WO2019240329A1 - Superparamagnetic nanoparticles for hyperthermia therapy - Google Patents

Abstract

The present invention relates to a composition for hyperthermia therapy, comprising Mn_xZn_1-xFe₂O₄ (0≤X≤1) superparamagnetic nanoparticles as an active ingredient. Superparamagnetic nanoparticles according to the present invention exhibit high magnetically-induced heat generation ability and biocompatibility, thereby enabling tumor growth to be effectively inhibited.

Description

Superparamagnetic Nanoparticles for Thermotherapy

The present invention relates to a composition for hyperthermia therapy comprising superparamagnetic nanoparticles as an active ingredient capable of selectively thermally treating tumors without affecting healthy tissues.

Magnetic nanoparticles have a self-induced exothermic property when exposed to alternating magnetic fields, and the hyperthermic effect specifically exposed to a desired site can be applied to various tumor treatments. Recently, a new type of treatment method that combines hyperthermia therapy, chemotherapy and radiation chemotherapy has been in the spotlight.

However, there are many factors to consider in order to apply the thermal therapy using magnetic nanoparticles in the body. The most important of these is the total amount of strength and frequency of the AC magnetic field applied to the body. Within this range, magnetic nanoparticles must be able to induce temperatures within the range of therapeutic effects and should not affect surrounding normal tissues and organs. In this respect, it can be said that the development of magnetic nanoparticles having sufficient self-induced exothermic characteristics in a magnetic or biologically safe range of magnetic fields is important.

An object of the present invention is to provide a composition for treating heat and a method of treating heat, comprising a superparamagnetic nanoparticle as an active ingredient, which is excellent in biocompatibility and enables effective heat treatment.

In order to achieve the above object,

The present invention provides a thermotherapy composition comprising the superparamagnetic nanoparticles represented by the following general formula 1 as an active ingredient and a method of preparing the superparamagnetic nanoparticles:

Formula 1

Mn _x Zn _1-x Fe ₂ O ₄ (0≤X≤1)

Specifically, X is a number between 0 and 1, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and the like.

The superparamagnetic nanoparticles of the present invention are preferably prepared by a modified HTTD method in which the chemical potential of the cation is changed by controlling the temperature in conventional high temperature thermal decomposition (HTTD). This preparation method is characterized by Fe (III) acetylacetonate and Mn (II) acetate tetrahydrate, Zn (II) acetate dihydrate, 1,2-hexadecanediol, oleic acid, oleylamine and benzyl, as shown in FIG. Mixing ether and stirring magnetically; Heating the magnetically stirred mixed solution to 200-203 ° C. over 20 minutes, and then maintaining the temperature at the temperature for 60 minutes to generate nuclei (nucleation step); After raising the temperature to 296 ° C. or the boiling point of benzyl ether over 8-10 minutes, maintaining the temperature at the temperature for 47 minutes to grow the nanoparticles (growth step), and separating the nanoparticles.

The superparamagnetic nanoparticles according to the present invention have an average diameter of 1 to 20 nm, preferably within 10 nm.

The size of such superparamagnetic nanoparticles is also important in achieving the thermal treatment desired in the present invention. As used herein, the term "superparamagnetic nanoparticles" means magnetic nanoparticles having a size of less than 20 nm and a saturation magnetization (MS) value within a certain range. The present invention allows the release of heat using very small magnetic nanoparticles, the small sized nanoparticles reduce the damage (damage) when penetrating cells and / or tissues than the larger nanoparticles, side effects It is more safe because it can be minimized.

In the present invention, the superparamagnetic nanoparticles also exhibit the highest exothermic properties in the solid state at 30-370 kHz frequency and 80-160 Oe magnetic field strength applicable to the human body, as shown in one experimental example, Eleven Mn _x Zn _1-x Fe ₂ O ₄ nanoparticles with eight different frequencies (31.9, 47.0, 98.9, 140.0, 168.1, 195.5, 239.9, 360.2 kHz) and five different magnetic field strengths (80,100,120,140,160 Oe) , May have magnetic induction heating properties, in particular, superparamagnetic Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles having about X of 0.5 may have the highest magnetic induction heating properties at all frequencies.

In the present invention, the superparamagnetic nanoparticles may be coated with a biocompatible polymer.

Biocompatible polymer materials useful for the human body that can be used in the present invention are biocompatible polymers that can be easily dissolved in various solvents and have a number average molecular weight, preferably from about 300 Daltons to about 100,000 Daltons, for example polyethylene glycol. , Polypropylene glycol, polyoxyethylene, polytrimethylene glycol, polylactic acid and derivatives thereof, polyacrylic acid and derivatives thereof, polyamino acid, polyvinyl alcohol, polyurethane, polyphosphazine, poly (L-lysine), polyalkyl Non-immunogenic polymeric materials selected from the group consisting of ethylene oxide, polysaccharides, dextran, polyvinyl pyrrolidone, polyacrylamide and two or more copolymers thereof. These biocompatible polymers are intended to include not only linear but also branched polymers.

In a preferred embodiment of the present invention, in order to increase biocompatibility, the superparamagnetic nanoparticles may be modified with Methoxy-polyethylene glycol (PEG: Poly Ethyleneglycol) -Silane.

The composition for thermotherapy comprising the superparamagnetic nanoparticles according to the present invention as an active ingredient can achieve therapeutic efficacy in a manner by direct injection.

As used herein, the term “thermotherapy” means exposing body tissues to a temperature slightly above normal temperature to kill lesion cells, including cancer cells, or to make them more susceptible to radiation therapy or anticancer agents. . According to the present invention, the composition of the present invention is to provide a thermal effect by the superparamagnetic nanoparticles, thereby achieving a therapeutic effect according to the self-induced fever specific to the desired site without affecting normal tissues and organs around can do.

The composition for thermal treatment of the present invention is usually provided as a pharmaceutical composition. Therefore, the composition for thermal treatment of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are conventionally used in the formulation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, poly Vinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995.

The composition for treating heat of the present invention is preferably administered parenterally. Parenteral administration may be by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, or intralesional injection. Suitable dosages of the compositions of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, patient's age, food, time of administration, route of administration, rate of excretion and response to reaction. have. The thermotherapy composition of the present invention comprises superparamagnetic nanoparticles that exhibit a therapeutically effective amount of a self-induced exothermic effect. The term "therapeutically effective amount" means an amount sufficient to treat a disease for treatment, generally 0.0001-100 mg / kg.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or it may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets, or capsules, and may further include a dispersing agent or a stabilizing agent.

The composition for treating heat of the present invention may be administered to a subject (patient) by a suitable route of administration, and then a magnetic field of 30-370 kHz frequency and 80-160 Oe intensity may be used.

According to a specific embodiment of the present invention, the disease treated with the composition for treating heat of the present invention is cancer (or tumor). The composition for treating heat of the present invention is particularly useful for treating cancer, for example, glioblastoma, malignant glioma, breast cancer, lung cancer, colon cancer, anal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer, liver cancer, testicular cancer, Cervical cancer, sarcoma, hemangioma, esophageal cancer, eye cancer, laryngeal cancer, oral cancer, mesothelioma, myeloma, oral cancer, rectal cancer, throat cancer, bladder cancer, uterine cancer, ovarian cancer, prostate cancer, colon cancer, pancreatic cancer, kidney cancer, stomach cancer, skin cancer, basal cell It can effectively induce cancer cell death in various cancer diseases such as cancer, melanoma, squamous cell carcinoma, oral squamous cell carcinoma, colorectal cancer, or endometrial cancer.

According to still another aspect of the present invention, the present invention relates to a thermotherapy comprising administering a composition for treating heat of the present invention to a subject (a subject in need of thermotherapy, including humans, animals, mice, etc.). A method of treating hyperthermia therapy and cancer is provided.

In the present invention, thermal therapy has many advantages compared to conventional thermal therapy such as ultrasound, focused ultrasound, microwave, radio frequency probe warming and radio frequency capacitance warming. For example, ultrasound and focused ultrasound can penetrate deep into the trachea, but have the disadvantage of affecting bones due to high energy absorption and excessive reflections from the surrounding air. Heat treatments, such as microwaves, have a weak penetration depth, and radio frequency probe thermal therapy has the disadvantage of insufficient access to deeply located tumors.

However, the composition for thermal treatment containing the nanoparticles according to the present invention as an active ingredient, in particular, can treat deep-seated tumors. Nanoparticles injected using intratumoral nanoparticle injection methods and heat induced in external magnetic fields enable the treatment of deep tumors such as brain tumors. In addition, the composition for treating heat containing the nanoparticles according to the present invention may selectively generate uniformly high temperature because it can continuously react with an external magnetic field when applying the magnetic nanoparticles dispersed in the target organ. That is, since a given external magnetic field can only react with the magnetic compound, it can be selectively focused on the tumor without affecting the surrounding healthy tissue, and can continuously generate the heating temperature.

Such methods of treatment may be performed alone or in combination with, or in addition to, conventional anticancer treatments.

Nanoparticles according to the present invention has a high magnetic induction heating properties and high biocompatibility in the range of 30-370 kHz and 80-160 Oe of the human body applicable, can be applied to the human body, and useful for heat treatment Can be used.

In addition, the composition for thermal treatment according to the present invention enables the treatment of deep tumors, and has the effect of effectively inhibiting the growth rate of tumors by selectively and uniformly generating high temperature without affecting surrounding healthy tissue.

On the other hand, the effects described above are merely exemplary, and effects predicted or expected from the detailed configuration of the present invention may be added to the effects unique to the present invention from the viewpoint of those skilled in the art.

1 is a flow chart showing a method for synthesizing superparamagnetic Mn _x Zn _1-x Fe ₂ O ₄ nanoparticles of the present invention.

Figure 2 is a superparamagnetic Mn _x Zn _1-x Fe ₂ O ₄ (x = 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1) nano synthesized by an embodiment of the present invention A graph showing the TEM and EDS results of manganese and zinc detected after the synthesis of the particles. (TEM and EDS results of synthesized ZnFe ₂ O ₄ (X = 0), TEM and EDS results of synthesized Mn _0.1 Zn _0.9 Fe ₂ O ₄ (X = 0.1), synthesized Mn _0.2 Zn _0.8 TEM and EDS results of Fe ₂ O ₄ (X = 0.2), synthesized Mn _0.3 Zn _0.7 TEM and EDS results of Fe ₂ O ₄ (X = 0.3), synthesized Mn _0.4 Zn _0.6 Fe ₂ O ₄ (X = 0.4) TEM and EDS results of), synthesized TEM and EDS results of synthesized Mn _0.5 Zn _0.5 Fe ₂ O ₄ (X = 0.5), TEM and EDS results of synthesized Mn _0.6 Zn _0.4 Fe ₂ O ₄ (X = 0.6), synthesis TEM and EDS results of Mn _0.7 Zn _0.3 Fe ₂ O ₄ (X = 0.7), TEM and EDS results of Mn _0.8 Zn _0.2 Fe ₂ O ₄ (X = 0.8), Mn _0.9 Zn _0.1 Fe ₂ O TEM and EDS results of ₄ (X = 0.9), and TEM and EDS results of synthesized MnFe ₂ O ₄ (X = 1).)

Figure 3 shows the EDS results of manganese and zinc detected after the synthesis of Mn _x Zn _1-x Fe ₂ O ₄ nanoparticles.

Figure 4 shows the average diameter of the synthesized Mn _x Zn _1-x Fe ₂ O ₄ nanoparticles.

5 is a graph showing alternating magnetic induction self-heating characteristics of the solid-state nanoparticles of the superparamagnetic Mn _x Zn _1-x Fe ₂ O ₄ nanoparticles synthesized by the embodiment of the present invention. (Sequential from left, self-induced self-exothermic properties of Mn _x Zn _1-x Fe ₂ O ₄ solid-state nanoparticles with Mn concentration at fixed 80 Oe (t = 600 seconds), Mn at fixed 100 Oe Mn _x Zn _1-x Fe ₂ O ₄ Depending on the concentration of self-induced self-heating (t = 600 seconds) of solid-state nanoparticles, Mn _x Zn _1-x Fe ₂ O ₄ according to Mn concentration at a fixed 120 Oe Self-induced self-heating properties of solid-state nanoparticles (t = 600 seconds), Mn _x Zn _1-x Fe ₂ O ₄ self-induced self-heating properties of solid state nanoparticles with fixed Mn concentration at 140 Oe (t = 600 seconds), Mn _x Zn _1-x Fe ₂ O ₄ solid-state nanoparticles with fixed Mn concentrations at fixed 160 Oe (t = 600 seconds), Mn with frequency at fixed 80 Oe Magnetically Induced Self-heating Characteristics of _x Zn _1-x Fe ₂ O ₄ Solid State Nanoparticles (t = 600 sec), Mn _x Zn _1-x Fe ₂ O ₄ Solid State Nanoparticles with Frequency at Fixed 100 Oe character Typically the self-heating characteristic induction (t = 600 sec.), With respect to the frequency at a fixed _{120 Oe Mn x Zn 1-x} Fe 2 O 4 the self-heating characteristic magnetically guided to a solid-state nanoparticles (t = 600 Sec), Mn _x Zn _1-x Fe ₂ O ₄ solid-state nanoparticles with Mn concentration at fixed 140 Oe (t = 600 seconds) and Mn with frequency at fixed 160 Oe _x Zn _1-x Fe ₂ O _{4 shows} the self-induced self-heating characteristics (t = 600 seconds) of solid-state nanoparticles.)

FIG. 6 is a graph showing cytotoxicity (cell viability) analysis results of PEG-coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles in various types of human glioblastoma cells and normal cortical cells.

Figure 7 is a PEG-coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles synthesized according to an embodiment of the present invention when treated in the same concentration on different commercial cancer cell lines / patient-derived tumor cells / normal cells The cells absorbed the nanoparticles and analyzed by transmission electron microscopy (TEM). ((a): TEM image of U87 with PEG-coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles, (b): TEM image of U118 with PEG-coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles (c): TEM image of U138 with PEG-coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles, (d): TEM image of U251 with PEG-coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles (e): TEM image of U373 with PEG-coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles, (f): TEM image of T98G with PEG-coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles , (g): TEM image of A172 with PEG-coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles, (h): GBL-28 with PEG-coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles TEM images, (i): PEG- coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nano GBL-37 TEM image of a particle, (j): PEG- having a coating Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles PEG- showing a TEM image of NSC09 having a coating Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles: TEM images, and (k) of NSC10 A.)

8 is a thermal image of a mouse tumor model exposed to AC magnetic field for 20 minutes after injection of PEG-coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles synthesized by an embodiment of the present invention.

9 is a graph showing the relative temperature difference between tumor and body temperature during superparamagnetic nanoparticle thermal treatment.

10 is a graph showing the relative tumor weight and body weight of the mouse tumor model during thermal treatment. (Relative tumor amount during heat treatment, control (black), tumor with only magnetic nanoparticles (red), heat treated tumors with blue nanoparticles (blue) (top), relative weight during heat treatment, control (black), Tumors with magnetic nanoparticles only (red), thermally treated tumors with blue nanoparticles (blue) (below).)

11 is a subcutaneous tumor image during heat treatment. (-) MNP, (-) AMF: no treatment attempt, no injection of nanoparticles and no magnetic field, Control Group; (+) MNP, (-) AMF: group injecting only nanoparticles into carcinoma; (+) MNP, (+) AMF: The group that underwent thermal treatment by applying a magnetic field after injection of nanoparticles.

In the present invention, a total of 11 kinds of superparamagnetic iron oxide nanoparticles Mn _x Zn _1-x Fe ₂ O _{4 having} different compositions (X = 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1), 8 different frequencies (31.9, 47.0, 98.9, 140.0, 168.1, 195.5, 239.9, 360.2 kHz) and 5 different magnetic field strengths (80, 100, 120, 140, 160 Oe) The magnetic induction exothermic properties of each nanoparticle were analyzed. In addition, Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles with the highest self-induced exothermic properties were modified to Methoxy-PEG-Silane to be suitable for living organisms. Various biocompatibility evaluation experiments were conducted to analyze the effects of thermotherapy using magnetic nanoparticles in animal tumor models.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Example 1 Preparation and Method of Compound

For the synthesis of the superparamagnetic nanoparticles according to the invention the material prepared the materials listed in Table 1.

TABLE 1

Fe (III) acetylacetonate (> 99.9%), Mn (II) acetate tetrahydrate (99.99%), Zn (II) acetate dihydrate (99.999%), oleic acid (90%), oleylamine (70%) , Benzyl ether (99%) was purchased from Aldrich Chemical Co., 1,2-hexadecanediol (> 98%) was purchased from Tokyo Chemical Industries Co.

Example 2 Synthesis and Surface Modification of Nanoparticles

2-1. Synthesis of Solid State Mn _x Zn _1-x Fe ₂ O ₄ Nanoparticles

For the synthesis of solid state Mn _x Zn _1-x Fe ₂ O ₄ nanoparticles, as shown in Table 2 below, Fe (2 mmol), Mn (0.1 ~ 0.9 mmol), Zn (0.1 ~ 0.9 mmol), 1, 2-hexadecanediol (10 mmol), oleic acid (6 mmol), oleylamine (6 mmol) and benzyl ether (20 mL) were mixed and magnetically stirred in a three necked round bottom flask.

TABLE 2

The existing HTTD method heats the mixed solution at 200 ° C.-203 ° C. for 30 minutes (˜6 ° C./minute, first ascent rate) and holds for another 30 minutes (nucleation step). The solution is then again heated (16 ° C./min to 14.5 ° C./min, second ascent rate) to 296 ° C., the boiling point of benzyl ether, again for 30 minutes (growing step).

However, in the modified HTTD method performed in the present invention, the initial heating rate and the heat treatment time in the nucleation step (process temperature: 200 ° C. to 203 ° C.) are respectively 10 ° C./min to 11.2 ° C./min, 58 in the conventional HTTD method. Adjusted from minutes to 60 minutes. The heat treatment time in the growth stage (process temperature: 296 ° C.) was adjusted to 46.5 minutes to adjust the average particle diameter of the nanoparticles to 10 nm or less. After removing the heat source, the mixed solution was cooled to room temperature and then ethanol (40 mL) was added to the solution under ambient conditions. The synthesized product (black or brown depending on the material) was washed, precipitated and separated using a centrifuge. The product was dissolved in hexane (15 mL) mixed with ethanol (30 mL) and magnetically stirred again for 30 minutes to remove organic residue. After centrifugation at 6500 rpm for 12.5 minutes, nanoparticles were obtained and dried at room temperature.

2-2. Surface Deformation of Synthetic Superparamagnetic Nanoparticles

The synthesized nanoparticles were coated with 500 Da (Dalton) Methoxy-PEG-Silane, a biocompatible polymer. To coat the PEG layer, the surface of the synthesized nanoparticles was first modified with oleic acid. Oleic acid (3 ml) and NH ₄ Cl (0.7 ml) were added to the ethanol solution with nanoparticles. After the mixture was stirred vigorously for 2 hours, the nanoparticles were precipitated by a permanent magnet and then washed with acetone to obtain nanoparticles coated with oleic acid. The nanoparticles coated with oleic acid were dispersed in toluene (7.5 mL) and then triethylamine (3.75 mL) and methoxy-PEG-silane 500 Da (0.75 mL) were added. The mixed solution was stirred well for 24 hours. The PEG-coated nanoparticles in the solution were washed with pentane and dispersed in water to produce a nanofluidic solution.

Example 3 Characterization of Synthesized Nanoparticles

3-1. Synthesis Results of Superparamagnetic Mn _x Zn _1-x Fe ₂ O ₄ Nanoparticles

The superparamagnetic Mn _x Zn _1-x Fe ₂ O ₄ nanoparticles used in the present invention were synthesized by a modified HTTD method in which the chemical potential of the cation was changed by controlling the temperature during the synthesis process. This synthesis method involves the decomposition of a metal precursor in the presence of a hot organic surfactant and a solvent. The basic principle of this method is to initiate a chemical reaction between reagents in a solvent and to control the nucleation and growth steps for synthesis. Mn _x Zn _1-x Fe ₂ O ₄ nanoparticles are produced by changing the thermal energy of a solution. The HTTD method provides important advantages, such as controlling the size and size distribution of the synthesized nanoparticles, helps to generate strong magnetically induced exothermic properties of the nanoparticles, which are explained by the improved magnetic properties, and enables mass production. . After the synthesis of Mn _x Zn _1-x Fe ₂ O ₄ , the total amount of solid nanoparticles obtained was on average about 130-150 milligrams. To prevent chemical changes or damping, the synthesized nanoparticles were stored in an air-tightened vacuum desiccator.

3-2. Confirmation of Pyroelectric Properties of Synthesized Mn _x Zn _1-x Fe ₂ O ₄ Nanoparticles

In order to confirm that the synthesized nanoparticles are superparamagnetic, each Mn _x Zn _1-x Fe ₂ O ₄ nanoparticles were analyzed by Transmission Electron Microscopy (TEM). The synthesis of 11 as a TEM _{Mn x Zn 1-x Fe 2} O 4 was measured nanoparticles (X = 0-1), each of _{Mn x Zn 1-x Fe 2} O 4 nanoparticles As shown in FIG. 4 The average diameter of was less than 10 nanometers. In addition, each of the synthesized Mn _x Zn _1-x Fe ₂ O ₄ nanoparticles to confirm the doping level of manganese and zinc were analyzed by EDS (Energy-dispersive X-ray Spectroscopy) as shown in Table 3 and FIG. Each sample analyzed by EDS was measured at three different sites in X50k magnification normal TEM mode, and the detected atomic weight percentages of manganese and zinc were found to be successfully doped with the dopant depending on the reagent added. Finally, magnetic properties such as major magnetic hysteresis loops and small magnetic hysteresis loops, initial magnetization slope, coercivity, and saturation magnetization values were measured with a Vibrating Sample Magnetometer (VSM), which means that all synthesized Mn _x Zn _1-x Fe ₂ O ₄ nanoparticles have pyroelectric properties.

TABLE 3

EDS results of manganese and zinc detected after synthesis of Mn _x Zn _1-x Fe ₂ O ₄ nanoparticles (atomic weight%)

3-3. Magnetically Induced Exothermic Measurement

The exothermic properties induced by the alternating (AC) magnetic field of solid-state Mn _x Zn _1-x Fe ₂ O ₄ nanoparticles and fluid-state PEG-coated nanoparticles can be transferred to AC coils, capacitors, DC power supplies, wave generators, and PC systems. It was measured using a specially designed AC magnetic field generation system constructed. AC magnetic field generation systems operate over a wide frequency range of 30-370 kHz with magnetic field strengths up to 170 Oe without changing capacitors or AC coils. Since nanoparticles have different AC heating capabilities depending on the material, particle size and size distribution, it is necessary to be able to evaluate the AC heating capacity of various nanoparticles over a wide range of frequencies and magnetic fields.

The total amount of solid nanoparticles measured for AC exothermic properties was fixed at 60 mg in an Eppendorf-tube. To design the measurement environment in an isolated system (to prevent AC heating measurement interference from the ambient atmosphere), each sample was placed in an insulating styrofoam in the center of the sample bed. The tip of the optical fiber was placed inside an Eppendorf tube containing Mn _x Zn _1-x Fe ₂ O ₄ nanoparticles in the solid state. Eleven Mn _x Zn _1-x Fe ₂ O ₄ nanoparticles with eight different frequencies (31.9, 47.0, 98.9, 140.0, 168.1, 195.5, 239.9, 360.2 kHz) and five different magnetic field strengths (80,100,120,140,160 Oe) Was investigated. The total magnetic field generation time is 600 seconds for each measurement. When the magnetic field was turned on, the AC heating temperature was measured with an optical thermometer and cooled when the magnetic field was turned off.

The maximum delta temperature value dTmax deviation of 11 kinds of Mn _x Zn _1-x Fe ₂ O ₄ nanoparticles at relatively low magnetic field strength (80, 100 Oe) seemed to be little difference in doping level and frequency of manganese and zinc. . However, at relatively high magnetic field strengths (120, 140, 160 Oe), Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles (X = 0.5 at the center of the X axis in the third graph of FIG. 5) exhibit the highest dTmax values and Mn _0.5 Zn _{The 0.5} Fe ₂ O ₄ nanoparticles produced the highest AC heating properties at all frequencies. In addition, the dTmax value of the AC heating characteristics at the frequencies of 140.0 kHz and 360.2 kHz was relatively high, and it was found that the nanoparticles used in the present invention reacted at a very low frequency compared to other groups.

Mn _0.5 Zn _0.5 Fe ₂ O ₄ particles were used to adjust the proper frequency and the appropriate magnetic field, which are known to be harmless to the human body when performing heat treatment to control or treat human diseases. In the present invention, the frequency and the magnetic field placed from outside Mn _0.5 Zn _0.5 Fe ₂ O lesion of particles is present in the _4, generates a thermal effect and around the Mn _0.5 Zn _0.5 Fe ₂ of the normal tissue cells, with no particles of the O ₄ The frequency is 140 kHZ or less, preferably 110 KHz or less, and the magnetic field is 190 Oe or less, preferably 140 Oe or less in order to be harmless and cause no side effects at all.

Example 4 Checking Biocompatibility of Nanoparticles

4-1. Cell Culture and In Vitro Cytotoxicity Analysis

Seven human glioblastoma cell lines (A172, T98G, U87, U118, U138, U251, U373) obtained from commercially available human glioblastoma cell lines from American Type Culture collection (ATCC) and Korean Cell Line Bank (KCLB), brain tumor ablation Primary cultured human glioblastoma cells (GBL-28 and GBL-37), and primary cultured human normal cortical cells (NSC10, NSC09) obtained in the course of brain tumor lobectomy were used.

All cells were cultured in 10% FBS and 100U / ml penicillin / streptomycin-containing DMEM humidified incubator at 5% CO2, 37 ℃ to (Dulbecco Modified Eagled Medium, WelGENE, LM001-05, Korea), using TrypLE ^TM Accessed by trypsinization every 7-8 days.

CCK-8 analysis (WST-8, Dojindolabs, Kumamoto, Japan) was used to assess the effect on the proliferation and viability of PEG-coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles. CCK-8 analysis was used according to the manufacturer's instructions. A172, T98G, U118, U138, U251, U373, U87, GBL-28, GBL-37, NSC09, NSC10 cells were seeded in a number of 3000 cells / well in 96-well plates. After 24 hours (inoculation time), PEG-coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles were obtained at 0, 3, 5, 10, 30, 50, 100, 300, and 5% CO ₂ , 37 ° C. humidity incubator. Treatment was performed for 24 hours at a concentration of 500 μg / mL. After incubation of PEG-coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles, each well was washed with phosphate buffered saline (PBS) and fresh medium was added to each well of a 96-well plate with CCK-8 solution. Plates were further incubated for 2 hours for reaction of the cells with CCK-8 solution. Finally, the absorbance at 450 nm was evaluated using a Multiscan MS spectrophotometer (Labsystems, Stockholm, Sweden).

4-2. In Vitro Cytotoxicity Assay of PEG-Coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ Nanoparticles

For thermotherapy applications with in vitro magnetic nanoparticles Mn _0.5 Zn _0.5 Fe ₂ O ₄ , cytotoxicity assays were performed using eleven different kinds of human derived cells, specifically human glioblastoma cell lines that are commercially available. 7 human glioblastoma cell lines (A172, T98G, U87, U118, U138, U251, U373) obtained from the American Type Culture collection (ATCC) and the Korean Cell Line Bank (KCLB), obtained during a brain tumor lobectomy procedure. Primary cultured human glioblastoma cells (GBL-28 and GBL-37), and primary cultured human normal cortical cells (NSC10, NSC09) were used.

As shown in FIG. 6, the relative cell viability of 11 cells within a nanoparticle concentration of 100 μg / ml exceeds 70% so that the nanoparticles themselves do not critically affect cell viability, and the nanoparticles It was found to be stable when incubated together.

4-3. Intracellular Uptake of PEG-Coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ Nanoparticles

TEM analysis of cells treated with PEG-coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles was performed to investigate cellular modifications, including degree of nanoparticle penetration into cells, cell death and nuclear fission.

Cells were incubated with 100 μg / mL nanoparticles for 24 hours. After incubation, the cells are washed and separated with TrypLE solution, followed by a mixture of 2.5% glutaraldehyde in 0.1M phosphate buffer (pH 7.2) and 2% paraformaldehyde in 2% 0.1M phosphate or cacodylate buffer (pH 7.2) Fixed overnight. The cells were post-fixed for 1.5 hours in 2% osmium trioxide in 0.1M phosphate or cacodylate buffer for 1.5 hours at room temperature. It was then simply washed with deuterated deuterated H2O2 through 50, 60, 70, 80, 90, 95, and 100% ethanol (X2) series in which propylene oxide and EPON epoxy resins were mixed and permeated (Embed 812, Nadic Poly Bed 812, dodecenylsuccinic anhydride, dimethylamino methyl phenol, Electron Microscopy Polysciences, USA), and finally only embedded with epoxy resin. The epoxy resin mixed sample was put in a capsule and polymerized at 37 ° C. for 12 hours and at 60 ° C. for 48 hours. Sections for optical microscopy were cut at 500 nm and stained for 45 seconds with 1% toluidine blue on a hot plate at 80 ° C. Thin sections were made using ultramicrotome (RMC MT-XL) and collected on a copper grid. Appropriate areas for thin incisions were cut at 65 nm and stained with saturated 6% uranyl acetate and 4% lead citric acid before being examined at 80 kV with transmission electron microscopy (JEM-1400; Japan).

4-4. Intracellular Uptake Behavior of PEG-Coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ Nanoparticles

100μg / mL PEG- the shape change caused by the intracellular absorption behavior of the coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles and PEG- coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nano-particles by 11 kinds of human-derived cells The nanofluid concentration of was investigated. Successful formation of high fever using magnetic nanoparticles requires localization of the absorbed or absorbed nanoparticles within cells without side effects such as inflammation, fission or cellular transformation.

As can be seen in FIG. 7, the absorbed nanoparticles are all located in the cytoplasmic region without disturbing or affecting the nucleus or nuclear membrane. When PEG-coated nanoparticles are injected into the tumor area and exposed to AC magnetic fields, the absorbed nanoparticles can directly destroy the cytoplasmic regions or cell membranes of malignant tumor cells and induce cell death by rising temperatures during magnetic nanoparticle warming treatment. .

4-5. Mouse tumor model

Animal treatment and subcutaneous fibrosarcoma cell infusion and in vivo high temperature increase experiments were performed according to the guidelines approved by the Seoul National University Hospital Animal Testing Committee (IACUC) (IACUC No. SNU-161129-2). All procedures were performed to reduce the amount of pain caused by Balb / c nude mice. For subcutaneous mouse tumor models, anesthetize the mice used in this experiment and use FSaLL cells (radiation-induced fibrosarcoma of C3H mice from Dr. Herman Suit laboratory at Massachusetts General Hospital, using early generation cells donated from Dr. Suit). ) (50 μL of 5 × 10 ⁶ cells) was administered by subcutaneous injection into the proximal thigh site of Balb / c nude mice.

4-6. Thermal treatment of magnetic nanoparticles in vivo

Subcutaneous FSALL injection mice were randomly divided into four groups (n = 5, control, n = 5, nanoparticle therapy without alternating magnetic field, n = 5, alternating magnetic field treatment without nanoparticles, n = 5, nanoparticles with alternating magnetic field) Treatment, ie thermotherapy group) The health status of the mice was checked daily. In vivo thermotherapy started 10 days after FSaLL cell injection. Solid tumor induced mouse models were anesthetized and injected 100 μL of PEG-coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanoparticles into the center of the induced tumor. Mice were placed in the center of the AC coil system for magnetic hyperthermia. The applied magnetic field strength and frequency were 140 Oe and 99.0 kHz (140 Oe is 11.14 kA / m and the total amount of magnetic field given is 1.103 × ^{10 9} A / m · s).

An alternating magnetic field was applied for 6 minutes of heat therapy for 20 minutes each time for 2 weeks of heat therapy. Mouse tumor size and body weight were checked every two days. Volume measurements of xenograft tumors were measured by Vernier caliper and the equation is:

Tumor volume (mm ³ ) = 1/2 (length Х width ² )

4-7. Analysis of Thermal Therapy Efficacy of Magnetic Nanoparticles in Vivo

To investigate the thermal therapeutic efficacy of magnetic nanoparticles in vivo, PEG-coated Mn _0.5 Zn _0.5 Fe ₂ O ₄ nanofluids were injected in the center of subcutaneous xenograft tumors induced by FSaLL cells. The alternating magnetic field was irradiated to mice and the surface temperature during magnetic nanoparticle thermal treatment was monitored using a thermal imaging camera (FIG. 8). The skin surface temperature of the tumor at self-heating exceeded 38.3 ° C., and it can be seen that the internal temperature or the center temperature of the tumor during the high temperature treatment may be higher than the skin temperature. In addition, the temperature difference between the tumor site and the normal body temperature was analyzed by a thermal imaging camera software called ResearchIR (FLIR, Wilsonville, USA). The temperature difference between the tumor and the body temperature was 5 ° C. or more as shown in FIG. 9.

After 6 minutes of AC magnetic field exposure, a high fever temperature was obtained in the tumor and the temperature was maintained during the thermotherapy. Tumor size was measured using Vernier caliper and volumetric volume was calculated using tumor volume calculation. The tumor size of the control group and the magnetic nanoparticle-only group increased significantly after the start of treatment, and the size increased more than 15 times. However, the hyperthermia group using magnetic nanoparticles and alternating magnetic fields showed slower tumor growth and smaller tumors than other groups. As shown in Figure 10, even in the tumor volume (Tumor Volume) it can be seen that the tumor amount in the hyperthermia treatment group shows the slowest increase or the smallest size, through which the present invention has a heat treatment effect . 11 shows a captured image of a subcutaneous tumor during heat treatment according to the present invention.

Although the present invention has been described in detail above, the scope of the present invention is not limited thereto, and various modifications and changes can be made without departing from the technical spirit of the present invention described in the claims. It will be self-evident to those who have knowledge of.

Claims (9)

1. Thermal treatment composition comprising a superparamagnetic nanoparticle represented by the general formula 1 as an active ingredient:

   Formula 1

   Mn _x Zn _1-x Fe ₂ O ₄ (0 ≦ _X ≦ 1).
2. The method of claim 1,

   The superparamagnetic nanoparticles have an average particle diameter of less than 10 nm, the composition for thermal treatment.
3. The method of claim 1,

   The superparamagnetic nanoparticles have a composition for thermal treatment, characterized in that it has a magnetic induction fever at 30 to 370 kHz frequency and 80 to 160 Oe magnetic field strength.
4. The method of claim 1,

   The superparamagnetic nanoparticles are heat treatment compositions, characterized in that the coating with a biocompatible polymer.
5. The method of claim 4, wherein

   The biocompatible polymer is a composition for thermal treatment, characterized in that the polyethylene glycol.
6. The method of claim 1,

   A composition for treating heat, characterized in that for treating cancer.
7. The method of claim 6,

   A composition for treating heat, characterized in that to inhibit the growth of cancer.
8. A method of thermal treatment comprising administering the composition of any one of claims 1 to 7.
9. A method of treating cancer, comprising administering the composition of any one of claims 1 to 7.

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