Ultra-small magnetic iron oxide nanoparticles taking small molecules as stabilizer as well as preparation method and application of ultra-small magnetic iron oxide nanoparticles

Abstract

The invention discloses ultra-small magnetic iron oxide nanoparticles taking small molecules as a stabilizer as well as a preparation method and application of the ultra-small magnetic iron oxide nanoparticles, and belongs to the technical field of nano materials. The magnetic iron oxide nanoparticles provided by the invention comprise magnetic iron oxide particles and hydrophilic small molecules connected to the surfaces of the magnetic iron oxide particles, the average molecular weight of the hydrophilic small molecules is less than 1000 Daltons. Hydrophilic small molecules are connected to the surfaces of the magnetic iron oxide particles, good stabilizing and dispersing effects can be achieved, and compared with existing magnetic iron oxide nanoparticles with macromolecules as a stabilizer, the magnetic iron oxide nanoparticles are lower in viscosity, higher in biocompatibility, higher in longitudinal relaxation r1 value and better in stability. The clinical practicability and the development prospect are better, and the preparation of a safe and reliable MRI contrast agent, especially a T1 weighted contrast agent, is facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials, and particularly relates to ultrasmall magnetic iron oxide nanoparticles stabilized by small molecules, a preparation method thereof, and an application thereof. Background Art

[0002] Magnetic resonance imaging (MRI) has received considerable attention in the past two decades as a clinical diagnostic technique. Although significant progress has been made in the diagnosis of major diseases by MRI, the ability to distinguish diseased tissues from healthy tissues still needs to be improved. Especially in the early stages of the development of many diseases, the resolution of current MRI technology is still unable to achieve accurate diagnosis of lesion tissues. To improve imaging sensitivity, contrast agents are used to accelerate the relaxation rate of water molecules, thereby increasing the contrast between specific tissues or specific organs.

[0003] There are mainly two types of contrast agents clinically used for MRI. One is a T1-weighted contrast agent based on gadolinium chelate, which presents a bright signal in the image; the other is a T2-weighted contrast agent based on magnetic iron oxide nanoparticles, which presents a dark signal in the image. For T1 contrast agents, the higher the r1 value, the better, and the lower the r2 / r1 ratio, the better; for T2 contrast agents, the higher the r2 value, the better, and the higher the r2 / r1 ratio, the better.

[0004] T2 contrast agents based on magnetic iron oxide nanoparticles have withdrawn from the market due to the following numerous defects. 1) T2 contrast agents generate dark signals, and the human eye is not sensitive to the intensity changes of dark signals, which is not conducive to clinical observation and is easily confused with dark signals caused by other pathogenic reasons, such as bleeding, calcification, metal deposition, etc. 2) The high magnetic moment of T2 contrast agents will cause magnetic susceptibility artifacts, destroying the background around the lesion and resulting in blurred images, causing local distortion of MRI images. 3) The particle size of T2 contrast agents is relatively large (16 - 180 nm), and the clearance rate in the body is relatively slow, with a long blood circulation time, and patients need to wait for a long time after administration to perform MRI. 4) Compared with T1-weighted MRI imaging, T2-weighted MRI imaging requires much longer processing time and more MRI machine time, which is not conducive to clinical application. 5) The liver specificity of T2 contrast agents based on magnetic iron oxide nanoparticles is a major advantage. The T1 contrast agent Primovist (gadoxetic acid disodium) was launched in 2008 and also has liver specificity. The emergence of Primovist has accelerated the elimination of T2-weighted contrast agents.

[0005] Due to the withdrawal of T2 contrast agents based on magnetic iron oxide nanoparticles from the market, currently, T1 contrast agents based on gadolinium chelate almost occupy the entire MRI contrast agent market. Including Magnevist Gadoteridol Dotarem Omniscan Gadobenate dimeglumine etc.

[0006] However, gadolinium chelate-based T1 contrast agents can cause nephrotoxicity, which can lead to nephrogenic fibrosis in some patients, and there is also brain deposition.

[0007] As an indispensable element in the human body, iron has far better biosafety than gadolinium. Ultrasmall superparamagnetic iron oxide nanoparticles with a particle size less than 5 nm can be used as T1-weighted MRI contrast agents, and their biosafety is better than that of gadolinium chelates. A particle size less than 5 nm and good dispersibility in the aqueous phase are necessary conditions for ultrasmall superparamagnetic iron oxide nanoparticles to be used as T1-weighted MRI contrast agents. However, ultrasmall superparamagnetic iron oxide nanoparticles are prone to aggregation in the aqueous phase, resulting in an increase in particle size and even precipitation, which will cause a sharp increase in the r2 value of the nanoparticles and is not conducive to T1 imaging. This requires a stabilizer to maintain the dispersibility of ultrasmall superparamagnetic iron oxide nanoparticles. Therefore, the properties of the stabilizer are the key to affecting the T1-weighted MRI effect of ultrasmall superparamagnetic iron oxide nanoparticles.

[0008] During the reaction, hydrophilic stabilizers coordinate with iron ions in the aqueous phase to form coordination compounds, and then an alkali is added to react with the coordination product to generate ultrasmall superparamagnetic iron oxide nanoparticles. To ensure good stability, the stabilizers of ultrasmall superparamagnetic iron oxide nanoparticles reported in the literature are all hydrophilic macromolecules. However, macromolecules will crosslink and entangle at high concentrations, resulting in a significant increase in viscosity and making intravenous injection impossible. Due to the high viscosity, the intravenous administration concentration of ultrasmall superparamagnetic iron oxide nanoparticle preparations stabilized by macromolecules is severely limited, and only by reducing the concentration and increasing the volume of the preparation can the dosage requirements for imaging be met. Therefore, intravenous bolus injection cannot be used clinically, and the drug can only be administered by intravenous drip. The intravenous drip administration method greatly increases the preparation time before MRI scanning, which is extremely inconvenient for both doctors and patients, thus limiting the clinical practicability of ultrasmall superparamagnetic iron oxide-based nanoparticles stabilized by macromolecules. Summary of the Invention

[0009] In order to overcome at least one of the above-mentioned problems existing in the prior art, one of the purposes of the present invention is to provide ultrasmall superparamagnetic iron oxide nanoparticles stabilized by small molecules, which have a high r1 value, a low r2 / r1 ratio, good water solubility, high stability, can maintain low viscosity at high concentrations, and have excellent biocompatibility.

[0010] The second purpose of the present invention is to provide a preparation method of the above-mentioned ultrasmall superparamagnetic iron oxide nanoparticles.

[0011] The third purpose of the present invention is to provide an application of the above-mentioned ultrasmall superparamagnetic iron oxide nanoparticles.

[0012] In order to achieve the above purposes, the technical solutions adopted by the present invention are as follows:

[0013] A first aspect of the present invention provides a magnetic iron oxide nanoparticle, comprising magnetic iron oxide particles and hydrophilic small molecules attached to the surface of the magnetic iron oxide particles; the average molecular weight of the hydrophilic small molecules is less than 1000 Daltons (Da).

[0014] In some specific embodiments of the present invention, the average molecular weight of the hydrophilic small molecules is greater than or equal to 80 Daltons and less than 1000 Daltons; for example, it can be any value among 80 Daltons, 100 Daltons, 150 Daltons, 200 Daltons, 250 Daltons, 300 Daltons, 500 Daltons, 800 Daltons or 950 Daltons or a range value between any two of them.

[0015] Preferably, the hydrophilic small molecules are selected from carboxylic acid-containing small molecules; more preferably, the number of carboxyl groups in the carboxylic acid-containing small molecules is ≥2; even more preferably, it is 2 - 10. In some embodiments of the present invention, the number of carboxyl groups in the carboxylic acid-containing small molecules is 2 - 4.

[0016] Preferably, the carboxylic acid-containing small molecules include at least one of citric acid (CA), malic acid (MA), tartaric acid (TA), aspartic acid (Asp), glutamic acid (Glu), glucaric acid (OAA), fumaric acid (FUM), maleic acid (MAE), malonic acid (Succ), tricarboxylic acid (Cit), butanetetracarboxylic acid (TCA) or ethylenediaminetetraacetic acid (EDTA); more preferably, the carboxylic acid-containing small molecules include one of citric acid (CA), malic acid (MA), tartaric acid (TA), aspartic acid (Asp), glutamic acid (Glu), glucaric acid (OAA), fumaric acid (FUM), maleic acid (MAE), malonic acid (Succ), tricarboxylic acid (Cit), butanetetracarboxylic acid (TCA) or ethylenediaminetetraacetic acid (EDTA).

[0017] Preferably, the magnetic iron oxide particles contain magnetite, hematite or a combination thereof; more preferably, the magnetic iron oxide particles contain magnetite. In some embodiments of the present invention, the magnetic iron oxide particles are selected from magnetite particles.

[0018] Preferably, the average particle size of the magnetic iron oxide nanoparticles is 1 - 10 nm; more preferably, it is 1.5 - 8 nm; even more preferably, it is 2 - 5 nm.

[0019] Preferably, the electrokinetic potential (Zeta potential) of the magnetic iron oxide nanoparticles is <0 mV; more preferably, it is -50 to -1 mV; even more preferably, it is -30 to -10 mV.

[0020] Preferably, the longitudinal relaxation rate r1 value of the magnetic iron oxide nanoparticles is ≥ 2 mM at a magnetic field strength of 3 T -1 s -1 ; more preferably, it is 2.5 - 10 mM -1 s -1 ; even more preferably, it is 3 - 8 mM -1 s -1 。

[0021] Preferably, the ratio r2 / r1 of the transverse relaxation rate r2 value to the longitudinal relaxation rate r1 value of the magnetic iron oxide nanoparticles is ≤ 12 at a magnetic field strength of 3 T; more preferably, it is 5 - 10; even more preferably, it is 6 - 9.

[0022] Preferably, the viscosity of the magnetic iron oxide nanoparticles is less than 5 CP at 25 ± 2 °C under the condition that the Fe concentration is greater than or equal to 100 mM; more preferably, it is 0.5 - 3 CP; even more preferably, it is 0.5 - 2 CP.

[0023] In some embodiments of the present invention, the viscosity of the magnetic iron oxide nanoparticles is less than 5 CP at 25 ± 2 °C under the condition that the Fe concentration is equal to 100 mM; specifically, it is 0.5 - 3 CP; more specifically, it is 0.5 - 2 CP.

[0024] The second aspect of the present invention provides a method for preparing the magnetic iron oxide nanoparticles as described in the first aspect of the present invention, comprising the following steps: making a mixed solution containing a hydrophilic small molecule, an iron source, and a solvent alkaline, and performing a coprecipitation reaction to obtain the magnetic iron oxide nanoparticles.

[0025] In the preparation method of the present invention, through the coprecipitation reaction, the iron source reacts to form magnetic iron oxide particles, and the hydrophilic small molecule is connected to the surface of the magnetic iron oxide particles, thereby obtaining the magnetic iron oxide nanoparticles.

[0026] Preferably, the iron source includes a trivalent iron source, or a trivalent iron source and a divalent iron source; more preferably, the iron source includes a trivalent iron source and a divalent iron source.

[0027] In some embodiments of the present invention, the divalent iron source is a water-soluble divalent iron salt; in some specific embodiments of the present invention, the water-soluble divalent iron salt includes at least one of ferrous chloride, ferrous nitrate, ferrous bromide, or ferrous sulfate; preferably, it is ferrous chloride.

[0028] In some embodiments of the present invention, the trivalent iron source is a water-soluble trivalent iron salt; in some specific embodiments of the present invention, the water-soluble trivalent iron salt includes at least one of ferric chloride, ferric nitrate, ferric bromide, or ferric sulfate; preferably, it is ferric chloride.

[0029] In some embodiments of the present invention, the solvent is selected from water.

[0030] In some embodiments of the present invention, the hydrophilic small molecule and the iron source are respectively mixed with the solvent first to obtain a hydrophilic small molecule solution and an iron source solution, and then the hydrophilic small molecule solution and the iron source solution are mixed to obtain a mixed solution.

[0031] In some embodiments of the present invention, the concentration of the hydrophilic small molecule solution is 8.0 - 150 mg / mL; specifically 16 - 128 mg / mL; for example, it can be any value among 16 mg / mL, 32 mg / mL, 64 mg / mL, 80 mg / mL, 100 mg / mL or 128 mg / mL or the range value between any two of them.

[0032] In some embodiments of the present invention, the iron source solution contains ferric ions; the concentration of ferric ions in the iron source solution is 100 - 1000 mM; specifically 200 - 750 mM; more specifically 350 - 500 mM. The ferric ions are introduced by a ferric source.

[0033] In some embodiments of the present invention, the iron source solution contains ferrous ions; the concentration of ferrous ions in the iron source solution is 50 - 500 mM; specifically 100 - 375 mM; more specifically 200 - 250 mM. The ferrous ions are introduced by a ferrous source.

[0034] In some embodiments of the present invention, the volume ratio of the hydrophilic small molecule solution to the iron source solution is (1 - 500):1; specifically (10 - 50):1.

[0035] Preferably, the pH value of the coprecipitation reaction is 8 - 10; for example, it can be 8, 8.5, 9, 9.5 or 10.

[0036] In the present invention, under alkaline conditions, especially when the pH value is 8 - 10, the coprecipitation reaction can cause the iron source to react to form magnetic iron oxide particles, and then the magnetic iron oxide nanoparticles are obtained.

[0037] In some embodiments of the present invention, the pH value of the coprecipitation reaction is adjusted by adding an alkaline reagent.

[0038] In some embodiments of the present invention, the alkaline reagent includes at least one of sodium hydroxide, aqueous sodium hydroxide solution, potassium hydroxide, aqueous potassium hydroxide solution or ammonia water; in some specific embodiments of the present invention, the alkaline reagent is selected from ammonia water. Specifically, the mass concentration of ammonia water is 1 - 20%; more specifically 1 - 8%; for example, it can be any value among 1%, 2%, 4%, 6% or 8% or the range value between any two of them.

[0039] In some embodiments of the present invention, the pH value of the basic reagent is 10 to 12; specifically 11 to 11.5.

[0040] In some embodiments of the present invention, the pH value of the mixed solution obtained by mixing the hydrophilic small molecule, the iron source and the solvent is 6.5 to 7.5, and after adding the basic reagent, the pH value of the reaction system is 8 to 10.

[0041] In some embodiments of the present invention, the basic reagent is in liquid form; the volume ratio of the basic reagent to the hydrophilic small molecule solution is 1:(2 to 10); specifically 1:(3 to 4).

[0042] Preferably, the temperature of the coprecipitation reaction is 25 to 120 °C; more preferably 50 to 110 °C; even more preferably 80 to 105 °C.

[0043] Preferably, the time of the coprecipitation reaction is ≥50 min; more preferably 50 to 300 min; even more preferably 120 to 300 min.

[0044] Preferably, the hydrophilic small molecule, the iron source and the solvent are first mixed for a coordination reaction, and then the mixed solution of the coordination reaction is adjusted to be alkaline to carry out a coprecipitation reaction.

[0045] In some embodiments of the present invention, the hydrophilic small molecule and the iron source are first mixed with the solvent respectively to obtain a hydrophilic small molecule solution and an iron source solution, then the hydrophilic small molecule solution is heated, and then the heated hydrophilic small molecule solution and the iron source solution are mixed for a coordination reaction, and then the mixed solution of the coordination reaction is adjusted to be alkaline to carry out a coprecipitation reaction. Specifically, the way to adjust the reaction system to be alkaline is to add a basic reagent.

[0046] In some embodiments of the present invention, the heating temperature for heating the hydrophilic small molecule solution is 25 to 120 °C; specifically 50 to 110 °C; more specifically 80 to 105 °C.

[0047] The third aspect of the present invention provides an MRI contrast agent, comprising the magnetic iron oxide nanoparticles described in the first aspect of the present invention.

[0048] Preferably, the MRI contrast agent is a longitudinal relaxation contrast agent (T1-weighted contrast agent).

[0049] The beneficial effects of the present invention are as follows: By connecting hydrophilic small molecules to the surface of magnetic iron oxide particles, the present invention can achieve good stabilization and dispersion effects. Compared with the existing magnetic iron oxide nanoparticles using macromolecules as stabilizers, the magnetic iron oxide nanoparticles of the present invention have lower viscosity, higher biocompatibility, and higher longitudinal relaxation r1 value, better clinical practicability and development prospects, which is conducive to the preparation of safe and reliable MRI contrast agents, especially T1-weighted contrast agents.

[0050] Specifically, compared with the prior art, the present invention has the following advantages:

[0051] 1. The magnetic iron oxide nanoparticles of the present invention have a small average particle size and a high r1 value (>2.0 mM -1 s -1 , 3.0 T), which can be comparable to clinically used gadolinium-based chelate contrast agents. As an iron-based MRI contrast agent, the present invention is synthesized by a coprecipitation method using hydrophilic small molecules as stabilizers, with good imaging effect, good water solubility, and good stability, which is conducive to blood circulation. At the same time, both the small molecule stabilizer and the iron-based material are highly biocompatible materials, and have higher biosafety compared with clinically used gadolinium chelate contrast agents.

[0052] 2. The ultrasmall magnetic iron oxide nanoparticles of the present invention use small molecules as stabilizers, and their dispersion can still maintain a low viscosity (≤5.0 CP, 25±2 °C) at high concentrations (C Fe >100 mM). Therefore, high-concentration preparations can be made, with a small required dose in clinical applications, and can be administered by intravenous bolus injection, greatly shortening the preparation time before MRI scanning, and having higher practicability and clinical transformation value compared with using macromolecule stabilizers. Description of the Drawings

[0053] Figure 1 It is the optimal relaxation map of the sample ES-MION-CA in Example 1.

[0054] Figure 2 It is the transmission electron microscope image of the sample ES-MION-CA in Example 1.

[0055] Figure 3 It is the hydrodynamic diameter map and Zeta potential map of the sample ES-MION-CA in Example 1.

[0056] Figure 4 It is the saturation magnetization map of the sample ES-MION-CA in Example 1.

[0057] Figure 5 It is the X-ray photoelectron spectroscopy map and X-ray diffraction map of the sample ES-MION-CA in Example 1.

[0058] Figure 6 IR absorption spectra of the samples ES-MION-CA and citric acid CA in Example 1.

[0059] Figure 7 T1-weighted MRI images of the samples ES-MION-CA in Example 1 and commercially available gadolinium-based contrast agents Gadovist and Magnevist, and their corresponding MRI signal intensity graphs.

[0060] Figure 8 Graphs of cytotoxicity experiment data of the samples ES-MION-CA in Example 1 and commercially available gadolinium-based contrast agent Gadovist.

[0061] Figure 9 MRI images and relative signal intensity graphs of the samples ES-MION-CA in Example 1 and commercially available gadolinium-based contrast agent Gadovist injected into tumor-bearing mice.

[0062] Figure 10 Viscosity graphs of the samples ES-MION-CA in Example 1 and ES-MION-PAA in Comparative Example 1 at a temperature of 25 ± 2°C.

[0063] Figure 11 Optimal relaxation graph of the sample ES-MION-MA in Example 2.

[0064] Figure 12 Optimal relaxation graph of the sample ES-MION-TA in Example 3.

[0065] Figure 13 Optimal relaxation graph of the sample ES-MION-Asp in Example 4.

[0066] Figure 14 Optimal relaxation graph of the sample ES-MION-Glu in Example 5.

[0067] Figure 15 Optimal relaxation graph of the sample ES-MION-OAA in Example 6.

[0068] Figure 16 Optimal relaxation graph of the sample ES-MION-FUM in Example 7.

[0069] Figure 17 Optimal relaxation graph of the sample ES-MION-MAE in Example 8.

[0070] Figure 18 Optimal relaxation graph of the sample ES-MION-Succ in Example 9.

[0071] Figure 19 Optimal relaxation graph of the sample ES-MION-Cit in Example 10.

[0072] Figure 20 Optimal relaxation map of the sample ES-MION-TCA in Example 11.

[0073] Figure 21 Optimal relaxation map of the sample ES-MION-EDTA in Example 12. Detailed implementation manners

[0074] The content of the present invention will be further described in detail through specific examples below. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art according to the principles described in the present invention all fall within the protection scope of the present invention. The specific process parameters and the like in the following examples are also only an example within a suitable range, that is, those skilled in the art can make selections within a suitable range through the description in this article, rather than being limited to the specific data in the following examples. The raw materials, reagents or devices used in the following examples and comparative examples can be obtained from conventional commercial channels or can be obtained by existing known methods without special instructions.

[0075] Example 1

[0076] This example provides ultra-small magnetic iron oxide nanoparticles (ES-MION-CA) with citric acid as a stabilizer. The specific preparation steps are as follows:

[0077] First, insert the inlet needle below the liquid level of the citric acid solution (16.0 mg / mL, 6.5 < pH < 7.5, 20 mL), introduce nitrogen for bubbling for one hour, and keep the air pressure in the reaction vessel positive to remove oxygen. While purging with nitrogen, heat the solution to 100 °C, and then raise the inlet needle above the liquid level. After starting magnetic stirring, add the iron ion mixed solution (0.4 mL, FeCl3·6H2O + FeCl2·4H2O) to the reaction system. When the color change of the solution is stable, quickly add ammonia water (6.0 mL, 1.5%). After reacting for 1.0 hour, stop the reaction and cool. Finally, purify the sample after the reaction to obtain the finished product ES-MION-CA.

[0078] Perform physical property characterization on the sample of Example 1. Calculate the iron recovery rate of the sample in Example 1 to be 90.7%, indicating high utilization rate of raw materials and effective cost reduction. Prepare 6 different concentrations of aqueous solutions of the sample in Example 1, Gadavist and Magnevist on the market respectively, and use a clinical medical 3.0T MRI system to perform in vitro imaging tests to obtain the longitudinal relaxation time T1 and the transverse relaxation time T2. Through the following formula (c is the concentration of the magnetic substance in the contrast agent, T iis the relaxation time, where i = 1 or 2) to calculate the longitudinal relaxation rate r1 and the transverse relaxation rate r2:

[0079]

[0080] Figure 1 is the optimal relaxation map of the sample ES-MION-CA in Example 1. Among them, (a) is the longitudinal relaxation map, and (b) is the transverse relaxation map; ES-MION-CA-1, ES-MION-CA-2, and ES-MION-CA-3 respectively represent 3 parallel samples; the slope of the fitting line is the corresponding relaxation rate (r1 = 7.03 ± 0.05 mM -1 s -1 , r2 = 52.18 ± 1.12 mM -1 s -1 ).

[0081] Figure 2 is the transmission electron microscopy image (TEM image) of the sample ES-MION-CA in Example 1. It can be seen from the electron microscopy image that the nanoparticles are of uniform size and evenly dispersed. Randomly select Figure 2 100 nanoparticles in it to statistically analyze their particle size distribution. The average particle size is about 4.90 nm. The average particle size of the sample ES-MION-CA is less than 5 nm, indicating the potential to become a T1 contrast agent in terms of particle size.

[0082] Figure 3 is the hydrodynamic diameter map and Zeta potential map of the sample ES-MION-CA in Example 1. Among them, (a) is the hydrodynamic diameter map, and (b) is the Zeta potential map. It can be seen from the figure that its hydrodynamic diameter is 9.41 nm and the Zeta potential is -19.49 ± 3.08 mV. Charge repulsion can be used to improve the aqueous phase dispersibility of the sample, which is beneficial to the blood circulation of the sample.

[0083] Figure 4 is the saturation magnetization map of the sample ES-MION-CA in Example 1. It can be seen from the figure that the sample in Example 1 is paramagnetic, which is in line with the properties of ultrasmall magnetic iron oxide nanoparticles.

[0084] Figure 5 is the X-ray photoelectron spectroscopy map and X-ray diffraction map of the sample ES-MION-CA in Example 1. Among them, (a) is the X-ray photoelectron spectroscopy map (XPS map), and (b) is the X-ray diffraction map (XRD map). It can be seen from the figure that the characteristic peaks of Fe 3+ (725.0 eV and 12.0 eV) and Fe 2+ (723.2 eV and 710.1 eV) are very obvious, indicating the simultaneous presence of trivalent iron and divalent iron, and Fe 3+ and Fe2+ The ratio is close to 2:1, proving that the sample of Example 1 is magnetite. The characteristic peaks of magnetite, including (220), (311), (400), (422), (511) and (440), are clearly shown in the XRD pattern, proving that the sample in Example 1 is magnetite with a crystal structure. The XRD pattern and the XPS pattern together prove the successful preparation of magnetite in Example 1. Combined with Figure 2 the electron microscopy images, it proves the successful preparation of magnetic magnetite nanoparticles in Example 1.

[0085] Figure 6 are the infrared absorption spectra of the sample ES-MION-CA and citric acid CA in Example 1. The peaks at 1589 cm -1 and 1395 cm -1 correspond to the asymmetric and symmetric stretching vibrations of the -COO- group in citric acid, respectively. In addition, compared with CA, a new C=O stretching vibration absorption peak appears at 1620 cm -1 for the carboxyl group of ES-MION-CA, and the Fe-O stretching vibration absorption peak appears at 443 cm -1 . These results indicate that the carboxyl group of citric acid coordinates with iron ions, making citric acid a stabilizer and coating the Fe3O4 nanoparticles.

[0086] Figure 7 are the T1-weighted MRI images and the corresponding MRI signal intensity images of the sample ES-MION-CA in Example 1 and commercially available gadolinium-based contrast agents Gadavist and Magnevist. Among them, (a) is the T1-weighted MRI image and (b) is the MRI signal intensity image; the magnetic field strength is 3.0 T, TE = 8.2 ms, TR = 200 ms, and △SNR represents the relative signal-to-noise ratio. From Figure 7 Figure (a), it can be seen that compared with pure water (completely black), the brightness of ES-MION-CA increases significantly with the increase in concentration. And compared with the commercially available gadolinium-based contrast agents Gadavist and Magnevist, the MRI signal intensity of ES-MION-CA is higher at the same concentration, and the relative signal-to-noise ratio is as high as 580.2 ± 15.4%. This demonstrates the excellent performance of iron oxide nanoparticles stabilized by small citric acid molecules as T1-weighted MRI contrast agents.

[0087] Figure 8Cell cytotoxicity experimental data graphs of the sample ES-MION-CA in Example 1 and the commercially available gadolinium-based contrast agent Gadavist, where (a) is murine breast cancer cells (4T1), (b) is human breast cancer cells (MCF-7), and (c) is human normal liver cells (LO-2). The above cells were used for in vitro cell culture and cytotoxicity experiments to obtain the above data. It can be seen from the figure that when the concentration of ES-MION-CA (iron ion concentration) is as high as 1600 mM, the cell survival rate is still as high as over 90%. In contrast, for the commercially available gadolinium-based contrast agent Gadavist, the cell survival rate is as low as below 80% at the same concentration (gadolinium ion concentration). This demonstrates that the combination of the small molecule stabilizer and the iron oxide core has extremely low biological toxicity and significantly higher biocompatibility compared to commercially available contrast agents.

[0088] Figure 9 MRI imaging graphs and relative signal intensity graphs of injecting the sample ES-MION-CA in Example 1 and the commercially available gadolinium-based contrast agent Gadavist into tumor-bearing mice, where (a) is the MRI imaging graph of ES-MION-CA, (b) is the MRI imaging graph of Gadavist, (c) is the relative signal intensity graph of ES-MION-CA, and (d) is the relative signal intensity graph of Gadavist. The injection dose is 15 mg / kg; 3.0T MRI imaging was performed at 0 h, 0.5 h, 1.0 h, 1.5 h, and 2.0 h after injecting into the mouse tail vein respectively in the figure. From Figure 9 In (a), it can be clearly seen that over time after injecting ES-MION-CA, the brightness of the tumor site gradually increases, reaches the brightest at 1.5 h, and then slowly fades. Through Figure 9 the relative signal intensity graph in (c), it can be seen that the relative signal-to-noise ratio of ES-MION-CA is as high as 80% at the brightest time (1.5 h). And from Figure 9 (b) and (d), it can be seen that Gadavist can also achieve an imaging effect, but its imaging time is shorter and reaches the brightest at 30 min. This proves that ES-MION-CA has good magnetic resonance imaging effects in animals, and the time window is appropriate, neither too long nor too short, with quite high practicality, and has significantly better imaging effects compared to the commercially available gadolinium-based contrast agent Gadavist. The results prove that Example 1 can be used as a T1-weighted MRI contrast agent with good imaging effects and good biocompatibility.

[0089] To compare the effect differences between using small molecules as stabilizers and using macromolecules as stabilizers, the following Comparative Example 1 was provided.

[0090] Comparative Example 1

[0091] This example provides ultrasmall magnetic iron oxide nanoparticles (ES-MION-PAA) stabilized with a macromolecular organic substance, polyacrylic acid (PAA). The specific preparation steps are different from those in Example 1 in that the citric acid solution is replaced with an equal amount and concentration of polyacrylic acid, and the other steps are the same as those in Example 1.

[0092] Figure 10 It is the viscosity graph of the sample ES-MION-CA in Example 1 and the sample ES-MION-PAA in Comparative Example 1 at a temperature of 25 ± 2°C. As can be seen from the graph, at a temperature of 25 ± 2°C, when the iron concentration reaches 100 mM, the viscosity of ES-MION-CA still remains below 1.5 CP, while that of ES-MION-PAA is close to 12 CP. It is proved that ES-MION-CA stabilized with small molecule citric acid can maintain an extremely low viscosity at high concentrations, can greatly increase the concentration of the preparation, reduce the dosage, and can be administered by intravenous bolus rather than by drip in clinical practice, greatly improving the clinical efficiency. Therefore, it has great potential for clinical translation.

[0093] The following provides different examples using different small molecules as stabilizers.

[0094] Example 2

[0095] This example provides ultrasmall magnetic iron oxide nanoparticles (ES-MION-MA) stabilized with malic acid. The specific preparation steps are as follows:

[0096] First, insert the inlet needle below the liquid level of the malic acid solution (32.0 mg / mL, 6.5 < pH < 7.5, 20 mL), introduce nitrogen gas to bubble for one hour, and keep the air pressure in the reaction vessel at positive pressure to remove oxygen. While bubbling with nitrogen, heat the solution to 100°C, and then raise the inlet needle above the liquid level. After starting magnetic stirring, add the iron ion mixed solution (0.4 mL, FeCl3·6H2O + FeCl2·4H2O) to the reaction system. When the color change of the solution is stable, quickly add ammonia water (6.0 mL, 2.0%). After reacting for 1.0 hour, stop the reaction and cool it. Finally, purify the sample after the reaction to obtain the finished product ES-MION-MA.

[0097] Perform physical property characterization and performance testing on the sample of Example 2. Calculate the iron recovery rate of the sample of Example 2 to be 94.8%, indicating high utilization rate of raw materials and cost savings. Prepare the sample of Example 2 into 6 aqueous solutions with different concentrations, and perform in vitro imaging testing under a 3.0T clinical MRI system (Philips, Ingenia) to obtain the longitudinal relaxation time T1 and the transverse relaxation time T2, and calculate the longitudinal magnetic relaxation rate r1 and the transverse magnetic relaxation rate r2.

[0098] Figure 11 Optimal relaxation maps of the sample ES-MION-MA in Example 2, where (a) is the longitudinal relaxation map and (b) is the transverse relaxation map; ES-MION-MA-1, ES-MION-MA-2, and ES-MION-MA-3 represent three parallel samples respectively; the slope of the fitting line is the corresponding magnetic relaxation rate (r1 = 6.89 ± 0.06 mM -1 s -1 , r2 = 45.47 ± 0.37 mM -1 s -1 ). The results prove that Example 2 can be used as a T1-weighted MRI contrast agent with good biocompatibility.

[0099] Example 3

[0100] This example provides an ultrasmall magnetic iron oxide nanoparticle (ES-MION-TA) with tartaric acid as a stabilizer, and the specific preparation steps are as follows:

[0101] First, insert the inlet needle below the liquid level of the tartaric acid solution (32.0 mg / mL, 6.5 < pH < 7.5, 20 mL), and introduce nitrogen gas to bubble for one hour while maintaining the positive pressure inside the reaction vessel to remove oxygen. While bubbling with nitrogen, heat the solution to 100 °C, and then raise the inlet needle above the liquid level. After starting magnetic stirring, add the iron ion mixed solution (0.4 mL, FeCl3·6H2O + FeCl2·4H2O) to the reaction system. When the color change of the solution becomes stable, quickly add ammonia water (6.0 mL, 3.0%). After reacting for 1.0 hour, stop the reaction and cool it. Finally, purify the sample after the reaction to obtain the finished product ES-MION-TA.

[0102] Perform physical property characterization and performance testing on the sample of Example 3. Calculate the iron recovery rate of the sample in Example 3 to be 91.2%, indicating high utilization rate of raw materials and cost savings. Prepare 6 aqueous solutions with different concentrations of the sample in Example 3, and perform in vitro imaging tests on a 3.0T clinical MRI system (Philips, Ingenia) to obtain the longitudinal relaxation time T1 and the transverse relaxation time T2, and calculate the longitudinal magnetic relaxation rate r1 and the transverse magnetic relaxation rate r2.

[0103] Figure 12 Optimal relaxation maps of the sample ES-MION-TA in Example 3, where (a) is the longitudinal relaxation map and (b) is the transverse relaxation map; ES-MION-TA-1, ES-MION-TA-2, and ES-MION-TA-3 represent three parallel samples respectively; the slope of the fitting line is the corresponding magnetic relaxation rate (r1 = 4.68 ± 0.18 mM -1 s -1 , r2 = 38.10 ± 0.96 mM-1 s -1 )。The result proves that Example 3 can be used as a T1-weighted MRI contrast agent with good biocompatibility.

[0104] Example 4

[0105] This example provides ultrasmall magnetic iron oxide nanoparticles (ES-MION-Asp) with aspartic acid as a stabilizer. The specific preparation steps are as follows:

[0106] First, insert the inlet needle below the liquid level of the aspartic acid solution (32.0 mg / mL, 6.5 < pH < 7.5, 20 mL), and introduce nitrogen gas to bubble for one hour, while maintaining a positive pressure in the reaction vessel to remove oxygen. While bubbling with nitrogen, heat the solution to 100 °C, and then raise the inlet needle above the liquid level. After starting magnetic stirring, add the iron ion mixed solution (0.4 mL, FeCl3·6H2O + FeCl2·4H2O) to the reaction system. When the color change of the solution is stable, quickly add ammonia water (6.0 mL, 2.0%). After reacting for 1.0 hour, stop the reaction and cool it. Finally, purify the sample after the reaction to obtain the finished product ES-MION-Asp.

[0107] Perform physical property characterization and performance testing on the sample of Example 4. Calculate the iron recovery rate of the sample of Example 4 to be 95.5%, indicating high utilization rate of raw materials and cost savings. Prepare the sample of Example 4 into 6 aqueous solutions with different concentrations, and perform in vitro imaging tests on a 3.0 T clinical MRI system (Philips, Ingenia) to obtain the longitudinal relaxation time T1 and the transverse relaxation time T2, and calculate the longitudinal magnetic relaxation rate r1 and the transverse magnetic relaxation rate r2.

[0108] Figure 13 is the optimal relaxation map of the sample ES-MION-Asp of Example 4, where (a) is the longitudinal relaxation map and (b) is the transverse relaxation map; ES-MION-Asp-1, ES-MION-Asp-2, and ES-MION-Asp-3 respectively represent 3 parallel samples; the slope of the fitted straight line is the corresponding magnetic relaxation rate (r1 = 6.43 ± 0.07 mM -1 s -1 , r2 = 48.86 ± 1.00 mM -1 s -1 ). The result proves that Example 4 can be used as a T1-weighted MRI contrast agent with good biocompatibility.

[0109] Example 5

[0110] This example provides ultrasmall magnetic iron oxide nanoparticles (ES-MION-Glu) with glutamic acid as a stabilizer. The specific preparation steps are as follows:

[0111] First, insert the inlet needle below the liquid level of the glutamic acid solution (64.0 mg / mL, 6.5 < pH < 7.5, 20 mL), introduce nitrogen gas for bubbling for one hour, and maintain the air pressure in the reaction vessel at a positive pressure to remove oxygen. While bubbling with nitrogen, heat the solution to 100 °C, and then raise the inlet needle above the liquid level. After starting magnetic stirring, add an iron ion mixed solution (0.4 mL, FeCl3·6H2O + FeCl2·4H2O) to the reaction system. When the color change of the solution is stable, quickly add ammonia water (6.0 mL, 2.0%). After reacting for 1.0 hour, stop the reaction and cool it. Finally, purify the sample after the reaction to obtain the finished product ES-MION-Glu.

[0112] Perform physical property characterization and performance testing on the sample of Example 5. Calculate the iron recovery rate of the sample of Example 5 to be 94.1%, indicating high utilization rate of raw materials and cost savings. Prepare the sample of Example 5 into 6 aqueous solutions with different concentrations, and perform in vitro imaging tests under a 3.0T clinical MRI system (Philips, Ingenia) to obtain the longitudinal relaxation time T1 and the transverse relaxation time T2, and calculate the longitudinal magnetic relaxation rate r1 and the transverse magnetic relaxation rate r2.

[0113] Figure 14 is the optimal relaxation map of the sample ES-MION-Glu of Example 5, where (a) is the longitudinal relaxation map and (b) is the transverse relaxation map; ES-MION-Glu-1, ES-MION-Glu-2, and ES-MION-Glu-3 respectively represent 3 parallel samples; the slope of the fitted line is the corresponding magnetic relaxation rate (r1 = 5.98 ± 0.09 mM -1 s -1 , r2 = 42.8 ± 2.69 mM -1 s -1 ). The results prove that Example 5 can be used as a T1-weighted MRI contrast agent with good biocompatibility.

[0114] Example 6

[0115] This example provides an ultrasmall magnetic iron oxide nanoparticle (ES-MION-OAA) using glucaric acid as a stabilizer, and the specific preparation steps are as follows:

[0116] First, insert the inlet needle below the liquid level of the glucaric acid solution (64.0 mg / mL, 6.5 < pH < 7.5, 20 mL), introduce nitrogen gas for bubbling for one hour, and maintain a positive pressure in the reaction vessel to remove oxygen. While bubbling with nitrogen, heat the solution to 100 °C, and then raise the inlet needle above the liquid level. After starting magnetic stirring, add the iron ion mixed solution (0.4 mL, FeCl3·6H2O + FeCl2·4H2O) to the reaction system. When the color change of the solution is stable, quickly add ammonia water (6.0 mL, 3.0%). After reacting for 1.0 hour, stop the reaction and cool it. Finally, purify the sample after the reaction to obtain the finished product ES-MION-OAA.

[0117] Perform physical property characterization and performance testing on the sample of Example 6. Calculate the iron recovery rate of the sample of Example 6 to be 92.2%, indicating high utilization rate of raw materials and cost savings. Prepare the sample of Example 6 into 6 aqueous solutions with different concentrations, and perform in vitro imaging tests under a 3.0 T clinical MRI system (Philips, Ingenia) to obtain the longitudinal relaxation time T1 and the transverse relaxation time T2, and calculate the longitudinal magnetic relaxation rate r1 and the transverse magnetic relaxation rate r2.

[0118] Figure 15 It is the optimal relaxation map of the sample ES-MION-OAA of Example 6, where (a) is the longitudinal relaxation map and (b) is the transverse relaxation map; ES-MION-OAA-1, ES-MION-OAA-2, and ES-MION-OAA-3 respectively represent 3 parallel samples; the slope of the fitted straight line is the corresponding magnetic relaxation rate (r1 = 5.97 ± 0.06 mM -1 s -1 , r2 = 46.18 ± 1.21 mM -1 s -1 ). The results prove that Example 6 can be used as a T1-weighted MRI contrast agent with good biocompatibility.

[0119] Example 7

[0120] This example provides an ultrasmall magnetic iron oxide nanoparticle (ES-MION-FUM) using fumaric acid as a stabilizer, and the specific preparation steps are as follows:

[0121] First, insert the inlet needle below the liquid level of the fumaric acid solution (128.0 mg / mL, 6.5 < pH < 7.5, 20 mL), bubble nitrogen through it for one hour, and maintain a positive pressure inside the reaction vessel to remove oxygen. While bubbling nitrogen, heat the solution to 100 °C, and then raise the inlet needle above the liquid level. After starting magnetic stirring, add the iron ion mixed solution (0.4 mL, FeCl3·6H2O + FeCl2·4H2O) to the reaction system. When the color change of the solution is stable, quickly add ammonia water (6.0 mL, 8.0%). After reacting for 1.0 hour, stop the reaction and cool it. Finally, purify the sample after the reaction to obtain the finished product ES-MION-FUM.

[0122] Perform physical property characterization and performance testing on the sample of Example 7. Calculate the iron recovery rate of the sample of Example 7 to be 97.6%, indicating high utilization rate of raw materials and cost savings. Prepare the sample of Example 7 into 6 aqueous solutions with different concentrations, and conduct in vitro imaging tests under a 3.0T clinical MRI system (Philips, Ingenia) to obtain the longitudinal relaxation time T1 and the transverse relaxation time T2, and calculate the longitudinal magnetic relaxation rate r1 and the transverse magnetic relaxation rate r2.

[0123] Figure 16 It is the optimal relaxation map of the sample ES-MION-FUM of Example 7, where (a) is the longitudinal relaxation map and (b) is the transverse relaxation map; ES-MION-FUM-1, ES-MION-FUM-2, and ES-MION-FUM-3 respectively represent 3 parallel samples; the slope of the fitted line is the corresponding magnetic relaxation rate (r1 = 4.32 ± 0.18 mM -1 s -1 , r2 = 29.09 ± 0.38 mM -1 s -1 ). The results prove that Example 7 can be used as a T1-weighted MRI contrast agent with good biocompatibility.

[0124] Example 8

[0125] This example provides an ultrasmall magnetic iron oxide nanoparticle (ES-MION-MAE) using maleic acid as a stabilizer, and the specific preparation steps are as follows:

[0126] First, insert the inlet needle below the liquid surface of the maleic acid solution (128.0 mg / mL, 6.5 < pH < 7.5, 20 mL), bubble nitrogen for one hour, and maintain a positive pressure inside the reaction vessel to remove oxygen. While bubbling nitrogen, heat the solution to 100 °C, and then raise the inlet needle above the liquid surface. After starting magnetic stirring, add an iron ion mixed solution (0.4 mL, FeCl3·6H2O + FeCl2·4H2O) to the reaction system. When the color change of the solution is stable, quickly add ammonia water (6.0 mL, 4.0%). After reacting for 1.0 hour, stop the reaction and cool it. Finally, purify the sample after the reaction to obtain the finished product ES-MION-MAE.

[0127] Perform physical property characterization and performance testing on the sample of Example 8. Calculate the iron recovery rate of the sample of Example 8 to be 96.6%, indicating high utilization rate of raw materials and cost savings. Prepare the sample of Example 8 into 6 aqueous solutions with different concentrations, and perform in vitro imaging tests under a 3.0T clinical MRI system (Philips, Ingenia) to obtain the longitudinal relaxation time T1 and the transverse relaxation time T2, and calculate the longitudinal magnetic relaxation rate r1 and the transverse magnetic relaxation rate r2.

[0128] Figure 17 It is the optimal relaxation map of the sample ES-MION-MAE of Example 8, where (a) is the longitudinal relaxation map and (b) is the transverse relaxation map; ES-MION-MAE-1, ES-MION-MAE-2, and ES-MION-MAE-3 respectively represent 3 parallel samples; the slope of the fitted straight line is the corresponding magnetic relaxation rate (r1 = 4.52 ± 0.03 mM -1 s -1 , r2 = 29.94 ± 0.72 mM -1 s -1 ). The results prove that Example 8 can be used as a T1-weighted MRI contrast agent with good biocompatibility.

[0129] Example 9

[0130] This example provides an ultrasmall magnetic iron oxide nanoparticle (ES-MION-Succ) with malonic acid as a stabilizer, and the specific preparation steps are as follows:

[0131] First, insert the inlet needle below the liquid level of the solution of malonic acid (64.0 mg / mL, 6.5 < pH < 7.5, 20 mL), and bubble nitrogen for one hour while maintaining a positive pressure inside the reaction vessel to remove oxygen. While bubbling nitrogen, heat the solution to 100 °C, and then raise the inlet needle above the liquid level. After starting magnetic stirring, add an iron ion mixed solution (0.4 mL, FeCl3·6H2O + FeCl2·4H2O) to the reaction system. When the color change of the solution is stable, quickly add ammonia water (6.0 mL, 8.0%). After reacting for 1.0 hour, stop the reaction and cool it. Finally, purify the sample after the reaction to obtain the finished product ES-MION-Succ.

[0132] Perform physical property characterization and performance testing on the sample of Example 9. Calculate the iron recovery rate of the sample of Example 9 to be 96.2%, indicating high utilization rate of raw materials and cost savings. Prepare the sample of Example 9 into 6 aqueous solutions with different concentrations, and perform in vitro imaging tests under a 3.0T clinical MRI system (Philips, Ingenia) to obtain the longitudinal relaxation time T1 and the transverse relaxation time T2, and calculate the longitudinal magnetic relaxation rate r1 and the transverse magnetic relaxation rate r2.

[0133] Figure 18 This is the optimal relaxation map of the sample ES-MION-Succ of Example 9, where (a) is the longitudinal relaxation map and (b) is the transverse relaxation map; ES-MION-Succ-1, ES-MION-Succ-2, and ES-MION-Succ-3 respectively represent 3 parallel samples; the slope of the fitted line is the corresponding magnetic relaxation rate (r1 = 4.66 ± 0.10 mM -1 s -1 , r2 = 37.80 ± 1.10 mM -1 s -1 ). The results prove that Example 9 can be used as a T1-weighted MRI contrast agent with good biocompatibility.

[0134] Example 10

[0135] This example provides an ultrasmall magnetic iron oxide nanoparticle (ES-MION-Cit) using citric acid as a stabilizer, and the specific preparation steps are as follows:

[0136] First, insert the inlet needle below the liquid level of the tricarboxylic acid solution (32.0 mg / mL, 6.5 < pH < 7.5, 20 mL), and bubble nitrogen for one hour while maintaining a positive pressure inside the reaction vessel to remove oxygen. While bubbling nitrogen, heat the solution to 100 °C, and then raise the inlet needle above the liquid level. After starting magnetic stirring, add an iron ion mixed solution (0.4 mL, FeCl3·6H2O + FeCl2·4H2O) to the reaction system. When the color change of the solution is stable, quickly add ammonia water (6.0 mL, 4.0%). After reacting for 1.0 hour, stop the reaction and cool it. Finally, purify the sample after the reaction to obtain the finished product ES-MION-Cit.

[0137] Perform physical property characterization and performance testing on the sample of Example 10. Calculate the iron recovery rate of the sample of Example 10 to be 92.5%, indicating high utilization rate of raw materials and cost savings. Prepare the sample of Example 10 into 6 aqueous solutions with different concentrations, and perform in vitro imaging tests under a 3.0T clinical MRI system (Philips, Ingenia) to obtain the longitudinal relaxation time T1 and the transverse relaxation time T2, and calculate the longitudinal magnetic relaxation rate r1 and the transverse magnetic relaxation rate r2.

[0138] Figure 19 is the optimal relaxation map of the sample ES-MION-Cit of Example 10, where (a) is the longitudinal relaxation map and (b) is the transverse relaxation map; ES-MION-Cit-1, ES-MION-Cit-2, and ES-MION-Cit-3 respectively represent 3 parallel samples; the slope of the fitted line is the corresponding magnetic relaxation rate (r1 = 5.17 ± 0.11 mM -1 s -1 , r2 = 32.52 ± 1.10 mM -1 s -1 ). The results prove that Example 10 can be used as a T1-weighted MRI contrast agent with good biocompatibility.

[0139] Example 11

[0140] This example provides ultrasmall magnetic iron oxide nanoparticles (ES-MION-TCA) stabilized with butane tetracarboxylic acid, and the specific preparation steps are as follows:

[0141] First, insert the inlet needle below the liquid level of a solution of butane tetracarboxylic acid (16.0 mg / mL, 6.5 < pH < 7.5, 20 mL), and bubble nitrogen through it for one hour while maintaining a positive pressure inside the reaction vessel to remove oxygen. While bubbling nitrogen, heat the solution to 100 °C, and then raise the inlet needle above the liquid level. After starting magnetic stirring, add an iron ion mixed solution (0.4 mL, FeCl3·6H2O + FeCl2·4H2O) to the reaction system. When the color change of the solution is stable, quickly add ammonia water (6.0 mL, 1.0%). After reacting for 1.0 hour, stop the reaction and cool it. Finally, purify the sample after the reaction to obtain the finished product ES-MION-TCA.

[0142] Perform physical property characterization and performance testing on the sample of Example 11. Calculate the iron recovery rate of the sample of Example 11 to be 94.8%, indicating high utilization rate of raw materials and cost savings. Prepare the sample of Example 11 into 6 aqueous solutions with different concentrations, and perform in vitro imaging tests under a 3.0T clinical MRI system (Philips, Ingenia) to obtain the longitudinal relaxation time T1 and the transverse relaxation time T2, and calculate the longitudinal magnetic relaxation rate r1 and the transverse magnetic relaxation rate r2.

[0143] Figure 20 is the optimal relaxation map of the sample ES-MION-TCA of Example 11, where (a) is the longitudinal relaxation map and (b) is the transverse relaxation map; ES-MION-TCA-1, ES-MION-TCA-2, and ES-MION-TCA-3 respectively represent 3 parallel samples; the slope of the fitted line is the corresponding magnetic relaxation rate (r1 = 6.78 ± 0.09 mM -1 s -1 , r2 = 43.86 ± 0.60 mM -1 s -1 ). The results prove that Example 11 can be used as a T1-weighted MRI contrast agent with good biocompatibility.

[0144] Example 12

[0145] This example provides an ultrasmall magnetic iron oxide nanoparticle (ES-MION-EDTA) using ethylenediaminetetraacetic acid as a stabilizer. The specific preparation steps are as follows:

[0146] First, insert the inlet needle below the liquid level of the ethylenediaminetetraacetic acid solution (16.0 mg / mL, 6.5 < pH < 7.5, 20 mL), introduce nitrogen gas for bubbling for one hour, and maintain a positive pressure inside the reaction vessel to remove oxygen. While bubbling with nitrogen, heat the solution to 100 °C, and then raise the inlet needle above the liquid level. After starting magnetic stirring, add an iron ion mixed solution (0.4 mL, FeCl3·6H2O + FeCl2·4H2O) to the reaction system. When the color change of the solution is stable, quickly add ammonia water (6.0 mL, 2.0%). After reacting for 1.0 hour, stop the reaction and cool it. Finally, purify the sample after the reaction to obtain the finished product ES-MION-EDTA.

[0147] Perform physical property characterization and performance testing on the sample of Example 12. Calculate the iron recovery rate of the sample of Example 12 to be 90.5%, indicating high utilization rate of raw materials and cost savings. Prepare the sample of Example 12 into 6 aqueous solutions with different concentrations, and conduct in vitro imaging tests under a 3.0T clinical MRI system (Philips, Ingenia) to obtain the longitudinal relaxation time T1 and the transverse relaxation time T2, and calculate the longitudinal magnetic relaxation rate r1 and the transverse magnetic relaxation rate r2.

[0148] Figure 21 is the optimal relaxation map of the sample ES-MION-EDTA of Example 12, where (a) is the longitudinal relaxation map and (b) is the transverse relaxation map; ES-MION-EDTA-1, ES-MION-EDTA-2, and ES-MION-EDTA-3 respectively represent 3 parallel samples; the slope of the fitted straight line is the corresponding magnetic relaxation rate (r1 = 5.08 ± 0.07 mM -1 s -1 , r2 = 32.45 ± 0.59 mM -1 s -1 ). The results prove that Example 12 can be used as a T1-weighted MRI contrast agent with good biocompatibility.

[0149] As can be seen from the above, the magnetic iron oxide nanoparticles prepared in the examples of the present invention have a small average particle size and a high r1 value (>2.0 mM -1 s -1 , 3.0T), which can be comparable to clinical medical gadolinium-based chelate contrast agents. As an iron-based MRI contrast agent, the examples of the present invention are synthesized by a coprecipitation method with hydrophilic small molecules as stabilizers, and have good imaging effects, good water solubility, and good stability, which is beneficial to blood circulation. At the same time, both the small molecule stabilizer and the iron-based material are highly biocompatible materials, and have higher biosafety compared with clinical medical gadolinium chelate contrast agents.

[0150] Moreover, the ultrasmall magnetic iron oxide nanoparticles of the embodiments of the present invention use small molecules as stabilizers, and their dispersion can still maintain a low viscosity (≤5.0 CP, 25±2 °C) at high concentrations (C Fe >100 mM). Therefore, a high-concentration preparation can be made, with a small required dose in clinical applications, and intravenous bolus injection can be used for administration, greatly shortening the preparation time before MRI scanning, and having higher practicability and clinical transformation value compared with using macromolecular stabilizers.

[0151] In summary, connecting hydrophilic small molecules to the surface of the magnetic iron oxide particles in the present invention can achieve good stabilization and dispersion effects. Compared with the existing magnetic iron oxide nanoparticles using macromolecules as stabilizers, the magnetic iron oxide nanoparticles of the present invention have lower viscosity, higher biocompatibility, and higher longitudinal relaxation r1 value, better clinical practicability and development prospects, and are beneficial to preparing a safe and reliable MRI contrast agent, especially a T1-weighted contrast agent.

Claims

1. A magnetic iron oxide nanoparticle, characterized in that, Comprising magnetic iron oxide particles and hydrophilic small molecules attached to the surface of the magnetic iron oxide particles; the average molecular weight of the hydrophilic small molecules is less than 1000 daltons.

2. The magnetic iron oxide nanoparticles according to claim 1, characterized in that, The hydrophilic small molecules are selected from carboxylic acid-containing small molecules.

3. The magnetic iron oxide nanoparticles according to claim 2, characterized in that, The carboxylic acid-containing small molecules include at least one of citric acid, malic acid, tartaric acid, aspartic acid, glutamic acid, glucaric acid, fumaric acid, maleic acid, malonic acid, tricarboxylic acid, butanetetracarboxylic acid or ethylenediaminetetraacetic acid.

4. The magnetic iron oxide nanoparticles according to claim 1, characterized in that, The magnetic iron oxide particles contain magnetite, ferric oxide or a combination thereof.

5. The magnetic iron oxide nanoparticles according to claim 1, characterized in that, The average particle size of the magnetic iron oxide nanoparticles is 1 to 10 nm; and / or, the zeta potential of the magnetic iron oxide nanoparticles < 0 mV; and / or, the longitudinal relaxation rate r1 value of the magnetic iron oxide nanoparticles is ≥ 2 mM at a magnetic field strength of 3T -1 s -1 ; and / or, the ratio r2 / r1 of the transverse relaxation rate r2 value to the longitudinal relaxation rate r1 value of the magnetic iron oxide nanoparticles at a magnetic field strength of 3 T ≤ 12; and / or, the viscosity of the dispersion of the magnetic iron oxide nanoparticles is less than 5 CP at 25 ± 2 °C under the condition that the Fe concentration is greater than or equal to 100 mM.

6. A method for preparing the magnetic iron oxide nanoparticles according to any one of claims 1 to 5, characterized in that, Comprising the following steps: Making a mixed solution containing hydrophilic small molecules, an iron source and a solvent alkaline and carrying out a coprecipitation reaction to obtain the magnetic iron oxide nanoparticles.

7. The preparation method according to claim 6, characterized in that, The iron source includes a trivalent iron source, or a trivalent iron source and a divalent iron source.

8. The preparation method according to claim 6, characterized in that, The pH value of the coprecipitation reaction is 8 to 10; and / or, the temperature of the coprecipitation reaction is 25 to 120 °C; and / or, the time of the coprecipitation reaction ≥ 50 min.

9. The preparation method according to claim 6, characterized in that, First, the hydrophilic small molecules, the iron source and the solvent are mixed to carry out a coordination reaction, and then the mixed solution after the coordination reaction is adjusted to be alkaline to carry out a coprecipitation reaction.

10. An MRI contrast agent, characterized in that, Comprising the magnetic iron oxide nanoparticles according to any one of claims 1 to 5.

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