Iron/shikonin nano-composite and use thereof and method for preparing the same by supermolecular self-assembly

Abstract

An iron/shikonin nano-composite and a use thereof, and a method for preparing the same by supermolecular self-assembly, belonging to the technical field of functional materials. The composite consists of shikonin and ferric ions, wherein shikonin is coordinated with the ferric ions, and the hydroxyl and carbonyl groups in shikonin are coordinated with the ferric irons to form a complex, which is then assembled by π-π stacking and hydrophobic interactions to form a nano-composite which exhibits glutathione response. The composite is obtained by the following steps: adding an aqueous solution of a ferric salt and an organic solvent solution of shikonin in sequence into water while stirring at ambient temperature, continuously stirring at ambient temperature, and centrifuging the resulting mixture to purify, thereby obtaining an iron/shikonin nano-composite in the resulting solution.

Claims

1. A method for preparing an iron/shikonin nano-composite, comprising, adding an aqueous solution of a ferric salt into water while stirring at ambient temperature; adding an organic solvent solution of shikonin, wherein the organic solvent is miscible with water; continuously stirring at ambient temperature; centrifuging the resulting mixture to purify, thereby obtaining an iron/shikonin nano-composite solution; and evaporating the iron/shikonin nano-composite solution to dryness to obtain an iron/shikonin nano-composite solid powder.

2. The method of claim 1, wherein the organic solvent is selected from the group consisting of: methanol, ethanol, isopropanol, acetonitrile, glycerol, acetone, dimethyl sulfoxide, tetrahydrofuran, and dimethylformamide.

3. The method of claim 1, wherein the ferric salt is selected from the group consisting of: ferric chloride, ferric nitrate, and ferric sulfate.

4. The method of claim 1, wherein a molar ratio of the ferric salt to shikonin is in a range of (0.1-12): 1; a concentration of shikonin in the organic solvent solution of shikonin is in a range of 1-10 mg/mL; and a concentration of the ferric salt in the aqueous solution of the ferric salt is in a range of 50-800 mg/mL.

5. The method of claim 1, wherein continuous stirring at ambient temperature comprises maintaining stirring for 30 min to 24 h.

6. An iron/shikonin nano-composite, which is prepared by the method of claim 1.

7. The iron/shikonin nano-composite of claim 6, wherein the iron/shikonin nano-composite has a diameter of 10-200 nm.

8. A method for treating cancer, comprising administering the iron/shikonin nano-composite of claim 6 to a subject in need thereof.

9. An iron/shikonin nano-composite, which is prepared by adding an aqueous solution of a ferric salt into water while stirring at ambient temperature; adding an organic solvent solution of shikonin, wherein the organic solvent is miscible with water; continuously stirring at ambient temperature; and centrifuging the resulting mixture to purify, thereby obtaining an iron/shikonin nano-composite in a resulting solution.

10. A method for treating cancer, comprising administrating the iron/shikonin nano-composite of claim 9 to a subject in need thereof.

11. The iron/shikonin nano-composite of claim 6, wherein the iron/shikonin nano-composite has a diameter of 30-90 nm.

12. The iron/shikonin nano-composite of claim 6, wherein the iron/shikonin nano-composite has a diameter of 50-70 nm.

13. The iron/shikonin nano-composite of claim 6, wherein the iron/shikonin nano-composite has a diameter of 60 nm.

14. The method of claim 4, wherein the molar ratio of the ferric salt to shikonin is 11.5:1, 4.3:1, 2.1:1, 0.4:1, or 0.2:1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a transmission electron microscope photograph of the iron/shikonin nano-composite as prepared in Example 1, with a size of 30 nm.

(2) FIG. 2 shows a transmission electron microscope photograph of the iron/shikonin nano-composite as prepared in Example 2, with a size of 50 nm.

(3) FIG. 3 shows a transmission electron microscope photograph of the iron/shikonin nano-composite as prepared in Example 3, with a size of 70 nm.

(4) FIG. 4 shows a transmission electron microscope photograph of the iron/shikonin nano-composite as prepared in Example 4, with a size of 90 nm.

(5) FIG. 5 shows a transmission electron microscope photograph of the iron/shikonin nano-composite as prepared in Example 5, with a size of 60 nm.

(6) FIG. 6 shows a transmission electron microscope photograph of the iron/shikonin nano-composite as prepared in Example 6, with a size of 50 nm.

(7) FIG. 7 shows ultraviolet-visible absorption spectra of the iron/Shikonin nano-composite (FSNPs) as prepared in Example 2, shikonin and ferric salt (Fe). It can be seen from the ultraviolet-visible absorption spectra that compared with shikonin, the newly formed iron/shikonin nano-composite (FSNPs) exhibits a new broad dispersion peak at 602 nm, which is attributed to the charge transfer transition caused by the coordination bond between the organic ligand shikonin and the central iron ion, confirming the formation of the iron/shikonin nano-composite.

(8) FIG. 8A and FIG. 8B show the response test curve of the iron/shikonin nano-composite as prepared in Example 7 to a glutathione solution with a concentration of 2 mM. FIG. 8A shows the ultraviolet-visible absorption spectra of nano-composite in a 2 mM glutathione solution at different times; FIG. 8B shows variations of the absorption value of the nano-composite in a glutathione solution with a concentration of 2 mM at 602 nm (peak position of nano-composite). It can be seen from FIG. 8A and FIG. 8B that the absorption value at 602 nm of the iron/shikonin nano-composite decreases gradually, indicating that the intensity of coordination peak gradually weakens and the nano-drugs are disassembled. FIG. 8C shows a transmission electron micrograph of the nano-composite after 1 hour in a glutathione solution with a concentration of 2 mM. It can be seen that the structure of the iron/shikonin nano-composite is broken up and the morphology thereof is changed, which further confirms the disassembly of the nano-drugs.

(9) FIG. 9 shows ultraviolet-visible absorption spectra of methylene blue, which shows effects of iron/shikonin nano-composite as prepared in Example 8 on the degradation of methylene blue under different conditions, showing that after redox reaction between ferric iron in the nano-drug and glutathione, the structure of the nano-drug is disassembled, thereby releasing ferrous irons; ferrous irons undergo Fenton reaction and generate hydroxyl radicals, which degrade methylene blue.

(10) FIG. 10 shows a cck8 cytotoxicity test chart of the iron/shikonin nano-composite as prepared in Example 9. It can be seen from this chart that the cell survival rate of cancer cells is dependent on the concentration of nano-drug, wherein the cell survival rate of cancer cells gradually decreases with an increase of the concentration of nano-drug, indicating that the nano-drug has a great ability of killing cancer cells.

DETAILED DESCRIPTION

(11) The present disclosure will be further illustrated with examples below, but these examples are not intended to limit the present disclosure.

Example 1

(12) Ferric chloride hexahydrate was dissolved in water to obtain a ferric chloride aqueous solution with a concentration of 100 mg/mL; shikonin was dissolved in ethanol to obtain a shikonin ethanol solution with a concentration of 5 mg/mL. 1 mL of the ferric chloride aqueous solution and 5 mL of the shikonin ethanol solution were added in sequence into 40.75 mL of water while stirring at ambient temperature. After stirring at ambient temperature for 1 hour, the resulting mixture was centrifuged at a rotation speed of 15000 rpm for 15 min, for 3 times in total, to obtain an iron/shikonin nano-composite solution, in which the nano-composite has an average size of 30 nm; finally the iron/shikonin nano-composite was dissolved in water to obtain a nano-composite solution with a concentration of 100 mg/mL.

Example 2

(13) Ferric chloride hexahydrate was dissolved in water to obtain a ferric chloride aqueous solution with a concentration of 100 mg/mL; shikonin was dissolved in ethanol to obtain a shikonin ethanol solution with a concentration of 5 mg/mL. 500 μL of the ferric chloride aqueous solution and 5 mL of the shikonin ethanol solution were added in sequence into 41.25 mL of water while stirring at ambient temperature. After stirring at ambient temperature for 1 hour, the resulting mixture was centrifuged at a rotation speed of 15000 rpm for 15 min, for 3 times in total, to obtain an iron/shikonin nano-composite solution, in which the nano-composite has an average size of 50 nm.

Example 3

(14) Ferric chloride hexahydrate was dissolved in water to obtain a ferric chloride aqueous solution with a concentration of 100 mg/mL; shikonin was dissolved in ethanol to obtain a shikonin ethanol solution with a concentration of 5 mg/mL. 100 μL of the ferric chloride aqueous solution and 5 mL of the shikonin ethanol solution were added in sequence into 41.65 mL of water while stirring at ambient temperature. After stirring at ambient temperature for 1 hour, the resulting mixture was centrifuged at a rotation speed of 15000 rpm for 15 min, for 3 times in total, to obtain an iron/shikonin nano-composite solution, in which the nano-composite has an average size of 70 nm.

Example 4

(15) Ferric chloride hexahydrate was dissolved in water to obtain a ferric chloride aqueous solution with a concentration of 100 mg/mL; shikonin was dissolved in ethanol to obtain a shikonin ethanol solution with a concentration of 5 mg/mL. 50 μL of the ferric chloride aqueous solution and 5 mL of the shikonin ethanol solution were added in sequence into 41.70 mL of water while stirring at ambient temperature. After stirring at ambient temperature for 1 hour, the resulting mixture was centrifuged at a rotation speed of 15000 rpm for 15 min, for 3 times in total, to obtain an iron/shikonin nano-composite solution, in which the nano-composite has an average size of 90 nm.

(16) It can be seen from Examples 1 to 4 that the size of iron/shikonin nano-composite can be adjusted by changing the amount of ferric salt. The size of the nano-particles decreased with an increase of the amount of ferric salt.

Example 5

(17) Ferric chloride hexahydrate was dissolved in water to obtain a ferric chloride aqueous solution with a concentration of 100 mg/mL; shikonin was dissolved in dimethylformamide to obtain a shikonin dimethylformamide solution with a concentration of 5 mg/mL. 1 mL of the ferric chloride aqueous solution and 5 mL of the shikonin dimethylformamide solution were added in sequence into 40.75 mL of water while stirring at ambient temperature. After stirring at ambient temperature for 1 hour, the resulting mixture was centrifuged at a rotation speed of 15000 rpm for 15 min, 3 times in total, to obtain an iron/shikonin nano-composite solution, in which the nano-composite has an average size of 60 nm.

Example 6

(18) Ferric nitrate nonahydrate was dissolved in water to obtain a ferric nitrate aqueous solution with a concentration of 400 mg/mL; shikonin was dissolved in ethanol to obtain a shikonin ethanol solution with a concentration of 5 mg/mL. 1 mL of the ferric nitrate aqueous solution and 5 mL of the shikonin ethanol solution were added in sequence into 40.75 mL of water under stirring at ambient temperature. After stirring at ambient temperature for 1 hour, the resulting mixture was centrifuged at a rotation speed of 15000 rpm for 15 min, for 3 times in total, to obtain an iron/shikonin nano-composite solution, in which the nano-composite has an average size of 50 nm.

Example 7

(19) The iron/shikonin nano-composite as prepared by Example 2 was dispersed in a glutathione solution with a concentration of 2 mM, and the ultraviolet-visible absorption spectra of the nano-composite at the initial, 1 min, 3 min, 5 min, 10 min, 20 min, 30 min and 60 min were recorded. After 60 min, a sample was taken for transmission electron microscope photos.

Example 8

(20) MB+FSNPs+GSH+H.sub.2O.sub.2 group: an iron/shikonin nano-composite (FSNPs) was dispersed in 2 mL of a glutathione (GSH) solution with a concentration of 2 mM for 1 h, such that the concentration of the iron/shikonin nano-composite is 50 μg/mL, and a centrifugation was performed, obtaining a mixture; 45 μL of a methylene blue (MB) solution with a concentration of 1 mg/mL and 20 μL of a hydrogen peroxide (H.sub.2O.sub.2) solution with a concentration of 10 mM were added to the mixture above; after standing for 15 min, the resulting mixture was subjected to an ultraviolet-visible absorption analysis and the spectrum was recorded.

(21) MB+FSNPs+GSH group: an iron/shikonin nano-composite (FSNPs) was dispersed in 2 mL of a glutathione (GSH) solution with a concentration of 2 mM for 1 h, such that the concentration of iron/shikonin nano-composite is 50 μg/mL, and a centrifugation was performed, to obtain a mixture; 45 μL of a methylene blue (MB) solution with a concentration of 1 mg/mL was added to the mixture above; after standing for 15 min, the resulting mixture was subjected to an ultraviolet-visible absorption analysis and the spectrum was recorded.

(22) MB+FSNPs+H.sub.2O.sub.2 group: 45 μL of a methylene blue (MB) solution with a concentration of 1 mg/mL and 20 μL of a hydrogen peroxide (H.sub.2O.sub.2) solution with a concentration of 10 mM were added to 2 mL of an iron/shikonin nano-composite (FSNPs) aqueous solution (the concentration of the iron/shikonin nano-composite is 50 μg/mL); after standing for 15 min, the resulting mixture was subjected to an ultraviolet-visible absorption analysis and the spectrum was recorded.

(23) MB+GSH+H.sub.2O.sub.2 group: 45 μL of a methylene blue (MB) solution with a concentration of 1 mg/mL and 20 μL of a hydrogen peroxide (H.sub.2O.sub.2) solution with a concentration of 10 mM were added to 2 mL of a glutathione solution with a concentration of 2 mM; after standing for 15 min, the resulting mixture was subjected to an ultraviolet-visible absorption analysis and the spectrum was recorded.

(24) MB+H.sub.2O.sub.2 group: 45 μL of a methylene blue (MB) solution with a concentration of 1 mg/mL and 20 μL of a hydrogen peroxide (H.sub.2O.sub.2) solution with a concentration of 10 mM were added to 2 mL of water; after standing for 15 min, the resulting mixture was subjected to an ultraviolet-visible analysis and the spectrum was recorded.

(25) MB+H.sub.2O group: 45 μL of a methylene blue (MB) solution with a concentration of 1 mg/mL was added to 2 mL of water; after standing for 15 min, the resulting mixture was subjected to an ultraviolet-visible analysis and the spectrum was recorded.

(26) It can be seen from Example 8 that compared with other groups, in the MB+FSNPs+GSH+H.sub.2O.sub.2 group, the methylene blue solution became colorless from blue, and the absorption peaks of the ultraviolet-visible absorption spectrum disappeared, indicating that after being disassembled in the glutathione solution, the iron/shikonin nano-composite could release ferrous ions, which underwent Fenton reaction with hydrogen peroxide and generated hydroxyl radicals, which degraded methylene blue.

Example 9

(27) 4T1 cells (purchased from Beyotime Biotechnology Co., Ltd.) were inoculated in a 96-well culture plate with an initial density of 1×10.sup.4 cells per well and incubated for 24 hours; then the cells were co-cultured with iron/shikonin nano-composite solutions with concentrations of 2 μg/mL, 5 μg/mL, 10 μg/mL, 12.5 μg/mL, 15 μg/mL, 20 μg/mL, 25 μg/mL and 30 μg/mL respectively for 24 hours; then, 10 μL of cck8 (Cell Counting Kit-8, purchased from bimake.cn) was added to each well, and the survival rate of cells in each well was tested with a microplate reader one hour later.

(28) The results of Example 9 showed that the survival rate of breast cancer cells in 4T1 mice gradually decreased with the increase of the concentration of the iron/shikonin nano-composite, and when the concentration reached 30 μg/mL, the survival rate of cells was only 20%, indicating that the nano-composite had a great ability of killing cancer cells.