present invention is to provide a method for preparing the above-mentioned Fe3O4@PHis@Regorafenib@2-DG / CB-839, which specifically comprises the following steps:

[0027] (1) Preparation of Fe3O4@PHis nanoparticles

[0028] Polyhistidine (PHis) is dispersed in phosphate buffer (PBS) according to a specific ratio and ultrasonically dispersed to obtain a PHis dispersion. Superparamagnetic Fe3O4 nanoparticles are then added to the PHis dispersion according to a specific ratio and stirred at room temperature for 1-3 hours. After the reaction is completed, the resulting product is ultrafiltered and centrifuged, washed, and obtained as Fe3O4@PHis nanoparticles.

[0029] (2) Preparation of Fe3O4@PHis@Regorafenib

[0030] Regorafenib is dispersed in a mixed solvent consisting of anhydrous ethanol and phosphate buffer saline (PBS) according to a ratio, and ultrasonically dispersed to obtain a Regorafenib dispersion; then the Fe3O4@PHis nanoparticles described in step (1) are added to the Regorafenib dispersion according to a ratio, and stirred in the dark for 2-6 hours; after the reaction, the obtained product is magnetically separated and washed to obtain Fe3O4@PHis@Regorafenib;

[0031] (3) Preparation of Fe3O4@PHis@Regorafenib@2-DG

[0032] 2-DG was dispersed in phosphate buffered saline (PBS) according to a specific ratio, and then EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) were added to the resulting 2-DG dispersion in sequence according to a specific ratio. The mixture was stirred at room temperature for 20-40 minutes to obtain an activated solution.

[0033] The Fe3O4@PHis@Regorafenib described in step (2) is added to the activation solution according to the ratio, and the reaction is stirred for 4-8 hours under light-proof conditions. After the reaction, the obtained product is magnetically separated and washed to obtain Fe3O4@PHis@Regorafenib@2-DG.

[0034] (4) Preparation of Fe3O4@PHis@Regorafenib@2-DG / CB-839

[0035] Polylactic acid (PLA) was dissolved in dichloromethane according to a certain ratio, and then the Fe3O4@PHis@Regorafenib@2-DG described in step (3) was added according to a certain ratio, and ultrasonic emulsification was performed for 5-15 minutes. After the ultrasonic emulsification, the obtained product was placed in a fume hood to evaporate the residual solvent, thereby obtaining PLA-coated Fe3O4@PHis@Regorafenib@2-DG.

[0036] CB-839 was dissolved in anhydrous ethanol according to a ratio to obtain a CB-839 solution; the CB-839 solution was then added dropwise to the PLA-coated Fe3O4@PHis@Regorafenib@2-DG according to a ratio, and the mixture was stirred in the dark for 2-8 hours; after the reaction, the mixture was centrifuged and washed to obtain the Fe3O4@PHis@Regorafenib@2-DG / CB-839.

[0037] Furthermore, in the above technical solution, the pH value of the phosphate buffer solution (PBS) in step (1) is 7.4.

[0038] Furthermore, in the above technical solution, the molecular weight of the polyhistidine PHis in step (1) is 5 kDa.

[0039] Furthermore, in the above technical solution, the mass ratio of the polyhistidine to the superparamagnetic Fe3O4 nanoparticles in step (1) is 1:1 to 3:1, preferably 2:1.

[0040] Furthermore, in the above technical solution, the stirring speed in step (1) is 600 to 1200 rpm.

[0041] Furthermore, in the above technical solution, the molecular weight cut-off of the ultrafiltration centrifuge tube used in the ultrafiltration centrifugation in step (1) is 50kD.

[0042] Furthermore, in the above technical solution, the usage ratio of the polyhistidine PHis to the phosphate buffer in step (1) is (5 to 15) parts by mass: 1 part by volume; wherein: the mass parts and volume parts are based on mg:mL.

[0043] Furthermore, in the above technical solution, in the mixed solvent of step (2), the volume ratio of anhydrous ethanol to phosphate buffer saline (PBS) is 1:2 to 2:1, preferably 1:1.

[0044] Furthermore, in the above technical solution, the pH value of the phosphate buffer solution (PBS) in step (2) is 7.4.

[0045] Furthermore, in the above technical solution, the usage ratio of Regorafenib to the mixed solvent in step (2) is (5-15) parts by mass: 1 part by volume; wherein: the parts by mass and parts by volume are based on mg:mL.

[0046] Furthermore, in the above technical solution, the mass ratio of Regorafenib to Fe3O4@PHis nanoparticles in step (2) is 0.5:1-2:1, preferably 1:1.

[0047] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the time of the light-proof stirring reaction in step (2) is 3 to 5 hours, preferably 4 hours.

[0048] Furthermore, in the above technical solution, the stirring speed in step (2) is 300 to 900 rpm.

[0049] Furthermore, in the above technical solution, the pH value of the PBS in step (3) is 6.5.

[0050] Furthermore, in the above technical solution, the usage ratio of the 2-DG to the phosphate buffer in step (3) is (5 to 15) parts by mass: 1 part by volume; wherein: the mass parts and volume parts are based on mg:mL.

[0051] Furthermore, in the above technical solution, the molar ratio of EDC to NHS in step (3) is 1-3:1, preferably 2:1.

[0052] Furthermore, in the above technical solution, the time for stirring the reaction under light-proof conditions in step (3) is 6 hours.

[0053] Furthermore, in the above technical solution, the molar ratio of the 2-DG in step (3) to the amino groups on the surface of Fe3O4@PHis@Regorafenib is 1:1-1:2.

[0054] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the weight average molecular weight Mw of the polylactic acid in step (4) is 10 kDa.

[0055] Furthermore, in the above technical solution, the usage ratio of the polylactic acid to dichloromethane in step (4) is (5 to 15) parts by mass: 1 part by volume; wherein: the ratio between the parts by mass and the parts by volume is based on mg:mL.

[0056] Furthermore, in the above technical solution, the mass ratio of PLA to Fe3O4@PHis@Regorafenib@2-DG in step (4) is 1:1.5-1:3, preferably 1:2.

[0057] Furthermore, in the above technical solution, the usage ratio of the CB-839 and anhydrous ethanol in step (4) is (5 to 15) parts by mass: 1 part by volume; wherein: the parts by mass and parts by volume are based on mg:mL.

[0058] Furthermore, in the above technical solution, the mass ratio of CB-839 to Fe3O4@PHis@Regorafenib@2-DG in step (4) is 10-20:100.

[0059] The third object of the present invention is to provide the use of the above-mentioned Fe3O4@PHis@Regorafenib@2-DG / CB-839 in the preparation of drugs for treating liver cancer.

[0060] The fourth object of the present invention is to provide a drug for treating liver cancer, comprising the above-mentioned Fe3O4@PHis@Regorafenib@2-DG / CB-839.

[0061] Compared with the prior art, the present invention has the following beneficial effects:

[0062] (1) The Fe3O4@PHis@Regorafenib@2-DG / CB-839 prepared in the present invention has an average hydrated particle size of 42±5 nm, a zeta potential of +5 to +10 mV, a regorafenib encapsulation efficiency of >85%, a drug loading rate of >11%, and a CB-839 loading efficiency of >80%.

[0063] (2) The drug carrier Fe3O4@PHis@Regorafenib@2-DG / CB-839 prepared by the present invention has a regorafenib release rate of ≥80% within 24 hours in a tumor microenvironment with a pH of 6.5-7.0, while the release rate in a normal physiological environment with a pH of 7.4 is less than 20%.

[0064] (3) The pH-responsive drug release mechanism of the present invention relies on the protonated swelling effect of polyhistidine in a weakly acidic environment, with a swelling volume change rate of 150%-200%. The PLA coating gradually degrades in the tumor microenvironment, controlling the sustained release rate of CB-839, with a cumulative release rate of 70%-85% over 48 hours.

[0065] (4) When the drug carrier Fe3O4@PHis@Regorafenib@2-DG / CB-839 prepared by the present invention is subjected to an external alternating magnetic field (frequency 100 kHz, field strength 10 kA / m), the temperature of the drug carrier rises to 42-45°C within 10 minutes, the release rate of Regorafenib increases by 2-3 times, and the magnetothermal effect directly induces tumor cell apoptosis.

[0066] (5) In the dual metabolic targeting mechanism, 2-DG blocks glycolysis by competitively inhibiting GLUT1 receptors, and CB-839 blocks glutamine metabolism by inhibiting glutaminase (GLS1), both of which cooperatively reduce the intracellular ATP level of tumor cells to 20%-30% of the normal value.

[0067] In summary, the beneficial effects of the present application are reflected in three aspects: first, the triple synergistic treatment mechanism - 2-DG competitively inhibits GLUT1 receptors to block glycolysis, CB-839 inhibits GLS1 enzyme to block glutamine metabolism, and the combination reduces the intracellular ATP of tumor cells to 20%-30% of the normal value; polyhistidine is protonated and swells (volume expansion 150%-200%) in the tumor microenvironment, the release rate of regorafenib is ≥80% (pH 6.5-7.0) within 24 hours and the release rate is <20% in the physiological environment (pH 7.4); in addition, the alternating magnetic field (100 kHz, 10 kA / m) makes the carrier temperature rise to 42-45℃ within 10 minutes, the drug release rate increases by 2-3 times and directly induces tumor cell apoptosis. Second, the high delivery performance - the average hydrated particle size of the drug carrier is 42±5nm, the Zeta potential is +5 to +10 mV, the encapsulation efficiency of regorafenib is >85%, the drug loading rate is >11%, the loading efficiency of CB-839 is >80%, the present application selects liver cancer cells HCC-LM3 as the experimental model, and the in vitro experiment shows that the IC50 of HCC-LM3 cells is reduced by 60% compared with free drugs, and the tumor inhibition rate of tumor-bearing mouse models reaches 75%-80%. Third, the process controllability - the particle size of Fe3O4, the thickness of PHis, the drug loading ratio and the coupling efficiency of targeting molecules can be accurately controlled by reaction parameters, and the batch stability RSD is <5%. The present application provides an efficient and accurate combined solution for liver cancer treatment. BRIEF DESCRIPTION OF DRAWINGS

[0068] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creating any inventive labor.

[0069] Figure 1 The structure diagram of the Fe3O4@PHis@Regorafenib@2-DG / CB-839 dual-targeting superparamagnetic nanomedicine carrier provided by the present application is shown.

[0070] Figure 2 The particle size distribution diagram of the Fe3O4@PHis@Regorafenib@2-DG / CB-839 dual-targeting superparamagnetic nanomedicine carrier prepared in Example 1 is shown.

[0071] Figure 3 This is a temperature rise curve of the Fe3O4@PHis@Regorafenib@2-DG / CB-839 dual-targeted superparamagnetic nanodrug carrier prepared in Example 1 under an external alternating magnetic field (frequency 100 kHz, field strength 10 kA / m).

[0072] Figure 4 This is a 24-hour drug release rate curve of the Fe3O4@PHis@Regorafenib@2-DG / CB-839 dual-targeted superparamagnetic nanodrug carrier prepared in Example 1 under three conditions: pH 6.5 without a magnetic field, pH 6.5 with a magnetic field, and pH 7.5 without a magnetic field.

[0073] Figure 5 This is a diagram showing the biological safety and anti-tumor effect of the Fe3O4@PHis@Regorafenib@2-DG / CB-839 dual-targeted superparamagnetic nanodrug carrier prepared in Example 1 evaluated by the CCK-8 method. DETAILED DESCRIPTION

[0074] The present invention provides a dual-targeted superparamagnetic nano-drug carrier, its preparation method, and its application in the treatment of liver cancer. Through structural design and functional integration, the drug carrier overcomes the problem of treatment failure caused by metabolic heterogeneity and microenvironmental barriers in liver cancer. The drug carrier is divided into three layers from the inside to the outside: a superparamagnetic Fe3O4 core synthesized by coprecipitation with a particle size of 5-10nm, surface-modified with citric acid to achieve both magnetic targeting and magnetic hyperthermia; a pH-sensitive middle layer encapsulates Fe3O4 with a molecular weight of 5kDa polyhistidine (PHis) and loads the chemotherapy drug regorafenib, triggering drug release by utilizing the weak acidity of the tumor microenvironment (pH 6.5-7.0); and a dual metabolic targeting outer layer covalently coupled with 2-deoxyglucose (2-DG) and the glutaminase inhibitor CB-839 to achieve synergistic blockade of glycolysis and glutamine metabolism.

[0075] The specific preparation method of the dual-targeted superparamagnetic nanodrug carrier provided in a preferred embodiment of the present invention includes the following steps: first, FeCl3·6H2O and FeCl2·4H2O are dissolved in deionized water in a mass ratio of 1:0.4, and ammonia water is added under nitrogen protection to induce a coprecipitation reaction to generate Fe3O4 nanoparticles, which are then modified with citric acid and purified; secondly, Fe3O4 is dispersed in PBS, PHis is added in a mass ratio of 2:1 to form a coating layer, and then regorafenib is loaded in a mass ratio of 1:1. The drug loading is completed by reacting in the dark for 4 hours; thirdly, under EDC / NHS activation, 2-DG is coupled to the carrier surface through an amide bond; finally, CB-839 is loaded through a PLA coating layer mediated by hydrophobic interaction to obtain the final product Fe3O4@PHis@Regorafenib-2-DG / CB-839.

[0076] The drug carrier prepared by the present invention achieves efficient treatment through a triple synergistic mechanism: ① Dual metabolic targeted blockade: 2-DG competitively inhibits glycolysis, and CB-839 specifically blocks glutamine metabolism, synergistically cutting off the energy supply of tumor cells; ② pH-responsive precise drug release: Polyhistidine protonates and swells in the tumor microenvironment (pH 6.5-7.0), triggering the rapid release of regorafenib; ③ Magnetothermal synergy: Fe3O4 generates local high temperature (42-45°C) under an external magnetic field, accelerating drug release and directly inducing tumor cell apoptosis. The average hydrated particle size of the prepared nanodrug carrier is 42±5nm, the regorafenib encapsulation efficiency is >85%, and the drug loading rate is >11%. In vitro experiments and tumor-bearing mouse models have confirmed that its inhibition rate on liver cancer cell proliferation is significantly better than traditional therapies, providing an innovative technical solution for the precise targeted treatment of liver cancer.

[0077] The present invention is further described in detail below through an implementation case. This implementation case is implemented based on the technology of the present invention. Detailed implementation methods and specific operating procedures are now given to illustrate the creativity of the present invention, but the scope of protection of the present invention is not limited to the following implementation case.

[0078] Based on the information contained in this application, it will be readily apparent to those skilled in the art that various changes can be made to the precise description of the present invention. It should be understood that the scope of the present invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are provided merely to illustrate specific aspects of the present invention.

[0079] In order to better understand the present invention and not to limit the scope of the present invention, all numbers used in this application to express amounts, percentages, and other numerical values ​​should be understood to be modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification are approximate values, which may be changed according to the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods.

[0080] The equipment and raw materials used in the present invention can be purchased from the market or are commonly used in the art. The methods in the following examples are all conventional methods in the art unless otherwise specified.

[0081] Example 1

[0082] The preparation method of a dual-targeted superparamagnetic nanoparticle drug carrier Fe3O4@PHis@Regorafenib@2-DG / CB-839 in this embodiment includes the following steps:

[0083] 1. Preparation of citric acid-modified Fe3O4 nanoparticles

[0084] 1.1 Preparation of precursor solution

[0085] Weigh 1.35 g of ferric chloride hexahydrate (FeCl3·6H2O, 5 mmol) and 0.5 g of ferrous chloride tetrahydrate (FeCl2·4H2O, 2.5 mmol) into a 500 mL three-necked flask. Add 200 mL of deionized water and stir magnetically until completely dissolved. Then, introduce nitrogen (N2) for 30 minutes to remove dissolved oxygen.

[0086] 1.2 Coprecipitation reaction

[0087] Under nitrogen, 10 mL of 25% aqueous ammonia (NH₃·H₂O) was added dropwise to the solution at a rate of 1 mL / min while maintaining a stirring rate of 1000 rpm. After 30 minutes of reaction, the solution turned from orange to black, forming Fe₃O₄ nanoparticles.

[0088] 1.3 Surface modification with citric acid

[0089] Add 0.5g of citric acid (C6H8O7, 2.6mmol) to the reaction system, heat to 80℃ and reflux for 2 hours to allow the carboxyl groups of citric acid to react with the Fe on the surface of Fe3O4. 3+ After the reaction is completed, the mixture is cooled to room temperature naturally.

[0090] 1.4 Purification and preservation

[0091] The Fe3O4nanoparticles were separated by an external magnet, the supernatant was discarded, and the unreacted ions and free citric acid were removed by washing with 50 mL of deionized water for 3 times. Finally, the product was dispersed in 50 mL of phosphate buffered saline (PBS, pH 7.4) and stored at 4°C for later use.

[0092] 2. Polyhistidine (PHis) coated Fe3O4nanoparticles

[0093] 2.1 Preparation of PHis solution

[0094] 200 mg of polyhistidine (PHis, molecular weight 5 kDa) was weighed and dissolved in 20 mL of PBS (pH 7.4) and sonicated (power 200 W, frequency 40 kHz) for 10 min until completely dissolved.

[0095] 2.2 Complexation of Fe3O4with PHis

[0096] 100 mg of citric acid-modified Fe3O4nanoparticles prepared in step 1 were added to the above PHis solution and stirred at 800 rpm at room temperature for 2 h to allow the PHis to form a coating layer of 2-5 nm in thickness by electrostatic adsorption and hydrophobic interaction.

[0097] 2.3 Purification

[0098] The reaction solution was transferred to an ultrafiltration centrifuge tube (molecular weight cut-off 50 kDa) and centrifuged at 10,000 rpm for 15 min to remove the unbound free PHis. After repeating the washing for 3 times, the Fe3O4@PHis complex was re-dispersed in 10 mL of PBS (pH 7.4).

[0099] 3. Loading of Regorafenib

[0100] 3.1 Preparation of drug solution

[0101] 100 mg of Regorafenib was weighed and dissolved in 10 mL of a mixed solvent (consisting of anhydrous ethanol and PBS (pH 7.4) at a volume ratio of 1:1) and sonicated (power 150 W, frequency 40 kHz) for 10 min until the drug was completely dissolved.

[0102] 3.2 Drug loading

[0103] 100 mg of Fe3O4@PHis nanoparticles prepared in step 2 were added to the above drug solution and stirred at 600 rpm in the dark for 4 h to allow the Regorafenib to be loaded into the carrier by hydrophobic interaction.

[0104] 3.3 Purification and storage

[0105] The drug-loaded nanoparticles were collected by magnetic separation and washed three times with PBS (pH 7.4) to remove unloaded regorafenib. The final product (Fe3O4@PHis@Regorafenib) was dispersed in 10 mL of PBS (pH 7.4) and stored at 4°C in the dark.

[0106] 4.2-DG covalent conjugation modification

[0107] 4.1 Carboxyl group activation

[0108] 50 mg of 2-deoxyglucose (2-DG) was weighed and dissolved in 5 mL of PBS (pH 6.5). 10 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 5 mg of NHS (N-hydroxysuccinimide) were added, and the mixture was stirred at room temperature for 30 minutes to activate the carboxyl groups to obtain an activated solution.

[0109] 4.2 Coupling reaction

[0110] 100 mg of Fe3O4@PHis@Regorafenib prepared in step 3 was added to the above activation solution and stirred at 500 rpm for 6 hours in the dark to covalently couple 2-DG to the support surface through amide bonds.

[0111] 4.3 Purification

[0112] Unreacted 2-DG was removed by magnetic separation and washed three times with PBS (pH 6.5) to obtain Fe3O4@PHis@Regorafenib@2-DG.

[0113] 5. Physical adsorption modification of CB-839

[0114] 5.1 Preparation of PLA coating

[0115] 50 mg of polylactic acid (PLA, Mw = 10 kDa) was weighed and dissolved in 5 mL of dichloromethane. 100 mg of Fe3O4@PHis@Regorafenib@2-DG was added and ultrasonic emulsification (power 300 W, frequency 40 kHz) was performed for 10 minutes. The solvent was then evaporated in a fume hood to form a PLA coating.

[0116] 5.2CB-839 load

[0117] 50 mg of CB-839 (glutaminase inhibitor) was dissolved in 5 mL of ethanol and added dropwise to the PLA-modified carrier suspension. The suspension was stirred at 600 rpm for 4 hours in the dark to embed CB-839 into the PLA layer through hydrophobic interaction.

[0118] 5.3 Purification and preservation

[0119] Free CB-839 was removed by centrifugation (8000 rpm, 10 min) and washed three times with PBS (pH 7.4). The final product (Fe3O4@PHis@Regorafenib@2-DG / CB-839) was dispersed in 10 mL of PBS (pH 7.4) and stored at 4°C.

[0120] Comparative Example 1 (single metabolic inhibitor 2-DG group)

[0121] The preparation method of Fe3O4@PHis@Regorafenib@2-DG in this comparative example is basically the same as that in Example 1, except that this comparative example does not include step 5, PLA and CB-839 are not modified, and the remaining steps are the same.

[0122] Comparative Example 2 (single metabolic inhibitor CB-839 group)

[0123] The preparation method of Fe3O4@PHis@Regorafenib@CB-839 in this comparative example is basically the same as that in Example 1, except that this comparative example does not include step 4 and 2-DG is not modified, and the remaining steps are the same.

[0124] Quality testing and performance verification

[0125] (1) Particle size and Zeta potential determination

[0126] The hydrated particle size and polydispersity index (PDI) of the target drug carrier Fe3O4@PHis@Regorafenib@2-DG / CB-839 prepared in Example 1 were determined by dynamic light scattering (DLS): a 0.1 mg / mL PBS dispersion of the nanoparticles (pH 7.4) was measured three times at 25°C using a dynamic light scattering instrument, and the average values ​​were reported as the particle size distribution (42±5 nm) and PDI value (<0.2). The zeta potential was determined by laser Doppler electrophoresis, and the surface potential value (+8 mV) was recorded under the same dispersion conditions. The relative standard deviation (RSD) of the parallel experiments was <5%. Figure 2 shown.

[0127] (2) Calculation of drug loading and encapsulation efficiency

[0128] Regorafenib: The drug loading efficiency (DL%) was 12.5±0.8%, and the encapsulation efficiency (EE%) was 85±3%, as determined by ultraviolet spectrophotometry (λ=260 nm).

[0129] CB-839: The loading efficiency was determined to be 82±2% by HPLC (C18 column, mobile phase acetonitrile / water = 60:40). The drug loading rate and encapsulation efficiency were calculated as follows:

[0130]

[0131] (3) In vitro drug release characteristics

[0132] Regorafenib: In a simulated tumor microenvironment at pH 6.5, the 24-hour release rate was 85±2%; in a normal environment at pH 7.4, the release rate was 18±3%.

[0133] CB-839: In a pH 6.5 environment, the cumulative release rate was 75±3% within 48 hours, and the PLA degradation rate was positively correlated with the release behavior.

[0134] (IV) Verification of magnetocaloric effect

[0135] Under the action of an external alternating magnetic field (frequency 100kHz, field strength 10kA / m):

[0136] Heating performance: The carrier temperature rises to 43±1℃ within 10 minutes (such as Figure 3 shown).

[0137] Accelerated drug release: The release rate of regorafenib increased by 2.5 times (24-hour release rate reached 95%).

[0138] (V) Regorafenib 24-hour release rate test

[0139] The in vitro drug release behavior of Fe3O4@PHis@Regorafenib@2-DG / CB-839 prepared in Example 1 was determined using a dialysis bag method (molecular weight cutoff 10 kDa): 10 mg of Fe3O4@PHis@Regorafenib@2-DG / CB-839 prepared in Example 1 was dispersed in 5 mL of PBS (pH 7.4) and sealed in a dialysis bag. The solution was then placed in 50 mL of release medium (simulating the tumor microenvironment pH 6.5 or the physiological environment pH 7.4) and incubated at 37°C with shaking (100 rpm). In the magnetothermal group, an alternating magnetic field (100 kHz, 10 kA / m) was applied throughout the process to maintain 43±1°C. Samples were collected at 1, 2, 4, 6, 8, 12, and 24 hours, and regorafenib concentrations were determined by UV spectrophotometry (λ = 260 nm). Three parallel groups were used, meeting the sink conditions, with relative standard deviations (RSDs) <5%.

[0140] (VI) Evaluation of the biosafety and anti-tumor effect of drug carriers using CCK-8 assay

[0141] Human normal hepatocytes LO2 and hepatoma cells HCC-LM3 were cultured at 5×10 3Cells were seeded in 96-well plates and culture medium containing 20 μg / mL (based on the mass of Fe3O4) of the drug carrier prepared in Example 1 was added. After 48 hours of culture, 10 μL of CCK-8 reagent was added to each well and incubated for 2 hours. The absorbance was measured at a wavelength of 450 nm, and the cell viability (%) was calculated. A blank control group (PBS) and three replicates were set up. Data are expressed as mean ± standard deviation. The survival rate formula is:

[0142]

[0143] This validated the drug carrier's safety against normal liver cells and its inhibitory activity against liver cancer cells. The experimental results showed that all materials had a survival rate of >95% for normal liver cells, demonstrating excellent biosafety and meeting clinical safety requirements. The intact drug carrier (Fe3O4@PHis@Regorafenib@2-DG / CB-839) achieved an inhibition rate of 71.5% against HCC-LM3, significantly superior to other groups (p<0.01), demonstrating the highly effective and selective killing of liver cancer cells by the Fe3O4@PHis@Regorafenib@2-DG / CB-839 prepared in Example 1, as well as its safety against normal liver cells.

[0144] (VII) Metabolic inhibition effect

[0145] ATP detection kit was used to quantitatively analyze the inhibitory effect of drug carrier on energy metabolism of liver cancer cells: HCC-LM3 cells were cultured at a density of 5×10 4 Cells were seeded in a 96-well plate and treated for 24 hours with culture medium containing 20 μg / mL of Fe3O4@PHis@Regorafenib@2-DG / CB-839 prepared in Example 1. After lysis of the cells, the ATP working solution was added according to the kit instructions. The intracellular ATP level (nmol / mg protein) was calculated using a standard curve using a chemiluminescence assay (RLU values ​​were detected using a microplate reader). The relative percentage (%) of ATP in the experimental group was calculated using the untreated group as 100% as the following formula:

[0146]

[0147] The experiment was conducted in triplicate, and the results confirmed that after HCC-LM3 cells were treated with the drug carrier, the ATP level dropped to 25±3% of the normal value, which was significantly better than the single metabolic inhibitor group (2-DG group: 45±5%; CB-839 group: 50±4%).

Claims

1. A dual-targeted superparamagnetic nanoparticle drug carrier, characterized by: It includes an inner core, an encapsulating layer and an outer layer in sequence; the inner core is superparamagnetic Fe3O4 nanoparticles; the encapsulating layer is a pH-sensitive polymer polyhistidine PHis, which is arranged on the surface of the inner core and loaded with chemotherapy drugs; the outer layer is composed of 2-deoxyglucose (2-DG) and the glutaminase inhibitor CB-839.

2. The dual-targeted superparamagnetic nanoparticle drug carrier according to claim 1, characterized in that: The particle size of the superparamagnetic Fe3O4 nanoparticles is 5-10 nm.

3. The dual-targeted superparamagnetic nanoparticle drug carrier according to claim 1, characterized in that: The thickness of the wrapping layer is 5 to 20 nm.

4. The dual-targeted superparamagnetic nanoparticle drug carrier according to claim 1, characterized in that: The superparamagnetic Fe3O4 nanoparticles are prepared by a co-precipitation method, and the steps are as follows: (A) At room temperature and under inert gas, ferric chloride hexahydrate (FeCl3·6H2O) and ferrous chloride tetrahydrate (FeCl2·4H2O) were dissolved in deionized water in the order specified. (B) under inert gas protection and stirring conditions, adding ammonia solution dropwise to the mixed solution 1 obtained in step (A) according to the ratio; after the dropwise addition is completed, the obtained mixed reaction solution 1 is continued to react under stirring conditions for 20 to 40 minutes; (C) After the reaction is completed, citric acid is added to the obtained product, and the obtained mixed reaction solution 2 is heated to 80° C. and refluxed for reaction for 2 hours; after the reaction is completed, the obtained product is subjected to magnetic separation and then washed to obtain the superparamagnetic Fe3O4 nanoparticles.

5. A method for preparing a dual-targeted superparamagnetic nanoparticle drug carrier Fe3O4@PHis@Regorafenib@2-DG / CB-839, characterized by: The method specifically comprises the following steps: (1) Preparation of Fe3O4@PHis nanoparticles Disperse polyhistidine PHis in phosphate buffer saline (PBS) according to the ratio, and perform ultrasonic dispersion to obtain PHis dispersion liquid; Then, superparamagnetic Fe3O4 nanoparticles are added to the PHis dispersion according to the ratio, and the mixture is stirred at room temperature for 1-3 hours. After the reaction, the obtained product is ultrafiltered and centrifuged, and washed to obtain Fe3O4@PHis nanoparticles. (2) Preparation of Fe3O4@PHis@Regorafenib Regorafenib is dispersed in a mixed solvent consisting of anhydrous ethanol and phosphate buffer saline (PBS) according to a ratio, and ultrasonically dispersed to obtain a Regorafenib dispersion; then the Fe3O4@PHis nanoparticles described in step (1) are added to the Regorafenib dispersion according to a ratio, and stirred in the dark for 2-6 hours; after the reaction, the obtained product is magnetically separated and washed to obtain Fe3O4@PHis@Regorafenib; (3) Preparation of Fe3O4@PHis@Regorafenib@2-DG 2-DG was dispersed in phosphate buffered saline (PBS) according to a specific ratio, and then EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) were added to the resulting 2-DG dispersion in sequence according to a specific ratio. The mixture was stirred at room temperature for 20-40 minutes to obtain an activated solution. The Fe3O4@PHis@Regorafenib described in step (2) is added to the activation solution according to the ratio, and the reaction is stirred for 4-8 hours under light-proof conditions. After the reaction, the obtained product is magnetically separated and washed to obtain Fe3O4@PHis@Regorafenib@2-DG. (4) Preparation of Fe3O4@PHis@Regorafenib@2-DG / CB-839 Polylactic acid (PLA) was dissolved in dichloromethane according to a certain ratio, and then the Fe3O4@PHis@Regorafenib@2-DG described in step (3) was added according to a certain ratio, and ultrasonic emulsification was performed for 5-15 minutes. After the ultrasonic emulsification, the obtained product was placed in a fume hood to evaporate the residual solvent, thereby obtaining PLA-coated Fe3O4@PHis@Regorafenib@2-DG. Dissolve CB-839 in anhydrous ethanol according to the ratio to obtain a CB-839 solution; Then, the CB-839 solution was added dropwise to the PLA-coated Fe3O4@PHis@Regorafenib@2-DG according to the ratio, and the mixture was stirred in the dark for 2-8 hours. After the reaction, the mixture was centrifuged and washed to obtain the Fe3O4@PHis@Regorafenib@2-DG / CB-839.

6. The preparation method according to claim 5, characterized in that: The mass ratio of the polyhistidine to the superparamagnetic Fe3O4 nanoparticles in step (1) is 1:1 to 3:

1.

7. The preparation method according to claim 5, characterized in that: The mass ratio of Regorafenib to Fe3O4@PHis nanoparticles in step (2) is 0.5:1-2:

1.

8. The preparation method according to claim 5, characterized in that: The molar ratio of the 2-DG in step (3) to the amino groups on the surface of Fe3O4@PHis@Regorafenib is 1:1-1:

2.

9. Use of the dual-targeted superparamagnetic nano-drug carrier according to claims 1 to 4 or the dual-targeted superparamagnetic nano-drug carrier Fe3O4@PHis@Regorafenib@2-DG / CB-839 prepared by the method according to claims 5 to 8 in the preparation of drugs for treating liver cancer.

10. A drug for treating liver cancer, characterized in that: The invention comprises the dual-targeted superparamagnetic nano drug carrier according to claims 1 to 4 or the dual-targeted superparamagnetic nano drug carrier Fe3O4@PHis@Regorafenib@2-DG / CB-839 prepared by the method according to claims 5 to 8.

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