Patent Application
20190023584
This application claims priority from KR 10-2017-0092955, filed Jul. 21, 2017, the contents of which application are incorporated herein by reference in their entireties for all purposes.
The present invention relates to iron oxide nanoparticles capable of gigantic AC self-heating characteristics in a biocompatible AC magnetic field and application to hyperthermia treatment.
Hyperthermia is a cancer treatment modality using characteristics of cancer cells, which are more sensitive to heat than normal cells due to abnormal environments around the cancer cells. This treatment modality maintains the surrounding temperature around the cancer cells in a warm range (41° C. to 45° C.) compared to conventional chemotherapy or radiotherapy, so that this treatment modality has medical advantages in that even extremely small cancer cells localized or deeply seated in living tissue can be selectively killed by heat without damage to normal cells.
Development of AC magnetic self-heating technique in in-vivo environments is required for realizing effective cancer treatment, and recently a great deal of research efforts have been paid to develop AC magnetic self-heating magnetic nanoparticles. In the cancer treatment of hyperthermia using magnetic nanoparticles, the cancer cells were killed by heat generated by magnetic nanoparticles in the AC magnetic field.
In addition, magnetic nanoparticles with superparamagnetism have no aggregation of particle when they are introduced into a living body. When an AC magnetic field is applied to the magnetic nanoparticles, it is possible to easily control heat generated by the applied AC magnetic field and magnetic nanoparticles can be introduced into a living body only by simple injection treatment without a surgery.
Although only bulk sized magnetic materials showed a self-heating effect, there are limitations for their practical application due to difficulty in continuously increasing the self-heating temperature and difficulty in introduction of the magnetic materials into a living body. However, recently, a collaborative research team of the National University of Singapore and the Yokohama National University of Japan has published a study on a new type of self-heating magnetic nanoparticles, and their heating effect in the cells is effective enough for practical application, thus it is possible to expect the realization of a new cancer treatment.
A paper entitled “Applications of NiFe2O4 nanoparticles for a hyperthermia agent in biomedicine” in Applied Physics Letters, Vol. 89, 252503 (2006) discloses the effectiveness of NiFe2O4 magnetic nanoparticles as an in vivo hyperthermia agent.
In addition, US Patent Publication No. 2005-0090732 discloses target-oriented hyperthermia treatment using iron oxide. However, most conventional hyperthermia treatment relate to iron oxide nanoparticles showing a heat emission effect at high frequencies and high magnetic field (or high AC magnetic field).
However, in the cancer treatment of high-frequency hyperthermia, red spots may appear around the skin, and area with high fat, some burns, wounds, inflammations, and cell necrosis may occur. Above all, side effects due to harmfulness of high-frequency electric fields to humans are unavoidable. Therefore, this treatment is prohibited for pregnant women, patients with severe inflammation, patients with implanted cardiac pacemakers, and patients with severe pleural effusion and ascites.
In addition, since heat needs to be irradiated to cancer tissues for a long, human bodies are exposed to high-frequency electromagnetic waves for a long time, and thus, there is a problem in that normal tissues can also be damaged.
To solve the aforementioned problems, it is required to develop the magnetic nanoparticles capable of self-heating in a biocompatible low AC magnetic field (or a safe AC magnetic field).
It is an object of the present invention to provide iron oxide nanoparticles capable of sufficient self-heating even in a biocompatible low (or safe) AC magnetic field.
In accordance with one aspect of the present invention, iron oxide nanoparticles are nanoparticles in which γ-Fe2O3 (maghemite) is doped with an alkali metal ion or alkali earth metal ion, specifically nanoparticles in which an Fe vacancy site of γ-Fe2O3 is doped with an alkali metal ion or alkali earth metal ion.
The alkali metal ion may include lithium (Li), sodium (Na) and potassium (K), and the alkali earth metal ion may include magnesium (Mg) or calcium (Ca).
The doping metal ion may include at least one alkali metal ion or alkali earth metal ion, preferably at least one selected from the group consisting of Li+, Na+, K+, Mg2+, and Ca2+.
The iron oxide nanoparticles may generate a gigantic heat even in a biocompatible low AC magnetic field of fappl·Happl of 3.0×109 Am−1s−1 or less, and may have an intrinsic loss power (ILP) of 13.5 nHm2/Kg to 14.5 nHm2/Kg in an AC magnetic field of fappl·Happl<1.8×109 Am−1s−1 (fappl<120 kHz, Happl<15.12 kA/m).
The iron oxide nanoparticles may be represented by Mx-γFe2O3 (M=Li, Na, K, Mg, and Ca), and x may satisfy 0.00<x≤0.30, preferably 0.10≤x≤0.25, more preferably 0.10≤x≤0.20.
The iron oxide nanoparticles may have an average particle diameter of about 7 nm to about 13 nm, without being limited thereto.
In accordance with another aspect of the present invention, a method of preparing iron oxide nanoparticles is applied to preparation of nanoparticles capable of being heated even in a biocompatible low AC magnetic field, and includes preparing iron oxide nanoparticles by mixing an Fe3+ precursor, an M+ or M2+ (M=Li, Na, K, Mg, and Ca) precursor, a surfactant, and a solvent in an oxygen atmosphere caused by high temperature thermal decomposition (HTTD).
The Fe3+ precursor and the M+ or M2+ (M=Li, Na, K, Mg, and Ca) precursor may include at least one selected from among metal nitrate, metal sulfate, metal acetylacetonate, metal fluoroacetoacetate, metal halide, metal perchlorate, metal alkyl oxide, metal sulfamate, metal stearate, and organic metal compounds, without being limited thereto. For example, for Mgx-γFe2O3 nanoparticles, magnesium (Mg) acetate tetrahydrate and iron (Fe) acetylacetonate was used.
The solvent may include benzene solvents, hydrocarbon solvents, ether solvents, polymer solvents, ionic liquid solvents, halogen hydrocarbons, alcohols, sulfoxide solvents, water, and the like, preferably at least one of benzene, toluene, halobenzene, octane, nonane, decane, benzyl ether, phenyl ether, hydrocarbon ethers, polymer solvents, diethylene glycol (DEG), water, and ionic liquid solvents, without being limited thereto. For example, for Mgx-γFe2O3 nanoparticles, benzyl ether was used.
In the method according to the present invention, the surfactant may be used to stabilize nanoparticles and may include at least one of organic acids (CnCOOH, Cn: hydrocarbon, 7≤n≤30) including oleic acid, lauric acid, stearic acid, myristic acid, and hexadecanoic acid, without being limited thereto. For example, for Mgx-γFe2O3 nanoparticles, oleic acid was used.
According to the present invention, the method of preparing iron oxide nanoparticles includes: (a) heating a mixed solution of an Fe3+ precursor, an M+ or M2+ (M=Li, Na, K, Mg, and Ca) precursor, a surfactant, and a solvent to a temperature less than a boiling point of the solvent in a mixed atmosphere of oxygen and argon, followed by maintaining the mixed solution at the temperature for a certain period of time; (b) heating the mixed solution again to the boiling point of the solvent in a mixed atmosphere of oxygen and argon, followed by maintaining the mixed solution at the boiling point for a certain period of time; (c) removing a heating source and cooling the mixed solution to room temperature; and (d) performing precipitation and separation of nanoparticle powder by adding a polar solvent to the mixed solution and then performing centrifugation.
In the method of preparing iron oxide nanoparticles according to the present invention, a doping level can be adjusted by adjusting an amount of the Fe3+ precursor or the M+ or M2+ (M=Li, Na, K, Mg, and Ca) precursor.
According to the present invention, the iron oxide nanoparticles can perform sufficient self-heating even in a biocompatible low AC magnetic field. Therefore, the iron oxide nanoparticles can be used for hyperthermia cancer treatment in a low AC magnetic field.
Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. It should be understood that the present invention is not limited to the following embodiments and may be embodied in different ways, and that the embodiments are provided for complete disclosure and thorough understanding of the invention by those skilled in the art.
According to the present invention, iron oxide nanoparticles are prepared by doping an Fe vacancy site of γ-Fe2O3 with an alkali metal ion or alkali earth metal ion, and generate gigantic heat even in a biocompatible low AC magnetic field. In hyperthermia, a biocompatible AC magnetic field generally has fappl·Happl of 5.0×109 Am−1s−1 or less, preferably 3.0×109 Am−1s−1 or less. The iron oxide nanoparticles according to the present invention can generate gigantic heat even in such a biocompatible low AC magnetic field.
As used herein, the expression “doped with . . . metal ion” means that a metal atom is doped and ion-bonded to surrounding atoms, and thus all of expressions “doped with . . . metal ion”, “doped with . . . metal atom”, and “doped with . . . metal” should be interpreted as having the same meaning.
The iron oxide nanoparticles according to the present invention generate heat in AC magnetic fields, and are preferably used in a biocompatible low AC magnetic field.
Hereinafter, as an example of the iron oxide nanoparticles according to the present invention, Mgx-γFe2O3 will be described.
Mgx-γFe2O3 is prepared by high-temperature thermal decomposition of an Fe3+ precursor and an Mg2+ precursor in an oxygen atmosphere, has a crystal structure in which an Fe vacancy site of γ-Fe2O3 is doped with Mg2+, and generates gigantic heat even in a biocompatible low AC magnetic field.
A process of preparing Mgx-γFe2O3 (x=0.13) will be described in more detail (see
To prepare Mg0.13-γFe2O3, 0.13 mmol of magnesium (Mg) acetate tetrahydrate, 2.0 mmol of iron (Fe) acetylacetonate, 1.2 mmol of oleic acid, and 20 mL of benzyl ether are mixed in a 50 mL round bottom flask and are magnetically stirred. The mixed solution is heated to 200° C. for 30 minutes (˜8° C./min, first ramping up rate) in a mixed atmosphere of oxygen and argon (flow rate of ˜100 mL/min) and is then maintained for 50 minutes (nucleation step). Next, the mixed solution is heated again to 296° C. (boiling point of benzyl ether) for 20 minutes (5° C./min, second ramping rate) and is then maintained for 60 minutes (growth step).
Next, a heating source is removed and the mixed solution is cooled to room temperature.
A polar solvent such as ethanol is added to the mixed solution, followed by centrifugation, thereby precipitating and separating black powder. Separated products (nanoparticles) are dispersed in a nonpolar solvent such as toluene.
To control the Mg2+ doping concentration (x) of Mgx-γFe2O3, the different amount of Mg2+/Fe3+ metal precursor are used under identical experimental conditions. For example, to synthesize the Mg0.10-γFe2O3 nanoparticles, 0.10 mmol of Mg acetate tetrahydrate and 2.0 mmol of Fe acetylacetonate are used under the identical experimental conditions.
In a conventionally synthesized nanoparticles, MgFe2O4 nanoparticles (Mg2+ doped Fe3O4 structure) are prepared.
To prepare MgFe2O4 nanoparticles, 1.0 mmol of MgCl2 and 2.0 mmol of Fe(acac)3 are placed in a 100 mL round bottom flask containing dibenzyl ether and a surfactant (oleic acid and oleylamine). 10.0 mmol of 1,2-hexadecandiol is used as a reductant.
The mixed solution is heated to 200° C. for 25 minutes in an argon atmosphere and maintained for 60 minutes (nucleation step).
Next, the mixed solution is heated again to 296° C. (boiling point of benzyl ether) for 30 minutes and maintained for 60 minutes. A heat source is removed and a reaction mixture is cooled to room temperature.
Ethanol is added to the reaction product, followed by centrifugation, thereby obtaining precipitated black powder. The obtained MgFe2O4 nanoparticles are dispersed in a nonpolar solvent such as toluene.
Upon preparation of existing nanoparticles, two chemical reagents, that is, oleic acid and oleylamine are used as size control factors, and oleylamine can be used as reducing agent. The crystal structure may be change γ-Fe2O3 into Fe3O4 during synthesis process thereof.
Oleylamine is mainly used in preparation of iron oxide nanoparticles. In order to investigate the role of surfactant, a control experiments were carried out with oleylamine instead of oleic acid. The nanoparticles synthesized with oleylamine showed a similar AC self-heating behavior to MFe2O4 (M=Fe3+, Co2+, Ni2+, Mg2+) nanoparticles due to the reduction of Fe3+ into Fe2+ that leads to the Fe3O4 lattice in the presence of oleylamine. This result confirms that Mg2+ ion doped Fe3O4 lattice has no contribution to the AC heating properties.
Mgx-γFe2O3, which corresponds to the iron oxide nanoparticles according to the present invention, is obtained by doping Fe vacancy sites of γ-Fe2O3 with Mg2+.
Unlike Fe3O4, γ-Fe2O3 has spaces (vacancy sites), which occupy about 12% of the total volume thereof (see
When such vacancy sites of γ-Fe2O3 is doped with an alkali metal or alkali earth metal, the doped γ-Fe2O3 demonstrate change in DC/AC magnetic softness and magnetic properties, specifically magnetic susceptibility, and thus responds to a low AC magnetic field, thereby generating heat (see
On the other hand, unlike the above case, in the case of a transition metal (Zn, Fe, Mn, Co, Ni, and the like), since it is energetically favorable in terms of thermodynamics that the transition metal is predominantly substituted with Fe3+ ions at an octahedral site (Oh) and a tetrahedral site (Th), not in vacancy site, doped γ-Fe2O3 exhibits reduced net magnetic properties and respond negligibly to a low AC magnetic field (see
The iron oxide nanoparticles according to the present invention are nanoparticles in which Fe vacancy sites of γ-Fe2O3 are doped with an alkali metal or alkali earth metal. According to the present invention, a doping metal includes any alkali metal or alkali earth metal without limitation. Preferably, the alkali metal is lithium (Li), sodium (Na), or potassium (K), and the alkali earth metal is magnesium (Mg) or calcium (Ca).
In addition, the iron oxide nanoparticles according to the present invention are doped with at least one alkali metal or alkali earth metal, preferably at least one metal ion selected from the group consisting of Li+, Na+, K+, Mg2+, and Ca2+.
The iron oxide nanoparticles according to the present invention may emit gigantic heat even in a biocompatible low AC magnetic field of fappl·Happl of 3.0×109 Am−1s−1 or less, and have an intrinsic loss power (ILP) of 13.5 nHm2/Kg to 14.5 nHm2/Kg in an AC magnetic field of fappl·Happl<1.8×109 Am−1s−1 (fappl<120 KHz, Happl<15.12 KA/m).
Further, the iron oxide nanoparticles according to the present invention may be represented by Mx-γFe2O3 (M=Li, Na, K, Mg, and Ca), and x may vary with a doping concentration of a metal. x satisfies 0.00<x≤0.30, preferably 0.10≤x≤0.25, more preferably 0.10≤x≤0.20.
The iron oxide nanoparticles according to the present invention have an average particle diameter of about 7 nm to about 13 nm, without being limited thereto, and may have various nanometer scale sizes.
The U87MG cells were incubated with 700 μg/mL of Mg0.13-γFe2O3 nanofluids and Resovist, as a control group, for cellular uptake. The cells were placed in the center of an AC magnetic coil, and a magnetic field of fappl=99 kHz and Happl=±155 Oe (Happl·fappl=1.22×109 Am−1s−1) was applied to the cells for 1500 seconds. Referring to the right-side graph of
Referring to
On the other hand,
Hep3B cells transfected with luciferase (for bioluminescence imaging, BLI) grew subcutaneously in mice (cancer-xenograft model)
A 100 μL of Mg0.13-γFe2O3 nanofluids (100 μL, 11.5 μg/μL) was intratumorally injected into cancer cells of the mice (˜1000 mm3) through soft tissue using a bent needle and optical thermometers were mounted in the cancer cells and rectum area to monitor the temperature.
For comparison, Resovist (100 μL, 11.5 μg/μL) and normal saline (100 μL, 11.5 μg/μL) were also intratumorally injected into the mice, respectively.
The mice were placed in the center of an AC magnetic coil and exposed to an AC magnetic field (fappl=99 kHz, Happl=±155 Oe, Happl·fappl=1.22×109 Am−1s−1) for 900 seconds.
The temperature of the rectum and Hep3B injected with Resovist were slightly increased from 34° C. to 36.37° C. and 37.14° C., respectively. However, the temperature of the Hep3B cells injected with the Mg0.13-γFe2O3 nanofluids was rapidly increased up to 50.2° C. (thermoablation temperature).
The activity of the Hep3B was analyzed by employing a bioluminescence imaging (BLI) technique. The Hep3B treated with Mg0.13-γFe2O3 nanofluids did not exhibit any BL-intensity from day 2 after magnetic nanofluid hyperthermia, while the control groups still exhibited strong BL-intensity after magnetic nanofluid hyperthermia. No BL-intensity means that the cancer cells was completely necrotized by magnetic nanofluid hyperthermia.
As described above, it was proven through both of the in-vitro and in-vivo tests that cancer cells could be completely killed using magnetic nanoparticles according to the present invention. Therefore, the iron oxide nanoparticles according to the present invention can be clinically used.
Heretofore, the present invention has been described with reference to some embodiments in conjunction with the accompanying drawings. Although specific terms are used herein, it should be understood that the terms are only for the purpose of describing the embodiments of the present invention and are not intended to limit the present invention. In addition, it should be understood that various modifications, changes, alterations, and equivalent embodiments can be made by those skilled in the art without departing from the spirit and scope of the present invention. Therefore, the scope of the present invention should be defined only by the accompanying claims and equivalents thereof.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1020170092955 | Jul 2017 | KR | national |
