Ferritin nano-composite with active targeting effect as well as preparation method and application of ferritin nano-composite

Abstract

The invention discloses a ferritin nano-composite with an active targeting effect as well as a preparation method and application thereof, and relates to the technical field of pharmacy. The ferritin nano-composite with the active targeting effect, provided by the invention, is loaded with the shikonin and the metal ions, and the ferritin nano-composite improves the water solubility of the shikonin and improves the bioavailability of the shikonin; cytotoxic medicine'warheads' can be selectively delivered to tumor cells, toxic and side effects are reduced, tumor killing power is improved, and precise treatment of hematologic tumors is achieved. The preparation method provided by the invention is mild in experimental condition, relatively small in damage to a protein structure and simple to operate, and the prepared compound is uniform in particle size, relatively good in repeatability, capable of being prepared on a large scale and suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a ferritin nanocomposite with active targeting effect, a preparation method and application thereof. Background Art

[0002] As a natural active molecule, shikonin can initiate the immunogenic death program of tumor cells by inducing endoplasmic reticulum stress and the production of reactive oxygen species, promote the maturation of dendritic cells and increase the infiltration of immunogenic cytokines into tumor sites, thereby inducing a strong immunogenic response. However, the defects of shikonin such as low water solubility, short blood circulation time, high toxicity to normal tissues, and poor tumor-specific accumulation have seriously restricted its potential to be developed as a clinical therapeutic drug.

[0003] The drug delivery system constructed based on nanocomplexes containing specific biomarkers can be used as a "targeting device" to selectively deliver cytotoxic drug "warheads" to tumor cells, and has good application prospects in tumor treatment. Among them, transferrin receptor CD71, as a core specific biomarker for malignant proliferation of blood tumor cells, can recruit signal molecules through the immunotyrosine inhibitory motif contained in the cell to regulate cell growth and differentiation. It has been proven that it is highly expressed on the surface of more than 95% of blood tumor cells and can be continuously internalized by cells; in addition, CD71 is almost not expressed on the surface of hematopoietic stem cells, mature granulocytes and other normal tissue cells, and is expected to become a new specific therapeutic target for the treatment of blood tumors.

[0004] How to improve the water solubility of shikonin, successfully combine the above substances, reduce drug loss and give full play to its synergistic effect in the treatment of blood tumors, and provide a feasible strategy for the treatment of acute myeloid leukemia (AML).

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a ferritin nanocomposite with active targeting effect, and its preparation method and application, which improves the water solubility of shikonin and increases its bioavailability; at the same time, shikonin is combined with ferritin to prepare a nanocomposite, which can selectively deliver cytotoxic drug "warheads" to tumor cells, while reducing toxic side effects and improving the lethality to tumors, thereby achieving precise treatment of blood tumors.

[0007] The present invention is achieved in that:

[0008] In a first aspect, an embodiment of the present invention provides a ferritin nanocomplex with active targeting effect, wherein the ferritin nanocomplex is loaded with shikonin and metal ions; wherein the metal ions are iron ions.

[0009] In a second aspect, an embodiment of the present invention provides a method for preparing the ferritin nanocomposite as described above, the preparation method comprising:

[0010] Mixing shikonin with a salt solution containing metal ions to prepare a shikonin / metal ion complex;

[0011] The shikonin / metal ion complex is mixed with ferritin for incubation to obtain a ferritin nanocomposite; the incubation temperature is 50° C.-65° C., and the time is 3 h-5 h.

[0012] In a third aspect, an embodiment of the present invention provides a use of a ferritin nanocomplex as described above or a ferritin nanocomplex prepared by the preparation method as described above in an in vitro anti-tumor activity study.

[0013] In a fourth aspect, an embodiment of the present invention provides a use of a ferritin nanocomplex as described above or a ferritin nanocomplex prepared by the preparation method as described above in targeted anti-tumor treatment.

[0014] The present invention has the following beneficial effects:

[0015] (1) The ferritin nanocomplex with active targeting provided in the embodiment of the present invention can accurately deliver the shikonin / metal ion complex to the tumor microenvironment, and intelligently respond to the tumor microenvironment to release shikonin and metal ions; wherein, after the metal ions enter the body, the Fenton reaction is initiated to increase the accumulation of intracellular lipid peroxidation and induce changes in the structure and function of the cell biomembrane; as a donor of metal iron ions, it continuously induces the occurrence of cell ferroptosis; the released shikonin, as a classic ferroptosis and cell immunogenic death inducer, can significantly kill cancer cells and is expected to be applied in the field of cancer treatment.

[0016] (2) The shikonin / metal ion complex provided in the embodiment of the present invention can significantly improve the solubility of shikonin in water and has good biocompatibility.

[0017] (3) The preparation method provided in the embodiment of the present invention has mild experimental conditions, less damage to the protein structure, simple operation, uniform particle size of the prepared complex, good reproducibility, can be prepared on a large scale, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 The appearance colors of Fe-SH and Fe-SH@Fn in Example 1;

[0020] Figure 2 SEM images of Fe-SH (A) and Fe-SH@Fn (B) in Example 1;

[0021] Figure 3 is the particle size distribution of Fe-SH and Fe-SH@Fn in Example 1;

[0022] Figure 4 is the potential distribution of Fe-SH and Fe-SH@Fn in Example 1;

[0023] Figure 5 XPS graphs of SH and Fe-SH and Fe-SH@Fn in Example 1;

[0024] Figure 6 FTIR images of SH, Fe-SH and Fe-SH@Fn in Example 1;

[0025] Figure 7 In vitro release curves of Fe-SH@Fn in PBS containing 1% Tween 80, pH = 5.4, 10 mmol GSH at pH = 7.4, and 10 mmol GSH at pH = 5.4;

[0026] Figure 8 is the in vitro release curve of Fe-SH@Fn at different GSH concentrations;

[0027] Fig. 9 To evaluate the ability of Fe-SH@Fn to generate reactive oxygen species under different conditions;

[0028] Fig.10 For cytotoxicity assay, where:

[0029] (A) Effects of different concentrations of iron ions on the proliferation of acute myeloid leukemia HL-60 cells;

[0030] (B) Effects of different concentrations of shikonin on the proliferation of HL-60 cells;

[0031] (C) Effects of different concentrations of shikonin in Fe-SH on the proliferation of HL-60 cells;

[0032] (D) Effects of different concentrations of shikonin in Fe-SH@Fn on the proliferation of HL-60 cells;

[0033] Fig.11The effects of FeCl3, shikonin, Fe-SH and Fe-SH@Fn on the proliferation of HL-60 cells at the same shikonin concentration;

[0034] Fig.12 It is the flow cytometric graph of cellular uptake of free drugs and Fn-labeled drugs by HL-60 cells;

[0035] Fig.13 The fluorescence images are of the cellular uptake of free drugs and Fn-labeled drugs by HL-60 cells. The scale bar is 50 μm. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0037] In a first aspect, an embodiment of the present invention provides a ferritin nanocomplex with active targeting effect, wherein the ferritin nanocomplex is loaded with shikonin and metal ions; wherein the metal ions are iron ions.

[0038] It should be noted that ferritin (Fn) is an iron storage protein widely present in organisms and is called the "biological element" of the human body. It consists of a protein shell and an internal iron core. The shell is a hollow cage-shaped protein molecule with a highly symmetrical structure composed of 24 identical or different subunits, and the iron core is composed of a large amount of iron hydroxide and phosphate, with strong acid-base tolerance and thermal stability. At the same time, its reversible dissociation and recombination characteristics have laid a good foundation for the application of ferritin nanocarriers as an embedding shell and in biomedicine and nano drug delivery. Because the inner surface of ferritin is rich in acidic amino acid residues such as glutamic acid and aspartic acid, it has a high negative charge density, and metal ions can be efficiently loaded into the inner cavity of ferritin through electrostatic attraction.

[0039] Ferritin complexes can target and enrich blood tumor cells by virtue of their affinity recognition with transferrin receptor CD71, and selectively release active drug ingredients in intracellular acidic lysosomes to efficiently kill tumor cells. So far, ferritin has been used as a new tumor-targeted carrier for the precise delivery of active ingredients such as small molecule drugs, siRNA and peptides to improve solubility, stability and cellular uptake, thereby achieving efficient targeted delivery of drugs.

[0040] In an embodiment of the present invention, the ferritin used is horse spleen ferritin. Horse spleen ferritin is well studied and has strong transferrin receptor targeting (targeting CD71). In other embodiments of the present invention, ferritins with different specificities can be selected according to actual needs.

[0041] Shikonin, a naphthoquinone active ingredient containing phenolic hydroxyl groups, is derived from the traditional Chinese medicinal plant Lithospermum officinale. It has been shown to exert anti-tumor activity in a variety of ways, involving the joint participation of multiple pathways, multiple targets, and multiple signaling pathways. Among them, phenolic hydroxyl groups, as plant active ingredients, can serve as chelating sites for metal ions, driving the formation of coordination bonds with metal ions, thereby constructing metal-polyphenol supramolecular self-assembled nanocomposites to enhance biocompatibility and response to the tumor microenvironment without losing the inherent biological properties of the active ingredients.

[0042] In an optional embodiment of the present invention, the salt of iron ions is selected from at least one of ferric chloride, ferric nitrate and ferric sulfate.

[0043] In a preferred embodiment of the present invention, the salt of iron ions is ferric chloride, further ferric chloride hexahydrate.

[0044] It should be noted that after entering the body, Fe(III) carried by the metal-polyphenol nanostructure is reduced to ferrous ion Fe(II) in a free state by the high content of GSH inside the tumor cells, which then initiates the Fenton reaction to increase the accumulation of intracellular lipid peroxidation and induce changes in the structure and function of the cell biomembrane, allowing Fe(III) to bypass the resistance of tumor cells to conventional programmed cell death and continue to induce cell ferroptosis as a donor of Fe(II). Therefore, based on the potential of shikonin and Fe(III) to form supramolecular self-assembled metal nanocomposites and their different regulatory effects on the tumor microenvironment, the synergistic mechanism of the two is innovatively analyzed, which can enhance the immunogenicity of tumor cells while inducing the occurrence of cell ferroptosis and synergistically amplify the immune killing effect, which may be an ideal strategy to overcome the insufficient clinical transformation of shikonin as an anti-tumor drug. The prepared shikonin / metal ion complex not only overcomes the shortcomings of shikonin's low bioavailability and high toxicity to normal tissues, but also integrates the therapeutic function of Fe(III).

[0045] Specifically, the ferritin nanocomplex prepared by the optional embodiment of the present invention has an active targeting effect and can accurately deliver the shikonin / iron ion complex to the tumor microenvironment, and respond intelligently in the tumor microenvironment to release shikonin and Fe (III). After Fe (III) enters the body, it initiates the Fenton reaction to increase the accumulation of intracellular lipid peroxidation (LPO) and induce changes in the structure and function of the cell biomembrane, and continuously induces the occurrence of cell ferroptosis as a donor of Fe (II); the released shikonin, as a classic ferroptosis and cell immunogenic death inducer, can significantly kill cancer cells.

[0046] In other embodiments of the present invention, different metal ions, such as calcium ions, potassium ions, magnesium ions and copper ions, can be selected according to actual needs to conduct experiments to match different needs. Specifically, the salt of the metal ion used can also select any form corresponding to the metal ion according to actual conditions, such as sulfate, nitrate or chloride.

[0047] In a second aspect, an embodiment of the present invention provides a method for preparing the ferritin nanocomposite as described above, the preparation method comprising:

[0048] Mixing shikonin with a salt solution containing metal ions to prepare a shikonin / metal ion complex;

[0049] The shikonin / metal ion complex is mixed with ferritin for incubation to obtain a ferritin nanocomposite; the incubation temperature is 50° C.-65° C., and the time is 3 h-5 h.

[0050] It should be noted that the shikonin / metal ion complex is a nanocomplex. After entering the body, the active ingredients and metal ions dissociate under the action of glutathione (GSH), resulting in the exposure of phenolic hydroxyl groups and promoting the continuity of its multifunctionality through Schiff base reaction and Michael addition reaction.

[0051] In an optional embodiment of the present invention, the molar ratio of shikonin to iron ions is 1:(0.5-12); the monomer concentration of shikonin is 0.02mmol / mL-0.03mmol / mL, and the concentration of the salt solution containing metal ions is 0.001mmol / mL-0.03mmol / mL.

[0052] In a preferred embodiment of the present invention, the monomer concentration of shikonin is 0.025 mmol / mL, and the concentration of the salt solution containing metal ions is 0.00125 mmol / mL-0.03 mmol / mL.

[0053] It should be noted that the molar ratio of shikonin to metal ions beyond the set range will reduce the loading rate of shikonin.

[0054] In an optional embodiment of the present invention, shikonin is dissolved in an organic solvent, and the organic solvent includes anhydrous ethanol; and the salt containing metal ions is dissolved in water.

[0055] It should be noted that the organic solvent used in the present invention is miscible with water in any proportion; in other embodiments of the present invention, the organic solvent can be selected according to actual needs and used any one of methanol, isopropanol, acetonitrile, glycerol, acetone, dimethyl sulfoxide, tetrahydrofuran, dimethylformamide, etc.; the water used to dissolve the salt of metal ions can be selected according to actual needs and used any one of distilled water, ultrapure water, deionized water and purified water, etc.

[0056] In an optional embodiment of the present invention, the preparation of shikonin / metal ion complex further comprises: performing solid-liquid separation and washing on the mixed product.

[0057] Furthermore, the solid-liquid separation method includes centrifugation, and the cleaning method includes dialysis.

[0058] It should be noted that the method for preparing the shikonin / metal ion complex is a physical mixing method. Specifically, the preparation steps of the shikonin / metal ion complex are as follows:

[0059] Firstly, shikonin is dissolved in anhydrous ethanol and ultrasonicated for 2-10 minutes to fully dissolve it; the salt of metal ions is dissolved in distilled water and put into a flask for later use; finally, the shikonin solution is added into the salt solution containing metal ions and stirred at room temperature for 1-2 hours. After the reaction is complete, the reaction liquid is collected and centrifuged at a speed of 11000-12000 rpm for 20-30 minutes, centrifuged three times, and then the supernatant is taken out to remove the incompletely reacted shikonin; finally, the lower layer after centrifugation is placed in a dialysis bag for dialysis treatment to remove the incompletely reacted metal ions.

[0060] In an optional embodiment of the present invention, the molar ratio of shikonin / metal ion complex to ferritin is (350-550):1.

[0061] It should be noted that if the molar ratio of shikonin / metal ion complex to ferritin exceeds this range, the loading rate of shikonin will be reduced.

[0062] In an optional embodiment of the present invention, the particle size of the prepared ferritin nanocomplex is in the range of 100 nm-150 nm, preferably 120 nm-130 nm.

[0063] It should be noted that the particle size of the ferritin nanocomplex prepared in the examples of the present invention is uniform and has good reproducibility.

[0064] In an optional embodiment of the present invention, the preparation of the ferritin nanocomplex further comprises: heating the ferritin solution to an incubation temperature and then adding the shikonin / metal ion complex solution.

[0065] It should be noted that heating the ferritin first in this operation is beneficial to open the cavity of the ferritin to facilitate the subsequent loading of drugs.

[0066] Specifically, the preparation steps of ferritin nanocomplex are as follows:

[0067] Using the temperature incubation method, the shikonin / metal ion complex solution is first placed at a temperature of 50°C-65°C, and then the ferritin solution is added and slowly stirred at 50°C-65°C for 3h-5h. After the reaction is complete, the reaction solution is placed in a dialysis bag for dialysis treatment to remove the unloaded shikonin / metal ion complex.

[0068] In a third aspect, an embodiment of the present invention provides a use of a ferritin nanocomplex as described above or a ferritin nanocomplex prepared by the preparation method as described above in an in vitro anti-tumor activity study.

[0069] In a fourth aspect, an embodiment of the present invention provides a use of a ferritin nanocomplex as described above or a ferritin nanocomplex prepared by the preparation method as described above in targeted anti-tumor treatment.

[0070] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0071] Example 1

[0072] This embodiment provides a ferritin nanocomposite, and the preparation method thereof comprises the following steps:

[0073] (1) Preparation of shikonin / iron ion complex:

[0074] The shikonin / iron ion complex was prepared by a physical mixing method. First, 0.05 mmol shikonin was dissolved in 2 mL of anhydrous ethanol (concentration of 0.025 mmol / mL) and ultrasonicated for 3 min to fully dissolve it; 0.4 mmol ferric chloride hexahydrate (108 mg) was dissolved in 20 mL of distilled water (concentration of 0.02 mmol / mL) and placed in a round-bottom flask. Finally, 2 mL of shikonin solution was added to 20 mL of ferric chloride hexahydrate solution and stirred at room temperature for 1.5 h. After the reaction was complete, the reaction solution was collected and centrifuged at 12000 rpm for 30 min, centrifuged three times, and the supernatant was taken to remove the unreacted shikonin. Finally, the unreacted ferric chloride hexahydrate was removed by dialyzing in a dialysis bag with a molecular weight of 200 Da for 3 d to obtain a shikonin / iron ion complex solution (denoted as Fe-SH).

[0075] (2) Preparation of ferritin nanocomplexes:

[0076] The temperature incubation method was used to prepare ferritin nanocomplexes loaded with shikonin / iron ions. First, 1.87 mL of shikonin / iron ion complex solution (shikonin mass was 0.655 mg) was placed at 60°C, and then 20 μL of horse spleen ferritin solution (100 mg / mL) was added and slowly stirred at 60°C for 4 h. After the reaction was complete, the reaction solution was placed in a dialysis bag with a molecular weight of 3500 Da and dialyzed for 3 days to remove the unloaded shikonin / iron ion complex to obtain a ferritin nanocomplex (denoted as Fe-SH@Fn).

[0077] Example 2

[0078] This embodiment provides a ferritin nanocomposite, which is different from embodiment 1 in that:

[0079] (1) Preparation of shikonin / iron ion complex: 0.6 mmol ferric chloride hexahydrate (162 mg) was dissolved in 20 mL distilled water (concentration: 0.03 mmol / mL).

[0080] (2) Preparation of ferritin nanocomplex: The amount of shikonin / iron ion complex solution (the mass of shikonin is 0.655 mg) used is 2.03 mL.

[0081] Example 3

[0082] This embodiment provides a ferritin nanocomposite, which is different from embodiment 1 in that:

[0083] (1) Preparation of shikonin / iron ion complex: 0.3 mmol of ferric chloride hexahydrate (81 mg) was dissolved in 20 mL of distilled water (concentration: 0.015 mmol / mL).

[0084] (2) Preparation of ferritin nanocomplex: The amount of shikonin / iron ion complex solution (the mass of shikonin is 0.655 mg) used is 2.25 mL.

[0085] Example 4

[0086] This embodiment provides a ferritin nanocomposite, which is different from embodiment 1 in that:

[0087] (1) Preparation of shikonin / iron ion complex: 0.2 mmol of ferric chloride hexahydrate (54 mg) was dissolved in 20 mL of distilled water (concentration: 0.01 mmol / mL).

[0088] (2) Preparation of ferritin nanocomplex: The amount of shikonin / iron ion complex solution (the mass of shikonin is 0.655 mg) used is 2.56 mL.

[0089] Example 5

[0090] This embodiment provides a ferritin nanocomposite, which is different from embodiment 1 in that:

[0091] (1) Preparation of shikonin / iron ion complex: 0.1 mmol ferric chloride hexahydrate (27 mg) was dissolved in 20 mL distilled water (concentration 0.005 mmol / mL).

[0092] (2) Preparation of ferritin nanocomplex: The amount of shikonin / iron ion complex solution (the mass of shikonin is 0.655 mg) used is 3.16 mL.

[0093] Example 6

[0094] This embodiment provides a ferritin nanocomposite, which is different from embodiment 1 in that:

[0095] (1) Preparation of shikonin / iron ion complex: 0.05 mmol of ferric chloride hexahydrate (13.5 mg) was dissolved in 20 mL of distilled water (0.0025 mmol / mL).

[0096] (2) Preparation of ferritin nanocomplex: The amount of shikonin / iron ion complex solution (the mass of shikonin is 0.655 mg) used is 3.91 mL.

[0097] Example 7

[0098] This embodiment provides a ferritin nanocomposite, which is different from embodiment 1 in that:

[0099] (1) Preparation of shikonin / iron ion complex: 0.025 mmol of ferric chloride hexahydrate (6.75 mg) was dissolved in 20 mL of distilled water (concentration: 0.00125 mmol / mL).

[0100] (2) Preparation of ferritin nanocomplex: The amount of shikonin / iron ion complex solution (the mass of shikonin is 0.655 mg) used is 7.25 mL.

[0101] Test Example 1

[0102] This test example tests the loading rate of the shikonin / iron ion complex and ferritin nanocomposite prepared in Examples 1-7. The test uses high performance liquid chromatography to establish a standard curve; the high performance liquid chromatograph model is: Waterse2695, the chromatographic column: SunFire C18 (5μm, 4.6×250mm), the column temperature: 30°C, the flow rate: 1.0mL / min, the detection wavelength: 515nm, the injection volume: 10μL, and the mobile phase accounts for 85% of methanol + 15% of 0.05mol / L phosphoric acid. The relevant results are shown in Table 1.

[0103] Table 1 Load rate data

[0104]

[0105]

[0106] It can be seen from Table 1 that the loading rate of shikonin / iron ion complex and the loading rate of ferritin nanocomposite first increase and then decrease with the increase of the molar ratio of shikonin:iron ion; among them, in Example 1, when the molar ratio of shikonin:iron ion is 1:8, the loading rate is the highest, and at this time the loading rate of ferritin nanocomposite is also the highest, therefore, this condition is the optimal condition.

[0107] Test Example 2

[0108] In this test example, the shikonin / iron ion complex and ferritin nanocomposite prepared in Example 1 were observed by appearance morphology and SEM. Figure 1 , SEM pictures are shown in Figure 2 .

[0109] It should be noted that the SEM test instrument is the German ZEISS GeminiSEM 300.

[0110] from Figure 1 It can be seen that the prepared shikonin / iron ion nanocomplex and ferritin nanocomplex are both stable solution systems with uniform color.

[0111] from Figure 2 It can be seen that Figure 2 (A) shows a shikonin / iron ion nanocomposite with uniform particle size of 60 nm; Figure 2 (B) shows ferritin nanocomplexes with a particle size of 130 nm.

[0112] Combination Figure 1 Appearance color and Figure 2 From the SEM image, it can be seen that ferritin successfully loaded with shikonin / iron ion nanocomplex.

[0113] Test Example 3

[0114] In this test example, the particle size distribution, potential distribution and dispersion coefficient of the shikonin / iron ion complex and ferritin nanocomposite prepared in Example 1 were measured.

[0115] The particle size distribution and dispersion coefficient were tested using a nanoparticle size and Zeta potential analyzer (Litesizer 500). Figure 3 ,from Figure 3 It can be seen that the particle size of shikonin / iron ion complex is concentrated at 69.15±2.57nm, and the particle size of ferritin nanocomplex is concentrated at 126.94±2.05nm. The PDI of shikonin / iron ion complex is 0.187±0.008, and the PDI of ferritin nanocomplex is 0.116±0.017. Figure 4 It can be seen that the Zata potential of the shikonin / iron ion complex is +47.92±2.45mV, and that of the ferritin nanocomposite is +37.19±0.56mV. Obviously, the nanoparticle size of the shikonin / iron ion complex formed by ferritin loading increases, the Zata potential decreases, and the dispersion coefficient is less than 0.3, which further indicates that the ferritin nanocomposite with uniform particle size and stability has been successfully prepared.

[0116] Test Example 4

[0117] In this test example, infrared spectrophotometer and X-ray diffraction analysis were used to determine the structure of the shikonin / iron ion complex and ferritin nanocomposite prepared in Example 1, and the binding mode of the shikonin / iron ion complex was determined; wherein, the XPS diagrams of Fe-SH and Fe-SH@Fn are shown in Figure 5 , FTIR chart see Figure 6 .

[0118] from Figure 5 It can be seen that the iron ions are successfully chelated with shikonin, and ferritin is successfully loaded with shikonin / iron ion complex. Figure 6 It can be seen that after shikonin integrates iron, the vibration value of shikonin changes from 3426cm -1 Move to 3428cm -1 1206cm -1 The intensity of the HO-C stretching vibration peak of shikonin was significantly reduced. These changes indicate that HO-C and Fe 3+ There is a coordination effect between them. In addition, the stretching vibration peak of C=O in shikonin increases from 1608cm -1 Moved to 1531cm -1 , proving that C=O and Fe 3+ The last 3396cm -1-OH stretching vibration peak at 1542cm -1 The characteristic peaks of shikonin after integrating iron, such as the C=O stretching vibration peak, are still retained, proving that the ferritin nanocomposite successfully relies on physical electrostatic adsorption to load the shikonin / iron ion nanocomposite.

[0119] Test Example 5

[0120] In this test example, the ferritin nanocomposite prepared in Example 1 was evaluated for its in vitro release characteristics. The specific process is as follows:

[0121] Take an appropriate amount (1 mL) of ferritin nanocomplex loaded with shikonin / iron ions and place it in an activated dialysis bag. Seal the dialysis bag with a sealing clip and suspend it in 9 mL of dissolution medium (PBS, pH = 5.4, 10 mmol GSH at pH = 7.4, 10 mmol GSH at pH = 5.4, and 1% Tween 80 in the dissolution medium). Place it in a 40°C incubator and shake it. Take 1 mL of the dissolution medium at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h. Measure the absorbance at a wavelength of 515 nm under a UV spectrophotometer and calculate the cumulative release rate at each time point. Draw a dissolution curve diagram, specifically Figure 7 In vitro release curves of Fe-SH@Fn in PBS containing 1% Tween 80, pH = 5.4, 10 mmol GSH at pH = 7.4, and 10 mmol GSH at pH = 5.4; Figure 8 In vitro release curves of Fe-SH@Fn at different GSH concentrations.

[0122] from Figure 7 It can be seen that the release of shikonin is limited under the conditions of pH = 7.4 and 5.4, while in the presence of GSH, most of the shikonin is released after 24 h. Under the condition of 10 mmol GSH at pH = 5.4, GSH is more able to promote the release of shikonin, and it enters a slow release period after 12 h, and the release rate reaches more than 90% after 24 h.

[0123] from Figure 8 It can be seen that as the concentration of GSH increases, the release rate of shikonin increases. In summary, the ferritin nanocomposite loaded with shikonin / iron ions prepared by the present invention has a GSH response and can intelligently respond to release shikonin in the case of high GSH in the tumor microenvironment.

[0124] Test Example 6

[0125] In this test example, the ferritin nanocomposite prepared in Example 1 was evaluated for the generation of active oxygen, and the specific process was as follows:

[0126] When ROS are generated, methylene blue will be degraded by using methylene blue as an indicator, and the color will gradually change from blue to colorless. Specific method: First, prepare PBS buffer solutions with pH values ​​of 5.4 and 7.4, followed by preparing methylene blue (MB, 200μg / mL) detection solution, hydrogen peroxide (H2O2, 40mM) stock solution, and PBS buffer solution of ferritin nanocomplex loaded with shikonin / iron ions. Test the change in absorbance of methylene blue at 665nm to evaluate the generation of ·OH. The specific results are shown in Fig. 9 .

[0127] from Fig. 9 It can be seen that in the presence of GSH, the absorbance of methylene blue at 665nm of the ferritin nanocomplex loaded with shikonin / iron ions decreased significantly, while in the absence of GSH, the absorbance remained basically unchanged, thus verifying that the ferritin nanocomplex loaded with shikonin / iron ions can significantly produce reactive oxygen species in the presence of GSH.

[0128] Test Example 7

[0129] This test example is a cytotoxicity test, as follows:

[0130] The CCK-8 method was used to detect the proliferation toxicity of shikonin, iron ions, shikonin / iron ion complex and ferritin nanocomplex on HL-60 cells in vitro.

[0131] HL-60 was plated in 96-well plates (3 × 10 6 The experimental groups were given iron ions, shikonin, shikonin / iron ion complex, and ferritin nanocomplex loaded with shikonin / iron ions (the concentrations of shikonin and iron ions were 0.25, 0.5, 1, 2, 4, 8, and 16 μg / mL, respectively) to HL-60 cells for logarithmic growth. A blank control group was set at the same time, with 5 multiwells in each group. After incubation for 24 hours, 20 μL of fresh CCK-8 solution was added to each well. Then, the cells were continuously cultured in an incubator for 1.5 hours, and the absorbance was measured at a wavelength of 450 nm on an ELISA reader to calculate the cell survival rate.

[0132] The results of cytotoxicity tests are shown in Fig.10 :(A) is the effect of different concentrations of iron ions on the proliferation of acute myeloid leukemia HL-60 cells; (B) is the effect of different concentrations of shikonin on the proliferation of HL-60 cells; (C) is the effect of different concentrations of shikonin in Fe-SH on the proliferation of HL-60 cells; (D) is the effect of different concentrations of shikonin in Fe-SH@Fn on the proliferation of HL-60 cells; Fig.11 The effects of FeCl3, shikonin, Fe-SH and Fe-SH@Fn on the proliferation of HL-60 cells at the same shikonin concentration.

[0133] Combination Fig.10 and Fig.11 It can be seen that with the increase of concentration, the cell survival rate of each group decreased slightly. 50 The value is 2.09μg / mL. However, when shikonin chelates with iron ions to form a shikonin / iron ion complex, its IC 50 The value was 0.58 μg / mL, which significantly reduced the IC of shikonin on HL-60 cells. 50 Finally, when ferritin was used to load the shikonin / iron ion complex, its IC 50 The value was 0.39 μg / mL, which further reduced the IC value of shikonin on HL-60 cells. 50 The results showed that the ferritin nanocomplex loaded with shikonin / iron ions prepared in this experiment could significantly inhibit the proliferation activity of HL-60.

[0134] Test Example 8

[0135] This test example is an in vitro tumor targeting evaluation, as follows:

[0136] Flow cytometry was used to evaluate the in vitro cell targeting of free drugs and ferritin-modified drugs.

[0137] Preparation of Dil-labeled Fn:

[0138] First, weigh 500 μg of Dil, add 1 mL of DMSO to make a Dil solution with a concentration of 0.5 mg / mL; take 50 μL of 100 mg / mL Fn and add 4.95 mL of PBS to make a protein solution with a concentration of 1 mg / mL. Finally, take 5 mL of protein solution and add 500 μL of Dil solution. Place the reaction tube in a dark place and incubate it at room temperature and shaking for 8 hours. Every 30 minutes, gently invert the reaction tube several times to fully mix the two reactants and improve the labeling efficiency. After the reaction is completed, centrifuge at 8000 rpm for 10 minutes to remove the unloaded Dil (lipid soluble) and store at 4°C for use.

[0139] Cellular uptake assay:

[0140] Two groups, Dil and Dil-Fn, were set up. The dosage per well of the 24-well plate was calculated based on the relative fluorescence intensity to ensure that the fluorescence intensity of each well was the same. Then, after administration at different times (1, 2, 4, 6, 8, 10, 12, 24 h), the fluorescence intensity of each well was measured using a flow cytometer.

[0141] Related results: Fig.12 The figure shows the cellular uptake of free drugs and Fn-labeled drugs by HL-60 cells; Fig.13 The fluorescence images are of the cellular uptake of free drugs and Fn-labeled drugs by HL-60 cells. The scale bar is 50 μm.

[0142] from Fig.12 It can be seen that the cell uptake of the ferritin-modified drug is significantly higher than that of the free drug. To further verify the ability of ferritin to target tumor cells, the cells were stained with DAPI and photographed under a fluorescence microscope after administration (1, 4, and 8 hours). Fig.13 As shown, the fluorescence intensity of the Dil dye modified with ferritin is the strongest at 8 hours, so it is concluded that the drug modified with ferritin can significantly enhance the cellular uptake of the drug.

[0143] According to the results of the above cell uptake experiments, it can be concluded that the ferritin nanocomplex prepared by the present invention has significant in vitro cell targeting.

[0144] In summary, the experimental conditions for preparing ferritin nanocomplexes of the present invention are mild, the damage to the protein structure is small, the operation is simple, the prepared complex has uniform particle size, good repeatability, can be prepared on a large scale, and is suitable for industrial production; the shikonin / iron ion complex prepared in the embodiment of the present invention can significantly improve the solubility of shikonin in water and has good biocompatibility; the prepared ferritin nanocomplex with active targeting effect can accurately deliver the shikonin / iron ion complex to the tumor microenvironment, and respond intelligently under the tumor microenvironment, has multiple tumor cell killing capabilities, and can be effectively applied to the field of cancer treatment; in addition, the ferritin nanocomplex with active targeting effect prepared by the present invention can also be doped with other metals to prepare shikonin / multi-metal ferritin nanocomplexes.

[0145] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A ferritin nanocomposite with active targeting effect, characterized in that: The ferritin nanocomposite is loaded with shikonin and metal ions; Wherein, the metal ions are iron ions.

2. The ferritin nanocomposite according to claim 1, characterized in that The salt of the iron ion is selected from any one of ferric chloride, ferric nitrate and ferric sulfate.

3. A method for preparing the ferritin nanocomposite according to any one of claims 1 to 2, characterized in that: The preparation method comprises: Mixing shikonin with a salt solution containing metal ions to prepare a shikonin / metal ion complex; The shikonin / metal ion complex is mixed with ferritin for incubation to obtain a ferritin nanocomposite; the incubation temperature is 50° C.-65° C., and the time is 3 h-5 h.

4. The preparation method according to claim 3, characterized in that: The molar ratio of shikonin to metal ions is 1:(0.5-12); Preferably, the monomer concentration of shikonin is 0.02 mmol / mL-0.03 mmol / mL, and the concentration of the salt solution containing metal ions is 0.001 mmol / mL-0.03 mmol / mL.

5. The preparation method according to claim 3, characterized in that: The shikonin is dissolved in an organic solvent, and the organic solvent includes anhydrous ethanol; Preferably, the salt containing metal ions is dissolved in water.

6. The preparation method according to claim 3, characterized in that: The preparation of the shikonin / metal ion complex also includes: solid-liquid separation and washing of the mixed product; Preferably, the solid-liquid separation method includes centrifugation, and the cleaning method includes dialysis; Preferably, the ferritin nanocomplex further comprises: heating the solution of the shikonin / metal ion complex to an incubation temperature and then adding the ferritin solution.

7. The preparation method according to claim 3, characterized in that: The molar ratio of the shikonin / metal ion complex to the ferritin is (350-550):

1.

8. The preparation method according to claim 3, characterized in that: The particle size of the ferritin nanocomposite is in the range of 100nm-150nm, preferably 120nm-130nm.

9. Use of the ferritin nanocomposite according to any one of claims 1 to 2 or the ferritin nanocomposite prepared by the preparation method according to any one of claims 3 to 8 in an in vitro anti-tumor activity study.

10. Use of the ferritin nanocomposite according to any one of claims 1 to 2 or the ferritin nanocomposite prepared by the preparation method according to any one of claims 3 to 8 in targeted anti-tumor treatment.

Images