COMPOSITION COMPRISING IRON OXIDE MAGNETIC PARTICLES FOR A TREATMENT OF LIVER CANCER

Abstract

Disclosed is a composition comprising iron oxide magnetic particles. The composition is delivered to a liver in a hepatocyte-specific manner to minimize damage to other organs, and is safely excreted from a body within a few weeks. Further, the particles have excellent hepatocyte targeting ability, and thus are used as a liver cancer treatment agent and a hepatocyte targeting carrier.

Claims

1. A composition for treating liver cancer, the composition comprising iron oxide magnetic particles, wherein each of the iron oxide magnetic particles contains: a core including an iron oxide derived from a composite of iron and at least one type of a. compound selected from a group consisting of an aliphatic hydrocarbon acid salt having 4 to 25 carbon atoms and an amine compound; MX.sub.n; and at least one selected from a group consisting of folate, glycyrrhetinic acid, and glucose, wherein M is selected from a group consisting of Cu, Sn, Pb, Mn, Ir, Pt, Rh, Re, Ag, Au, Pd and Os, wherein X is selected from a group consisting of F, Cl, Br and I, wherein n is an integer of 1 to 6, wherein the iron oxide magnetic particle has an average particle diameter of 6 nm to 20 nm.

2. The composition of claim 1, wherein the iron oxide includes at least one selected from a group consisting of Fe.sub.13O.sub.19, Fe.sub.3O.sub.4 (magnetite), γ-Fe.sub.2O.sub.3 (maghemite), α-Fe.sub.2O.sub.3 (hematite), β-Fe.sub.2O.sub.3 (beta phase), ε-Fe.sub.2O.sub.3 (epsilon phase), FeO (Wstite), FeO.sub.2 (iron dioxide). Fe.sub.4O.sub.5, Fe.sub.5O.sub.6, Fe.sub.5O.sub.7, Fe.sub.25O.sub.32, Ferrite type and delafossite.

3. The composition of claim 1, wherein X includes a radioactive isotope of X or a mixture of radioactive isotopes of X.

4. The composition of claim 1, wherein the composite is an iron-oleic acid composite.

5. The composition of claim 1, wherein MX.sub.n is CuI.

6. The composition of claim 1, wherein the iron oxide magnetic particle includes the glycyrrhetinic acid.

7. The composition of claim 1, wherein the core is coated with a hydrophilic ligand.

8. The composition of claim 1, wherein MX.sub.n is contained in an amount of 1 to 13 mol % based on 100 mol % of the composite.

9. The composition of claim 1, wherein the composition is used in a magnetic field having. a low frequency of 1 kHz to 1 MHz and an intensity of 20 Oe (1.6 kA/m) to 200 Oe (16.0 kA/m).

10. The composition of claim 7, wherein the hydrophilic ligand includes at least one from a group consisting of polyethylene glycol, polyethyleneamine, polyethyleneimine, polyactylic acid, polymaleic anhydride, polyvinyl alcohol, polyvinylpyrrolidone, polyvinylamine, polyacrylamide, polyethylene glycol, phosphoric acid-polyethylene glycol, polybutylene terephthalate, polylactic acid, polytrimethylene carbonate, polydioxanone, polypropylene oxide, polyhydroxyethyl methacrylate, starch, dextran derivatives, sulfonic amino acids, sulfonic acid peptide, silica and polypeptide.

11. A hepatocyte targeting carrier comprising iron oxide magnetic particles, wherein each of the iron oxide magnetic particles contains: a core including an iron oxide derived from a composite of iron and at least one type of a compound selected from a group consisting of an aliphatic hydrocarbon acid salt having 4 to 25 carbon atoms and an amine compound; MX.sub.n; and at least one selected from a group consisting of folate, glycyrrhetinic acid, and glucose, wherein M is selected from a group consisting of Cu, Sn, Pb, Mn, tr, Pt, Rh, Re, Au, Pd and Os, wherein X is selected from a group consisting of F, Cl, Br and I, wherein n is an integer of 1 to 6, wherein the iron oxide magnetic particle has an average particle diameter of 6 nm to 20 nm.

12. The hepatocyte targeting carrier of claim 11, wherein the hepatocyte targeting carrier further contains an active iruzredient for treatment of liver cancer.

13. A method for treating liver cancer, comprising administering the composition of claim 1 to a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The above and other aspects, features, and advantages of certain embodiments of the disclosure will be more apparent from the following description taken in conjunction with the accompanying drawings, in which:

[0062] FIG. 1 is a schematic diagram of a preparation process and a structure of a nanoparticle according to an embodiment of the disclosure;

[0063] FIG. 2 is a graph showing uptake efficiency of nanoparticles into liver cancer cells according to an embodiment of the disclosure;

[0064] FIG. 3 is a graph showing biotoxicity test results against liver and kidney of nanoparticles according to an embodiment of the disclosure;

[0065] FIG. 4 is a graph showing biodistribution in an animal model of nanoparticles according to an embodiment of the disclosure;

[0066] FIG. 5 is a graph showing delivery efficiency of nanoparticles according to an embodiment of the disclosure to liver cancer cells; and

[0067] FIG. 6 is a graph showing liver cancer treatment effect of nanoparticles according to an embodiment of the disclosure,

DETAILED DESCRIPTION

[0068] Hereinafter, in order to help understanding of the disclosure, Example will be described in detail. However, Examples according to the disclosure may be changed into various other forms, and the scope of the disclosure should not be construed as being limited to the following Examples. Examples of the disclosure are provided to more fully explain the disclosure to a person with average knowledge in the art.

[0069] Present Example 1: Preparation of Iron Oxide Magnetic Particles Into Which GA (glycyrrhetinic acid) Is Introduced

[0070] (a) Formation of iron-oleic acid or iron-oleyl amine composite

[0071] FeCl.sub.3.6H.sub.2O 6.218 g (60 mmol), sodium oleate 54.79 g (180 mmol), hexane 224 ml, ethanol 120 ml, and deionized water 90 ml reacted with each other at 50° C. for about 4 hours at 900 rpm while vigorously stirring the mixture. After the reaction solution was cooled to room temperature, a transparent lower layer was removed using a separatory funnel. 100 ml of water was mixed with a brown upper organic layer, and the mixture was shaken, and a lower water layer was removed again. This was repeated 3 times. A remaining brown organic layer was transferred to a beaker which was heated at 110° C. overnight to evaporate hexane therefrom, thereby obtaining iron-oleic acid composite as iron oxide core particles.

[0072] (b) Synthesis of iron oxide magnetic particles containing CuF.sub.2

[0073] The iron-oleic acid composite as prepared above 4.5 g (5 mmol) and oleic acid 0.8 ml (2.5 mmol) were mixed with each other, and CuF.sub.230.5 mg (0.3 mmol) and 1-octadecene 15 ml were added thereto and mixed therewith. The mixture was placed in a round bottom flask and heated to 90° C. in a vacuum for 30 minutes to remove gas and moisture therefrom. Nitrogen was injected thereto and a temperature was raised to 200° C. Thereafter, the temperature was raised to 320° C. at a rate of 3.3° C./renin and then reaction occurred for 30 minutes. After cooling the reaction solution, the cooled solution was transferred to a 50 ml conical tube, and then 30 ml of ethanol and hexane were injected thereto in a 2:1 ratio, and then centrifugation was carded out to precipitate the particles. The precipitated particles were washed with 25 ml of ethanol and 15 ml of hexane, and the resulting precipitate was dispersed in hexane. Then, the dispersion was dispensed into a 50 ml vial. The solvent was evaporated therefrom, and then a resulting product was redispersed in toluene such that the iron oxide had a concentration of 25 mg/ml.

[0074] (c) Introduction of 1 and glycyrrhetinic acid to iron oxide magnetic particles containing CuF.sub.2

[0075] 10 mg of iron oxide magnetic particles containing CuF.sub.2 were dispersed in 1 mL of chloroform. The dispersion, 2 mL of deionized water, 20 mg of NaI, and DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]-2000), and DSPE-PEG2000-Glycyrrhetinic acid (1,2-distearoyl-sn-glycero-3-phosphoethariolamine -N-(polyethylene glycol)-glycyrrhetinic acid) at a density of 8 per a particle surface area. (1 nm.sup.2) and in a weight ratio of 9:1 were put into a 50 mL vial. For one minute, the vial was subjected to an operation of a microwave 2.4 GHz 1000W.

[0076] After removing the solution using an evaporator, 3 ml of deionized water was added thereto and a resulting solution was dispersed via sonication for 5 minutes. After the dispersing, the dispersion, and ethanol and deionized water in a ratio of 2:8 were input to Amicon 100k and centrifugation was carried out (5,000 rpm, 5 m). Deionized water was input to Amicon 100k and centrifugation (5,000 rpm, 5 m) was conducted to obtain iron oxide nanoparticles. An average particle diameter of the prepared nanoparticies was 10 nm.

[0077] Present Example 2: Preparation of Iron Oxide Magnetic Particles To Which Folate Is Introduced

[0078] A process was performed in the same manner as in Present Example 1, except that a step of introducing I and folate to the iron oxide magnetic particles containing CuF.sub.2 of Present Example 1-(c), and subsequent steps were performed as follows.

[0079] 10 mg of iron oxide magnetic particles containing CuF.sub.2 were dispersed in of chloroform. The dispersion, 2 mL of deionized water, 20 mg of NaI, and DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]-2000) and DSPE-PEG2000-Folate (1,2-distearoyl-sn-glycero-3-phosphoethanolarnine-N-(polyethylene glycol)-folate) at a density of 8 per a particle surface area (1nm.sup.2) and in a weight ratio of 9:1 were put into a 50 mL vial. For one minute, the vial was subjected to an operation of a microwave 2.4 GHz 1000W, A subsequent procedure was performed in the same manner as in Present Example 1. The average particle diameter of the prepared nanoparticles was 10 nm.

[0080] Present Example 3: Preparation Of Iron Oxide Magnetic Particles To Which Glu (Glucose) Is Introduced

[0081] A process was performed in the same manner as in Present Example 1, except that a step of introducing I and glucose to the iron oxide magnetic particles containing CuF.sub.2 of Present Example 1-(c), and subsequent steps were performed as follows,

[0082] 10 mg of iron oxide magnetic particles containing CuF.sub.2 were dispersed in 1 mL of chloroform. The dispersion, 2 mL of deionized water, 20 mg of NaI, and DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-knethoxy(polyethylene glycol)-2000), and DSPE-PEG2000-glucose (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(polyethylene glycol)-glucose) at a density of 8 per a particle surface area (1 nm.sup.2) and in a weight ratio of 9:1 were put into a 50 mL, vial. For one minute, the vial was subjected to an operation of a microwave 2.4 GHz 1000W. A subsequent procedure was performed in the same manner as in Present Example 1. The average particle diameter of the prepared nanoparticles was 10 nm.

[0083] Present Example 4: Preparation Of Iron Oxide Magnetic Particles To Which GA And .sup.131I Are Introduced

[0084] A process was performed in the same manner as in Present Example 1, except that a step of introducing GA and .sup.131I to the iron oxide magnetic particles containing CuF.sub.2 of Present Example 1-(c), and subsequent steps were performed as follows.

[0085] 10 mg of iron oxide magnetic particles containing CuF.sub.2 were dispersed in 1 mL of chloroform. The dispersion, 2 mL of deionized water, 1 mL of NaI.sup.131 (185 MBq(5mCi)), DSPE-PEG2000(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]-2000), and DSPE-PEG2000-glycyrrhetinic acid) (1,2-distearoyl-sn-alycero-3-phosphoethanolamine-N-(polyethylene glycol)-glycyrrhetinic acid)) at a density of 8 per a particle surface area (1 nm.sup.2) and in a weight ratio of 9:1 were put into a 50 mL vial. For one minute, the vial was subjected to an operation of a microwave 24 GHz 1000W. A subsequent procedure was performed in the same manner as in Present Example 1.

[0086] The average particle diameter of the prepared nanoparticles was 10 nm. When the iron oxide magnetic particles to which GA and .sup.131I were introduced as prepared in the above experiment had a radiation dose of 50 MBq (1.35 mCi) as measured with a gamma-counter.

[0087] Comparative Example 1: Preparation of Iron Oxide Magnetic Particles to which Only I is Introduced

[0088] A process was performed in the same manner as in Present Example 1, except that a step of introducing I to the iron oxide magnetic particles containing CuF.sub.2 of Present Example 1-(c), and subsequent steps were performed as follows.

[0089] 10 mg of iron oxide magnetic particles containing CuF.sub.2 were dispersed in 1 mL of chloroform. The dispersion, 2 mL of deionized water, 1 ml of NaI, and SSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]-2000) at a density of 8 per a particle surface area (1 nm.sup.2) were put into a 50 mL vial. For one minute, the vial was subjected to an operation of a microwave 2.4 GHz 1000W. A subsequent procedure was performed in the same manner as in Present Example 1.

[0090] Comparative Example 2: Preparation of Iron Oxide Magnetic Particles to which Only .sup.131I is Introduced

[0091] A process was performed in the same manner as in Present Example 1, except that a step of introducing .sup.131I to the iron oxide magnetic particles containing CuF.sub.2 of Present Example 1-(c), and subsequent steps were performed as follows.

[0092] 10 mg of iron oxide magnetic particles containing CuF.sub.2 were dispersed in 1 mL of chloroform. The dispersion, 2 mL of deionized water, linL of NaI.sup.131 (185MBq(5mCi)), and DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]-2000) at a density of 8 per a particle surface area (1 nm.sup.2) were put into a 50 mL vial. For one minute, the vial was subjected to an operation of a microwave 2.4 GHz 1000W. A subsequent procedure was performed in the same manner as in Present Example 1.

[0093] Test Example 1: Uptake Test Into Live Cancer Cells (in vitro)

[0094] The uptake of the iron oxide magnetic particles according to the disclosure into the liver cancer cells was examined to evaluate an ability of the particle to deliver the active ingredient to liver cancer cells. Specifically, hepG2 cells, which are liver cancer cells, were treated with the iron oxide magnetic particles at 200 μug/mL. Then, extracellular iron oxide magnetic particles were removed at each timing, and then the cells were decomposed with an acidic solution and 4% potassium ferrocyanide solution was added thereto. Then, a delivery rate of the iron ions into the hepatocarcinoma cells over time was measured based on UV absorbance value using Prussian blue staining.

[0095] The results are shown in FIG. 2. Present Examples 1 to 3 exhibited a higher delivery rate into hepatocellular carcinoma, compared to Comparative Example 1. Among Present Examples 1 to 3, Present Example 1 exhibited the highest delivery rate into hepatocellular carcinoma.

[0096] Test Example 2: In Vivo Toxicity Test

[0097] It was tested Whether the iron oxide magnetic particles according to the disclosure have biotoxicity. Specifically, after administering each of Present Examples 1 to 3 to 100 mg/kg to Balb/c nude mice, orbital blood was collected for liver and kidney enzymes before the administration and on 1st, 7th, 14th, and 28th days after the administration. Then, blood biochemical levels were tested. In a control group, only water for injection as used for administering Present Examples 1 to 3 thereto was administered thereto.

[0098] The results are shown in FIG. 3. Based on a result of the test, it was identified that for all of Present Examples 1 to 3, enzyme levels related to liver and kidney among all observed organs were within a normal range.

[0099] Test Example 3: Biodistribution Test in Animal Model

[0100] Animal experiments were conducted to evaluate delivery effect of the iron oxide magnetic particles according to the disclosure into the liver. Specifically, after 100 mg/kg of Presort Example I was administered to Balb/c nude mice at the tail vein thereof, the distribution of the particles in each organ in the body and the change thereof over time were identified based on iron ion analysis via hourly ICP-MS analysis.

[0101] The results are shown in FIG. 4. Based on a result of observing distribution of Present Example 1 in each organ tissue, it was identified that the iron oxide magnetic particles were specifically delivered only to the liver, but were hardly delivered to the kidneys or lungs, and almost all of the iron oxide magnetic particles delivered to the liver were excreted within about 2 weeks.

[0102] Test Example 4: Delivery Test Into Liver Cancer Cell

[0103] Animal experiments were conducted to evaluate the delivery effect of iron oxide magnetic particles according to the disclosure into the liver cancer cells. The animal model as used was a xenograft mouse model, which was produced by transplanting human liver cancer cells into buttocks of Balblc nude mice to induce the liver cancer. After administering Present Example 1 and general iron oxides as a control at 100 mg/kg to the tail vein of the produced xenograft mouse model, the delivery rate to the liver cancer cells, the distribution in the body and change thereof over time were identified based on iron ion analysis via ICP-MS analysis based on timings.

[0104] The results are shown in FIG. 5. Based on a result of observing the distribution of Present Example 1 in each organ tissue, it was identified that the iron oxide magnetic particles initially delivered to the liver were accumulated into liver cancer cells over time, and the maximum amount had been accumulated in the liver cancer cells after 1 week, and almost all thereof were excreted after about 2 weeks. Further, it was identified that the particles were hardly delivered to the kidneys or lungs. On the other hand, it was observed that it the conventional iron oxide particle was not transferred to the liver cancer cells after about 2 weeks, and most thereof were accumulated in the liver and were not excreted.

[0105] Test Example 5: Liver Cancer Treatment Test

[0106] An animal experiment was conducted to evaluate the liver cancer treatment effect of the iron oxide magnetic particles according to the disclosure. Specifically, we induced the liver cancer in Balblc nude mice. The liver cancer treatment effect of each of Present Example 4 (with GA) as a magnetic drug carrier which contained GA and was doped with I.sup.131, and Comparative Example 2 (w/o GA) as a magnetic drug carrier which was free of GA and was doped with I.sup.131 was identified. Further, the liver cancer treatment effect of Present Example 4 was identified based on a varying radiation dose of I.sup.131.

[0107] The results are shown in FIG. 6. Based on a result of measuring a tumor size at intervals of 3, 7, 10, and 14 days, it was identified that the liver cancer treatment effect of Present Example 4 (with GA) as a magnetic drug carrier which contained GA and was doped with was higher than that of each of the PBS control and Comparative Example 2. Further, the liver cancer treatment effect was higher at a larger radiation dose.

[0108] According to the disclosure, the composition comprising the nanoparticles according to one embodiment is delivered specifically to the liver, the composition may act on the liver cancer cells without damaging other organs.

[0109] Further, the nanoparticles according to one embodiment remain in the body for a certain period of time and are excreted outside the body within a few weeks. Thus, there is little risk of side effects such as organ damage caused by the accumulated iron oxide.

[0110] Further, nanoparticles according to one embodiment include the iron oxide magnetic particles, and thus have high responsiveness to external stimuli such as radiation, magnetic field and radio waves, and thus may be used for hyperthermia.

[0111] While the disclosure has been shown and described with reference to various embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the appended claims and their equivalents.