Superparamagnetic iron oxide nanoparticles for MRI contrast enhancement and method for manufacturing same

Abstract

The present invention is directed to the field of medical imaging diagnosis, and relates to a magnetic resonance imaging (MRI) contrast agent composition comprising superparamagnetic iron oxide nanoparticles (SPIONs) having excellent magnetic properties, a computed tomography (CT) contrast agent composition, a method for manufacturing the superparamagnetic iron oxide nanoparticles, and superparamagnetic iron oxide nanoparticles obtained by the manufacturing method.

Description

Superparamagnetic iron oxide nanoparticles for MRI contrast imaging and method for manufacturing the same

[0001] The present invention relates to the field of medical imaging diagnostics and to a Magnetic Resonance Imaging (MRI) contrast agent composition comprising superparamagnetic iron oxide nanoparticles (SPION) having excellent magnetic properties; a Computed Tomography (CT) contrast agent composition; a method for manufacturing the superparamagnetic iron oxide nanoparticles; and superparamagnetic iron oxide nanoparticles manufactured by the method.

[0002]

[0003] In modern medicine, non-invasive imaging diagnostic technologies play an essential role in the early diagnosis of diseases and the monitoring of treatment. Magnetic Resonance Imaging (MRI), a representative imaging device, offers the advantages of excellent soft tissue contrast and no radiation exposure; however, its low sensitivity necessitates the use of contrast agents. Conventional gadolinium (Gd)-based contrast agents are primarily used for T1-weighted imaging, but they have disadvantages such as concerns regarding side effects like renal systemic fibrosis (NSF) and a short retention time in the body.

[0004] Meanwhile, Computed Tomography (CT) offers high spatial resolution and excellent imaging capabilities for bone and calcified tissues, but it has low soft tissue contrast. Consequently, there is an increasing demand for dual-mode imaging diagnostics that combines the advantages of MRI and CT.

[0005] Superparamagnetic iron oxide nanoparticles (SPIONs) have primarily been studied as T2-weighted MRI contrast agents; however, existing commercially available SPIONs (e.g., Feridex) have limitations, such as requiring high doses or causing reduced image clarity due to their low T2 relaxivity. Additionally, large particle sizes can lead to toxicity issues as they are difficult to excrete from the body.

[0006] Accordingly, the inventors have completed the present invention by developing a new nanoparticle that controls the particle size to a level suitable for renal excretion while maximizing magnetic properties through metal ion doping to enhance the MRI T2 contrast effect and simultaneously possesses a CT contrast effect.

[0007]

[0008] The present invention aims to solve the aforementioned problem and other related problems.

[0009] One exemplary objective of the present invention is to provide a magnetic resonance imaging (MRI) contrast agent composition comprising superparamagnetic iron oxide nanoparticles represented by the following chemical formula 1.

[0010] [Chemical Formula 1]

[0011] Mn X Zn 1-X Fe2O4(0.4 ≤ X ≤ 0.6).

[0012] Another exemplary object of the present invention is to provide a method for preparing the superparamagnetic iron oxide nanoparticles comprising the following steps: (a) mixing Fe(III) acetylacetonate, Mn(II) acetate tetrahydrate, and Zn(II) acetate dihydrate in a solvent; (b) heating the mixture at a heating rate of 1.0 °C / min to 1.5 °C / min to reach a first temperature range of 190 °C to 210 °C and then maintaining it to induce nucleation; and (c) heating the nucleation-induced mixture to a second temperature range of 290 °C to 310 °C and maintaining it to grow particles.

[0013] Another exemplary objective of the present invention is to provide superparamagnetic iron oxide nanoparticles produced by the above-described manufacturing method.

[0014] Another exemplary objective of the present invention is to provide a computed tomography (CT) contrast agent composition comprising superparamagnetic iron oxide nanoparticles represented by the above chemical formula 1.

[0015] The technical problems to be solved according to the technical concept of the invention disclosed in this specification are not limited to those for solving the problems mentioned above, and other unmentioned problems will be clearly understood by a person skilled in the art from the description below.

[0016]

[0017] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application should not be considered limited by the specific descriptions provided below.

[0018] As one embodiment for achieving the above objective, the present invention provides a contrast agent composition comprising superparamagnetic iron oxide nanoparticles represented by the following chemical formula 1:

[0019] [Chemical Formula 1]

[0020] Mn X Zn 1-X Fe2O4(0 ≤ X ≤ 1).

[0021] In the present invention, "superparamagnetic iron oxide nanoparticles" refers to nano-sized iron oxide particles that do not exhibit magnetism in the absence of an external magnetic field but exhibit strong magnetism when an external magnetic field is applied. Specifically, in Chemical Formula 1 above, 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, or 1.0, and preferably 0.4 to 0.6. More specifically, the superparamagnetic iron oxide nanoparticles are Mn 0.5 Zn 0.5It may be Fe2O4. Through doping with manganese and zinc ions, it has magnetic properties that are significantly improved compared to pure iron oxide (Fe3O4), with a saturation magnetization value (Ms) of about 78.6 emu / g.

[0022] In the present invention, a "contrast agent" is a substance used to enhance the contrast of structures or fluids within the body in medical imaging. The contrast agent includes, but is not limited to, magnetic resonance imaging (MRI) contrast agents and computed tomography (CT) contrast agents.

[0023] In the present invention, "MRI contrast agent" refers to a substance that improves image contrast by artificially changing the difference in the magnetic relaxation rate of hydrogen nuclei within tissues during magnetic resonance imaging (MRI).

[0024] In the present invention, the MRI contrast agent composition may exhibit a negative contrast effect in T2-weighted images during MRI imaging. Specifically, the nanoparticles of the present invention may provide a negative contrast effect that images a target area (e.g., a tumor) darker than surrounding tissue in T2-weighted images by effectively shortening the spin-spin relaxation time (T2 relaxation time) of surrounding water molecules through a high magnetic moment, but are not limited thereto.

[0025] In the present invention, the superparamagnetic iron oxide nanoparticles are 1000 mM under a 3.0 T magnetic field. - ¹s -It may have a T2 relaxation rate (r2 relaxivity) of 1 or greater. T2 relaxation rate (r2 relaxivity) is a measure of the efficiency of a contrast agent and refers to the change in the transverse relaxation velocity (1 / T2) of water molecules per contrast agent concentration (mM). The nanoparticles of the present invention are 1000 mM under a 3.0 T magnetic field. - ¹s - ¹ or higher, 1100 mM - ¹s - ¹ or more, specifically about 1193 mM - ¹s - It exhibits a T2 relaxation rate of ¹, which is significantly higher than that of existing commercial contrast agents, meaning that clear diagnosis is possible even with a small amount.

[0026] In the present invention, "CT contrast agent" refers to a substance that artificially increases the difference in X-ray absorption between tissues during computed tomography to enhance image contrast.

[0027] In the present invention, the CT contrast agent composition may exhibit a contrast enhancement effect during CT scanning, and specifically, may exhibit a contrast enhancement effect in CT images through X-ray attenuation by metal ions upon X-ray irradiation. More specifically, the nanoparticles of the present invention may exhibit a positive contrast / contrast enhancement effect that images the target area brighter than the surrounding tissue through an X-ray attenuation effect caused by the high electron density of constituent metal ions (Mn, Zn, Fe), but are not limited thereto.

[0028] In the present invention, the MRI contrast agent composition may be a dual-mode contrast agent that is simultaneously used as a Computed Tomography (CT) contrast agent. In the present invention, a dual-mode contrast agent refers to a contrast agent that is applicable to two or more imaging diagnostic devices using a single substance. The composition of the present invention simultaneously provides MRI images with a high T2 relaxation rate and CT images with high X-ray attenuation capability, thereby enabling the complementary utilization of the advantages of MRI and CT.

[0029] In the present invention, the average diameter of the nanoparticles may be 5 nm to 10 nm, and preferably may have a uniform size of about 7 nm. The average diameter refers to the physical size of the core portion of the nanoparticles and may be measured by a transmission electron microscope (TEM), but is not limited thereto.

[0030] In the present invention, the composition may be cleared from the body within 4 weeks after administration. Specifically, the clearance may be renal clearance. The size of the nanoparticles of the present invention corresponds to a size of approximately 10 nm or less, which is the threshold for renal glomerular filtration, and is the optimal size for excretion in urine through the kidneys after administration into the body. In the present invention, the clearance refers to the characteristic that the administered nanoparticles are not permanently accumulated in internal organs (liver, spleen, etc.) but are eliminated from the body through metabolic processes. In one embodiment, it was confirmed that the nanoparticles of the present invention are mostly eliminated from major organs within 4 weeks after administration, thus possessing high biosafety without concerns regarding long-term toxicity.

[0031] In the present invention, the superparamagnetic nanoparticles may have a surface coated with a biocompatible polymer. The biocompatible polymer material useful to the human body that can be used in the present invention is a polymer that is easily soluble in various solvents, and may be one or more selected from the group consisting of, for example, Poly Ethyleneglycol (PEG), poly(lactide-co-glycolide) (PLGA), Poly(DL-lactide-co-glycolide) (PDLGA), poly(hydroxybutyrate), and Polycaprolactone (PCL), and specifically may be Poly Ethyleneglycol (PEG) or Silane-PEG.

[0032] In the present invention, the composition may be used for the diagnosis of cancer tissue. Specifically, the nanoparticles of the present invention are effectively accumulated in cancer tissue upon intravenous injection or injection into a tumor, and simultaneously provide a negative contrast effect that makes the cancer tissue appear darker than surrounding normal tissue during MRI imaging, and a contrast enhancement effect that makes the cancer tissue appear brighter during CT imaging. This enables the accurate identification of the location, size, and boundaries of cancer, such as brain tumors (Glioblastoma), at high resolution.

[0033] In the present invention, the cancer may be, but is not limited to, fibrosarcoma, brain cancer, lung cancer, colorectal cancer, liver cancer, breast cancer, stomach cancer, ovarian cancer, skin cancer, pancreatic cancer, prostate cancer, kidney cancer, or thyroid cancer.

[0034] The contrast agent composition according to the present invention can be effectively accumulated in cancer tissue through a passive targeting mechanism based on the optimized physicochemical properties of nanoparticles, even without the binding of a separate target ligand.

[0035] Specifically, unlike normal blood vessels, the neovascularization of rapidly growing cancer tissue is characterized by wide gaps between endothelial cells (leaky) and an incomplete drainage system through the lymphatic system. Since the nanoparticles of the present invention have a fine size of approximately 7 nm (hydrodynamic diameter approximately 13.77 nm), they can easily penetrate (permeability) into the cancer tissue by passing through these loose blood vessel walls, and the effect of long-term retention within the tissue due to inefficient lymphatic drainage can be maximized. In addition, the surface of the nanoparticles of the present invention is coated with polyethylene glycol (PEG), a biocompatible polymer. This PEG layer inhibits the opsonization of proteins in the blood onto the particle surface, thereby allowing the nanoparticles to evade phagocytosis by the reticuloendothelial system (RES), which is part of the body's immune system. Through this, the nanoparticles can perform the function of increasing the probability of reaching the cancer tissue while circulating in the bloodstream for a long time.

[0036] In the present invention, the composition can simultaneously perform magnetic hyperthermia by generating heat under an alternating magnetic field. That is, the composition of the present invention can be used as an MRI and / or CT contrast agent to diagnose cancer, and, if necessary, can simultaneously perform the function of generating therapeutic temperatures (42°C or higher) required for cancer cell death or mild hyperthermia that induce immune activation by applying an external alternating magnetic field (AMF). This can be used as a theranostic agent capable of performing therapy and diagnosis simultaneously. This treatment method can be performed as a standalone treatment method, in conjunction with conventional treatment methods, or as an adjunct.

[0037] Specifically, in the present invention, the nanoparticles may exhibit an intrinsic loss power (ILP) of 0.5 nHm² / kg or more, specifically 0.5 nHm² / kg to 1.0 nHm² / kg, under an alternating magnetic field. The intrinsic loss power (ILP) is an indicator representing the heating efficiency of the nanoparticles and signifies the intrinsic heating performance of the nanoparticles that does not depend on the frequency (f) and strength (H) of the external magnetic field.

[0038] In one embodiment, the nanoparticles of the present invention exhibited an ILP value of about 0.82 nHm² / kg, which is about five times higher than that of conventional commercially available iron oxide nanoparticles (Fe3O4, about 0.15 nHm² / kg). This high ILP value suggests that the nanoparticles of the present invention can produce a sufficient therapeutic effect even under low frequency and magnetic field strength conditions that are safe for the human body.

[0039] The contrast agent composition of the present invention may be administered parenterally depending on the diagnostic purpose and site, preferably via intravenous injection or intratumoral injection. In particular, the nanoparticles of the present invention are manufactured in the form of a nanofluid stably dispersed in an aqueous medium, circulate throughout the body without aggregation even when administered intravascularly, and are suitable for intravenous administration as they have a size that allows for renal excretion.

[0040] When the contrast agent composition of the present invention simultaneously performs a thermotherapy function, it may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995.

[0041] In addition, the above composition may be prepared in a unit volume form by formulation using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by a person skilled in the art to which the invention belongs, or by being contained in a multi-volume container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0042]

[0043] As another embodiment for achieving the above objective, the present invention provides a method for producing the superparamagnetic iron oxide nanoparticles comprising the following steps: (a) mixing Fe(III) acetylacetonate, Mn(II) acetate tetrahydrate, and Zn(II) acetate dihydrate in a solvent; (b) heating the mixture at a heating rate of 1.0 ℃ / min to 1.5 ℃ / min to reach a first temperature range of 190℃ to 210℃ and then maintaining it to induce nucleation; and (c) heating the nucleation-induced mixture to a second temperature range of 290℃ to 310℃ and maintaining it to grow particles.

[0044] The above superparamagnetic iron oxide nanoparticles are as described above.

[0045] In the present invention, a modified high-temperature thermal decomposition (mHTTD) method with precise control of the heating rate is provided to produce superparamagnetic iron oxide nanoparticles having monodisperse size and high crystallinity.

[0046] In the present invention, step (a) is a step of preparing a reaction solution by mixing a metal precursor and a solvent.

[0047] Specifically, Fe(III) acetylacetonate, an iron (Fe) source, Mn(II) acetate tetrahydrate, a manganese (Mn) source, and Zn(II) acetate dihydrate, a zinc (Zn) source, can be mixed in a solvent according to a specific doping ratio (e.g., Mn:Zn:Fe = 0.5:0.5:2 molar ratio).

[0048] As the solvent for step (a) above, an organic solvent capable of high-temperature reaction with a high boiling point may be used, and preferably, benzyl ether may be used. Benzyl ether has a high boiling point of about 298°C, which can provide a reaction environment suitable for high-temperature synthesis around 300°C where crystallization of particles occurs.

[0049] In step (a) above, surfactants and reducing agents such as oleic acid, oleylamine, and 1,2-hexadecanediol may be additionally mixed to increase the dispersion stability of the particles and control their size.

[0050] In the present invention, step (b) is a step of heating the reaction mixture to induce nucleation of particles.

[0051] In step (b) above, the mixture is heated from room temperature at a very slow rate of 1.0°C / min to 1.5°C / min, preferably at a heating rate of about 1.2°C / min, to reach a first temperature range of 190°C to 210°C, preferably about 200°C. This is distinguished from the conventional rapid heating method (about 10°C / min or more), and the particle size distribution can be improved by gradually decomposing the precursor without rapid thermal shock and inducing nucleation at a uniform time.

[0052] After reaching the first temperature range, the temperature may be maintained for 30 to 90 minutes, specifically for 40 to 80 minutes, and more specifically for 50 to 70 minutes, preferably for 60 minutes. During this maintenance time, sufficient and uniform nucleation is completed, which allows the final particles to have a fine and uniform size of 7 nm.

[0053] In the present invention, step (c) is a particle growth step, which is a step of inducing crystal growth by further increasing the temperature after nucleation is completed.

[0054] In step (c) above, the mixture is heated to a second temperature range of 290°C to 310°C, preferably about 300°C. At this time, the heating rate may be 8°C / min to 12°C / min, specifically about 10°C / min, but is not limited thereto.

[0055] After reaching the second temperature range, the temperature may be maintained for 20 to 80 minutes, specifically for 30 to 70 minutes, and more specifically for 40 to 60 minutes. During this process, the spinel structure of the nanoparticles is completed and magnetic properties can be enhanced. Additionally, by controlling the maintenance time of this step, excessive growth of the particles can be prevented, and a size (about 7 nm) optimized for renal excretion can be obtained.

[0056] Since the surface of nanoparticles immediately after synthesis is hydrophobic, a surface modification process can be performed to convert them to hydrophobic for in vivo application.

[0057] Specifically, in the present invention, the manufacturing method may further include a step of coating the nanoparticles synthesized after step (c) with oleic acid (first coating step). In this way, by coating the particle surface with a uniform hydrophobic ligand, the efficiency of the subsequent silane substitution reaction can be increased.

[0058] In addition, in the present invention, the manufacturing method may further include a step of reacting the coated nanoparticles with methoxy-polyethylene glycol-silane (methoxy-PEG-silane) after the coating step to convert them into a water-soluble form (second coating step). At this time, the silane group can form a strong covalent bond with the hydroxyl group (-OH) on the surface of the iron oxide to fix the PEG chain to the particle surface, and the introduced PEG layer (preferably MW 500 to 1000) prevents aggregation between particles to ensure dispersion stability in an aqueous solution, and can impart stealth and long-circulation characteristics by inhibiting plasma protein adsorption when administered into the body.

[0059]

[0060] In another embodiment for achieving the above objective, the present invention provides superparamagnetic iron oxide nanoparticles produced by the above manufacturing method.

[0061] The above superparamagnetic iron oxide nanoparticles are as described above.

[0062] The nanoparticles of the present invention obtained by the above manufacturing method have an average diameter of 5 nm to 10 nm, specifically about 5.5 nm to 8.5 nm, and are very fine and uniform, so that when administered into the body, they can be excreted through the kidneys (renal clearance).

[0063] In addition, the nanoparticles obtained by the above manufacturing method are 1000 mM under a 3.0 T magnetic field - ¹s - ¹ or more, specifically about 1193 mM - ¹s - It has the characteristic of enabling high-resolution MRI imaging by exhibiting a T2 relaxation rate of ¹.

[0064] In addition, the nanoparticles obtained by the above manufacturing method exhibit an intrinsic loss power (ILP) of 0.5 nHm² / kg or more, specifically 0.5 nHm² / kg to 1.0 nHm² / kg, under an alternating magnetic field, thereby demonstrating effective thermotherapy performance.

[0065]

[0066] In another embodiment for achieving the above objective, the present invention provides a use for superparamagnetic iron oxide nanoparticles represented by the following chemical formula 1 to be used as a magnetic resonance imaging (MRI) contrast agent.

[0067] [Chemical Formula 1]

[0068] Mn X Zn 1-X Fe2O4(0 ≤ X ≤ 1).

[0069] In another embodiment for achieving the above objective, the present invention provides a use for superparamagnetic iron oxide nanoparticles represented by the above chemical formula 1 to be used as a computed tomography (CT) contrast agent.

[0070] As another embodiment for achieving the above objective, the present invention provides a magnetic resonance imaging (MRI) method using superparamagnetic iron oxide nanoparticles represented by the above chemical formula 1.

[0071] As another embodiment for achieving the above objective, the present invention provides a computed tomography (CT) method using superparamagnetic iron oxide nanoparticles represented by the above chemical formula 1.

[0072]

[0073] Mn according to the present invention 0.5 Zn 0.5Fe2O4 nanoparticles (MnZn-SPION-7) exhibit a significantly higher T2 relaxation rate compared to commercial contrast agents, resulting in excellent MRI contrast enhancement. They also provide contrast enhancement effects during CT scanning, making it possible to perform both MRI and CT diagnoses simultaneously with a single drug. Furthermore, they have excellent in vivo dispersibility due to their uniform size of approximately 7 nm and PEG coating. They are also safe as they are non-cytotoxic and excreted from the body after a certain period of time.

[0074]

[0075] FIG. 1 shows the synthesis results of superparamagnetic iron oxide nanoparticles (MnZn-SPION-7) according to Example 1 of the present invention, comprising (A) a graph of average particle size according to doping ratio, and (B) optimized Mn 0.5 Zn 0.5 This is a transmission electron microscope (TEM) image of Fe2O4 nanoparticles and a control (Fe3O4).

[0076] FIG. 2 illustrates the nanoparticle manufacturing process and composition optimization process of the present invention, FIG. 2a shows the device configuration and temperature profile (heating rate control) of the modified high-temperature pyrolysis method (mHTTD), FIG. 2b shows the energy-dispersive X-ray spectroscopy (EDS) spectrum, and FIG. 2c to 2n are a collection of TEM images comparing changes in particle size and distribution according to changes in the doping ratio of Mn and Zn (X = 0 to 1).

[0077] Figure 3 is a graph showing the results of vibrating sample magnetometer (VSM) measurements evaluating the magnetic properties of the nanoparticles of the present invention, including (A) the saturation magnetization curve (Major Loop) under a magnetic field of 15 kOe, (B) the magnetic hysteresis curve (Minor Loop) in the low magnetic field region, and (C) the dynamic magnetization curve under an alternating magnetic field.

[0078] Figure 4 shows the energy loss characteristics of the nanoparticles of the present invention under an alternating magnetic field (AMF), (A) a graph of the temperature of heat over time in the powder state, and (B) a graph comparing the intrinsic loss power (ILP) and specific absorption rate (SAR) in the powder state and (C) the solution state with a control group.

[0079] Figure 5 shows the surface modification and dispersion characteristics of the nanoparticles of the present invention in an aqueous solution, including (A) a schematic diagram of the PEGylation process, (B) FT-IR spectra before and after surface modification, (C) hydrodynamic diameter and distribution map using dynamic light scattering (DLS), (D) a photograph of magnetic reactivity in an aqueous solution, and (E) a graph of the exothermic characteristics of the alternating magnetic field reaction in an aqueous solution.

[0080] Figure 6 shows the results of evaluating the MRI contrast performance of the nanoparticles of the present invention (PEG-MnZn-SPION-7), including T2-weighted phantom images at various concentrations and a graph of the corresponding T2 relaxation rate (r2 relaxivity) measurements (compared to the control group PEG-Fe3O4-SPION).

[0081] Figure 7 shows the in vivo MRI contrast efficacy using a mouse model of a brain tumor (Glioblastoma), and is a T2-weighted image showing a clear negative contrast effect of the tumor area in a 9.4 T MRI after injection of the nanoparticles of the present invention.

[0082] Figure 8 shows the efficacy of in vivo MRI and CT contrast imaging using a subcutaneous tumor mouse model, with (A) 3.0 T MRI contrast images in the subcutaneous tumor model and (B) computed tomography (CT) contrast images in the same model over time (before injection, 10 minutes, 60 minutes, 4 hours, etc.).

[0083] Figure 9 is a TEM image showing the results of evaluating the cellular-level safety and uptake of the nanoparticles of the present invention, (A) a graph of CCK-8 cytotoxicity evaluation on various cell lines, (B) cancer cells, and (C) the uptake (internalization) of nanoparticles into normal cells.

[0084] Figure 10 shows the results of evaluating the in vivo distribution and biosafety of the nanoparticles of the present invention, including (A) H&E stained tissue images of major organs (liver, kidney, spleen, etc.), (B) images confirming the distribution of iron particles through Prussian blue staining, (C) H&E stained images of heart, muscle, brain, and eye tissues, and (D) Prussian blue stained images of heart, muscle, brain, and eye tissues over time (2 hours to 4 weeks).

[0085]

[0086] The structure and effects of the present invention will be explained in more detail below through examples. These examples are intended solely to illustrate the present invention, and the scope of the present invention is not limited by them.

[0087]

[0088] Example 1: Synthesis and Optimization of Superparamagnetic Iron Oxide Nanoparticles (MnZn-SPION-7)

[0089] The nanoparticles according to the present invention are superparamagnetic iron oxide nanoparticles doped with manganese (Mn) and zinc (Zn), and were synthesized using modified high-temperature thermal decomposition (mHTTD). Specifically, to confirm the change in properties according to the doping ratio of Mn and Zn, Mn x Zn {1-x} Particles were synthesized with various compositions of Fe2O4 (X=0~1). As a result, Mn 0.5 Zn 0.5It was confirmed that the Fe2O4 composition simultaneously satisfied the best magnetic properties (saturation magnetization value and heating efficiency) and a uniform micro-size optimized for renal excretion, and this was determined as the optimal ratio (Fig. 1A, Fig. 2).

[0090] The modified high-temperature thermal decomposition (mHTTD), which is the optimal synthesis process, is as follows. Fe(III) acetylacetonate (2 mmol), Mn(II) acetate tetrahydrate (0.5 mmol), Zn(II) acetate dihydrate (0.5 mmol), oleic acid (6 mmol), oleylamine (6 mmol), and 1,2-hexadecanediol (10 mmol) were mixed in benzyl ether (20 mL) and stirred in a three-necked flask. The reaction solution was slowly heated from room temperature to approximately 200°C at a rate of 1.2°C / min and maintained for 60 minutes to induce uniform nucleation. Subsequently, particle growth was induced by increasing the temperature to approximately 300°C (approx. 10°C / min) and maintaining for 50 minutes. The synthesized particles were purified by precipitating with ethanol, centrifuging, and redispersing in hexane.

[0091] Transmission electron microscopy (TEM) analysis results of the synthesized Mn 0.5 Zn 0.5 Fe2O4SPION (hereinafter MnZn-SPION-7) was a uniform spherical particle with an average diameter of 7.0 ± 1.5 nm (Fig. 1B), which has a size similar to that of conventional Fe3O4SPION (6.5 ± 0.7 nm) and possesses micro-size characteristics favorable for renal clearance.

[0092]

[0093] Example 2: Evaluation of Magnetic Properties and Magnetic Reactivity

[0094] The magnetic properties of the nanoparticles according to the present invention were evaluated using a vibrating sample magnetometer (VSM; Toei Kogyo, VSM-5) according to the method disclosed in Lee et al. (Theranostics 15(7), 2883-2902, 2025). As a result, MnZn-SPION-7 exhibited superparamagnetic behavior with almost no coercivity at room temperature, and the saturation magnetization value under a magnetic field of 15 kOe was measured to be approximately 78.6 emu / g (Fig. 3A, B, C). This value is significantly higher than that of the control group, Fe3O4-SPION, at 65.3 emu / g, suggesting that Mn and Zn ions substituted Fe ions within the spinel structure and enhanced the magnetic moment.

[0095] In addition, to confirm the magnetic reactivity of the particles, the energy loss (heat generation) characteristics under an alternating magnetic field (AMF, 100 kHz, 140 Oe) were evaluated according to the method disclosed in Lee et al. (Theranostics 15(7), 2883-2902, 2025). As a result, MnZn-SPION-7 was found to have superior magnetic reactivity, exhibiting an intrinsic loss power (ILP) of 0.82 nHm² / kg, which is about 5 times higher than that of Fe3O4-SPION (0.15 nHm² / kg) (Fig. 4A, B, C). This high magnetic sensitivity serves as the physical basis for maximizing the T2 signal attenuation effect as an MRI contrast agent.

[0096]

[0097] Example 3: Surface Modification (PEGylation) and Dispersibility of Nanoparticles

[0098] To ensure in vivo stability and dispersibility and to increase utility as a contrast agent, the surface of the synthesized MnZn-SPION-7 was coated with the biocompatible polymer methoxy-PEG-silane (molecular weight 500 Da) (Fig. 5A).

[0099] First, the synthesized nanoparticles were reacted with oleic acid and NH4Cl in an ethanol solution to replace the surface with oleic acid. The mixture was stirred for 2 hours and then washed with acetone. Afterward, the nanoparticles were precipitated using a permanent magnet to obtain nanoparticles coated with oleic acid. Next, the coated nanoparticles were dispersed in toluene (7.5 mL), followed by the addition of 3.75 mL of triethylamine and 0.75 mL of methoxy-PEG-silane 500 Da, and the mixture was stirred for 24 hours. After the reaction, the nanoparticles were washed with pentane and redispersed in distilled water to prepare PEGylated MnZn-SPION-7 (hereinafter PEG-MnZn-SPION-7) in the form of a water-soluble nanofluidic solution.

[0100] Subsequently, characterization of PEG-MnZn-SPION-7 confirmed that PEG molecules were successfully bound to the surface of the nanoparticles through Fourier Transform Infrared (FT-IR) analysis (Fig. 5B). Dynamic Light Scattering (DLS) analysis revealed a hydrodynamic diameter of approximately 13.77 nm and a Polydispersity Index (PDI) of 0.26, indicating excellent monodispersity in an aqueous solution (Fig. 5C). Furthermore, the particles reacted immediately to a magnet even in an aqueous solution (10 mg / mL) (Fig. 5D), and showed a temperature increase of approximately 19.6°C under an alternating magnetic field, confirming that they maintain excellent magnetic reactivity even in a liquid state (Fig. 5E). This implies that the particles of the present invention can exhibit sufficient MRI contrast performance even when injected into the body in the form of an injectable.

[0101]

[0102] Example 4: In Vitro MRI Contrast Performance Evaluation (T2 Relaxation Rate)

[0103] To quantitatively confirm the performance of PEG-MnZn-SPION-7 of the present invention as an MRI contrast agent, T2 relaxivity (r2) was measured using a 3.0 T MRI system (SIMENS, Berlin, Germany) according to the method disclosed in Lee et al. (Theranostics 15(7), 2883-2902, 2025). Eight nanoparticle solutions of different concentrations (0.001, 0.003, 0.006, 0.013, 0.025, 0.05, 0.1, and 0.2 mmol) were prepared, T2-weighted images were acquired, and the relaxivity was calculated.

[0104] As a result of the experiment, the T2 relaxation rate (r2) of PEG-MnZn-SPION-7 was 1193 mM - ¹s - It was measured as ¹ (Fig. 6). This is 603 mM, which is the r2 value of the control PEG-Fe3O4-SPION measured under the same conditions. - ¹s - This value is approximately twice as high as ¹, and is comparable to the r2 values ​​of other previously reported commercial SPION contrast agents (typically 100–400 mM). - ¹s - This is a remarkably high value even when compared to ¹). This demonstrates that MnZn-SPION-7, despite its small size (7 nm), has a high saturation magnetization value due to Mn / Zn doping, which effectively shortens the T2 relaxation time of surrounding water molecules and provides a very sharp negative contrast effect.

[0105]

[0106] Example 5: Evaluation of In Vivo MRI and CT Dual-Mode Contrast Efficacy

[0107] The in vivo MRI and CT contrast performance was evaluated using animal models.

[0108] 5-1. MRI Contrast in Brain Tumor Models

[0109] PEG-MnZn-SPION-7 was injected into the tumor site of an orthotopic xenograft mouse model of brain tumor transplanted with U87MG cells, and imaging was performed using a 9.4 T MRI system (Agilent Technology Inc, Santa Clara, USA) according to the method disclosed in Lee et al. (Theranostics 15(7), 2883-2902, 2025). After injection, a strong negative contrast effect was observed in the T2 and T2-weighted images, in which the tumor site appeared clearly dark, allowing for clear identification of the tumor boundaries and location (Fig. 7).

[0110] 5-2. MRI and CT Contrast Imaging in Subcutaneous Tumor Models (Dual Mode)

[0111] Using a model in which fibrosarcoma (FSaII) was implanted subcutaneously in nude mice, PEG-MnZn-SPION-7 was injected intratumorally or intravenously, and then 3.0 T MRI and CT scans were performed according to the method disclosed in Lee et al. (Theranostics 15(7), 2883-2902, 2025).

[0112] When analyzing images taken at 10 minutes, 1 hour, and 4 hours after nanoparticle injection during MRI (3.0 T) imaging, a distinct reduction in T2 signal (turning dark) was confirmed in the tumor area where the nanoparticles were located, demonstrating an excellent MRI contrast effect (Fig. 8A).

[0113] As a result of performing CT (Computed Tomography) imaging on the same model, a distinct contrast enhancement effect due to X-ray attenuation was observed in the tumor site injected with nanoparticles (Fig. 8B).

[0114] These results demonstrate that the nanoparticles of the present invention are imaging diagnostic agents with dual-mode capabilities, capable of simultaneously obtaining soft tissue contrast of MRI and high-resolution images of CT with a single drug injection. Of particular note is that the nanoparticles of the present invention effectively accumulated in tumor tissue after intravenous injection and exhibited a clear contrast effect, even though they were not bound to a separate target ligand such as an antibody. This may be because the optimized micro-size (approx. 7 nm) and PEG surface modification of the nanoparticles of the present invention extended the residence time in the blood and maximized the passive targeting effect, allowing the particles to penetrate through the loose tumor blood vessel walls. Therefore, the composition of the present invention has the advantage of being able to precisely target and diagnose cancer tissue solely through the control of physicochemical properties, without the need for a complex target substance conjugation process.

[0115]

[0116] Example 6: Evaluation of Biosafety and In vivo Distribution

[0117] 6-1. Evaluation of Cytotoxicity and Uptake

[0118] CCK-8 analysis was performed on various glioblastoma cell lines (9 types including U87 and T98G) and normal cerebral cortical cells (NSC09, NSC10) according to the method disclosed in Lee et al. (Theranostics 15(7), 2883-2902, 2025). As a result, no significant change in cell viability was observed even when PEG-MnZn-SPION-7 was treated at a high concentration of 500 μg / mL, confirming that the cytotoxicity was very low (Fig. 9A). In addition, TEM observation of internalization confirmed that the nanoparticles were successfully incorporated into the cells without causing morphological abnormalities or damage (Fig. 9B, C).

[0119] 6-2. Tissue Toxicity and Biodistribution

[0120] After intravenously injecting nanoparticles into mice, major organs (liver, kidney, spleen, lung, heart, brain, etc.) were excised and H&E staining and Prussian blue staining were performed. Histological analysis revealed no tissue damage in the major organs caused by the nanoparticle injection. The nanoparticles were primarily observed in the liver and spleen during the initial period of injection, but it was confirmed that most of them were cleared from the body after 4 weeks (Fig. 10). This suggests that the particles of the present invention are safe and have a low risk of long-term toxicity due to accumulation in vivo, as they have a fine size of approximately 7 nm.

[0121]

[0122] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

1. Magnetic Resonance Imaging (MRI) contrast agent composition comprising superparamagnetic iron oxide nanoparticles represented by the following chemical formula 1: [Chemical Formula 1] Mn X Zn 1-X Fe2O4(0.4 ≤ X ≤ 0.6).

2. In Paragraph 1, The above nanoparticles are a contrast agent composition having an average diameter of 5 nm to 10 nm.

3. In Paragraph 1, The above nanoparticles are contrast agent compositions in which the surface is coated with polyethylene glycol (PEG) or silane-polyethylene glycol (Silane-PEG).

4. In Paragraph 1, The above nanoparticles are 1000 mM under a 3.0 T magnetic field - ¹s - A contrast agent composition having a T2 relaxation rate (r2 relaxivity) of ¹ or more.

5. In Paragraph 1, The above composition is a contrast agent composition that is cleared from the body within 4 weeks after administration.

6. In Paragraph 1, The above composition is a contrast agent composition used for the diagnosis of cancer tissue.

7. In Paragraph 1, The above composition is a contrast agent composition that simultaneously performs magnetic hyperthermia by generating heat under an alternating magnetic field.

8. In Paragraph 7, A contrast agent composition in which the above nanoparticles exhibit an intrinsic loss power (ILP) of 0.5 nHm² / kg or more under alternating magnetic field conditions.

9. A method for manufacturing superparamagnetic iron oxide nanoparticles of claim 1, comprising the following steps: (a) a step of mixing Fe(III) acetylacetonate, Mn(II) acetate tetrahydrate and Zn(II) acetate dihydrate in a solvent; (b) a step of heating the above-mentioned mixed mixture at a heating rate of 1.0 ℃ / min to 1.5 ℃ / min to reach a first temperature range of 190℃ to 210℃ and then maintaining it to induce nucleation; and (c) A step of growing particles by heating the mixture in which nucleation is induced and maintaining it in a second temperature range of 290°C to 310°C.

10. In Paragraph 9, A method of preparation in which the solvent of step (a) above is benzyl ether.

11. In Paragraph 9, A manufacturing method in which the maintenance of step (b) above is performed for 50 to 70 minutes.

12. In Paragraph 9, A manufacturing method in which the maintenance of step (c) above is performed for 40 to 60 minutes.

13. In Paragraph 9, A manufacturing method comprising the step of coating the synthesized nanoparticles with oleic acid after step (c) above.

14. In Paragraph 13, A manufacturing method comprising, after the above coating step, an additional step of reacting the coated nanoparticles with methoxy-polyethylene glycol-silane (Methoxy-PEG-silane) to convert them into a water-soluble form.

15. Computed Tomography (CT) contrast agent composition comprising superparamagnetic iron oxide nanoparticles represented by the following chemical formula 1: [Chemical Formula 1] Mn X Zn 1-X Fe2O4(0.4 ≤ X ≤ 0.6).

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