A controllable size and surface group hydrophilic magnetic ferroferric oxide nanoparticle and a preparation method thereof

Abstract

The application provides a controllable size and surface group hydrophilic magnetic ferroferric oxide nanoparticle and a preparation method thereof, and belongs to the technical field of biomedical materials. The application uses benzyl ether, polyethylene glycol modifier and acetylacetone iron as raw materials, adopts a hydrophilic ligand type high-temperature thermal decomposition method, and can obtain ferroferric oxide nanoparticles with different particle sizes by changing the heating rate. The nanoparticles do not need to be subjected to surface modification treatment or ligand replacement, have good hydrophilic performance, and have good stability in water. The magnetic ferroferric oxide nanoparticles prepared by the method have a single modified group, a same double modified group or a different double modified group on the surface, and provide a convenient chemical modification way for subsequent application. In addition, the application uses a fluorescent small molecule AF647 NHS activated ester and BSA protein as model drugs respectively, and verifies the feasibility of the hydrophilic magnetic ferroferric oxide nanoparticles as small molecule and biological macromolecule delivery carriers.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a hydrophilic magnetic iron oxide nanoparticle with controllable size and surface groups and its preparation method. Background Technology

[0002] Magnetic nanoparticles (MNPs) typically refer to nanoparticles with a size between 1 and 100 nm, generally composed of single or combined nanoscale metal oxide particles such as iron, cobalt, and nickel. Among them, iron(II,III) oxide nanoparticles are widely used in the biomedical field due to their advantages such as low toxicity, excellent stability, good biocompatibility, and high saturation magnetization, including drug delivery, magnetic resonance imaging (MRI), and magnetic hyperthermia therapy (MHT).

[0003] Currently, the main methods for preparing iron oxide nanoparticles include coprecipitation, hydrothermal methods, and high-temperature thermal decomposition. While nanoparticles prepared by coprecipitation and hydrothermal methods are water-soluble, they exhibit poor particle size distribution, weak magnetic properties, and a tendency to aggregate, hindering their widespread application in the biomedical field. Oil-phase nanoparticles prepared by conventional high-temperature thermal decomposition methods (using oleic acid or oleylamine as stabilizers) typically possess advantages such as narrow particle size distribution and high saturation magnetization, but their hydration process presents several challenges. First, it requires ligand substitution with dopamine-PEG or DSPE-PEG encapsulation, resulting in complex processes and poor batch-to-batch stability. Second, the hydrophilic ligands (dopamine-PEG or DSPE-PEG) interact only with the nanoparticle surface through coordination or hydrophobic interactions, easily dissociating in the in vivo environment and affecting the reliability of subsequent in vivo applications.

[0004] In recent years, high-temperature thermal decomposition using polyols as reducing agents has made significant progress in improving the water dispersibility and stability of iron oxide nanoparticles. However, this method still relies on subsequent chemical modification to introduce surface functional groups, making the operation complex and inefficient. Furthermore, the nanoparticles prepared by high-temperature thermal decomposition using polyols as reducing agents have a uniform particle size. The particle size of nanoparticles is a key factor determining their physicochemical properties, thus significantly affecting their application performance in drug delivery, magnetic separation, catalysis, and other fields. For example, in the biomedical field, smaller iron oxide nanoparticles (<15 nm) exhibit good in vivo circulation characteristics, making them suitable as drug delivery carriers for in vivo applications; while larger iron oxide nanoparticles (>50 nm) demonstrate stronger magnetic responsiveness, making them more suitable for magnetic separation applications such as cell sorting and protein enrichment.

[0005] Therefore, achieving controllable particle size preparation and controllable surface group modification of hydrophilic magnetic iron oxide nanoparticles is of great significance for meeting the diverse application needs in the biomedical field. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a hydrophilic magnetic iron oxide nanoparticle with controllable size and surface functional groups, and a method for preparing the same. The preparation method provided by this invention is simple to operate and can achieve the controllable synthesis of iron oxide nanoparticles with hydrophilicity, adjustable particle size, and functional groups on the surface.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for preparing hydrophilic magnetic iron oxide nanoparticles with controllable size and surface functional groups, comprising the following steps:

[0009] A mixture of acetylacetone iron, benzyl ether, and polyethylene glycol modifier was preheated to obtain a preheated mixture.

[0010] The preheated mixture is heated to the thermal decomposition temperature of acetylacetone iron to carry out a high-temperature thermal decomposition reaction, and the high-temperature thermal decomposition reaction product is obtained.

[0011] The high-temperature thermal decomposition reaction products were subjected to centrifugation, magnetic separation and membrane filtration to obtain hydrophilic magnetic iron oxide nanoparticles.

[0012] The polyethylene glycol modifier includes a polyethylene glycol molecular structure and modifying groups located at one or both ends of the polyethylene glycol molecular structure.

[0013] The preheating temperature is 80~150℃;

[0014] The high-temperature thermal decomposition reaction temperature is 200~300 ℃;

[0015] The heating rate from the preheating temperature to the thermal decomposition temperature is 1~10 ℃ / min.

[0016] Preferably, the polyethylene glycol modifier includes one or more of the following: polyethylene glycol modifiers containing a single modifier group, polyethylene glycol modifiers containing the same dual modifier group, and polyethylene glycol modifiers containing different dual modifier groups.

[0017] Preferably, the polyethylene glycol modifier containing a single modifying group includes one or a combination of methoxy-PEG-maleimide (mPEG-Mal), methoxy-PEG-amino (mPEG-NH2), methoxy-PEG-carboxyl (mPEG-COOH), methoxy-PEG-hydroxy (mPEG-OH), methoxy-PEG-azide (mPEG-N3), and methoxy-PEG-mercapto (mPEG-SH).

[0018] Preferably, the polyethylene glycol modifier containing the heterodimodified groups includes one or a combination of carboxyl-PEG-maleimide (COOH-PEG-Mal), amino-PEG-maleimide (NH2-PEG-Mal), carboxyl-PEG-amino (COOH-PEG-NH2), hydroxy-PEG-maleimide (HO-PEG-Mal), carboxyl-PEG-hydroxy (COOH-PEG-OH), and amino-PEG-hydroxy (NH2-PEG-OH).

[0019] Preferably, the polyethylene glycol modifier containing the same dual modifying groups includes one or a combination of carboxyl-PEG-carboxyl (COOH-PEG-COOH), amino-PEG-amino (NH2-PEG-NH2), hydroxy-PEG-hydroxy (HO-PEG-OH), and maleimide-PEG-maleimide (Mal-PEG-Mal).

[0020] Preferably, the PEG unit in the polyethylene glycol modifier has a molecular weight of 1~5 kDa.

[0021] Preferably, the molar ratio of iron acetylacetone to polyethylene glycol modifier is 1~4: 0.5~3;

[0022] The volume ratio of the iron in the acetylacetone to the benzyl ether is 0.5~1 mmol: 2~4 mL.

[0023] Preferably, the preheating holding time is 30~100 min;

[0024] The high-temperature thermal decomposition reaction takes place at a temperature of 200-300 °C for 1-4 h.

[0025] Preferably, the magnetic separation includes the following steps:

[0026] The centrifuged product was added to an LS magnetic separation column and eluted with water.

[0027] Remove the LS magnetic separation column from the magnetic pole, add water to the magnetic separation column, and use a plunger to squeeze out the hydrophilic magnetic iron oxide nanoparticle solution.

[0028] The filter membrane used for the membrane filtration has a pore size of 0.1 μm.

[0029] This invention provides hydrophilic magnetic iron tetroxide nanoparticles prepared by the above preparation method.

[0030] This invention provides a method for preparing hydrophilic magnetic iron oxide nanoparticles with controllable size and surface functional groups. Using benzyl ether, polyethylene glycol modifier, and iron acetylacetone as raw materials, the invention employs a hydrophilic ligand-based high-temperature thermal decomposition method. By varying the heating rate, iron oxide nanoparticles of different sizes can be obtained without further surface modification, exhibiting good hydrophilicity and stability in water. The magnetic iron oxide nanoparticles prepared by this method possess single-modified groups, homo- and hetero-modified groups, or different-modified groups on their surface, providing a convenient chemical modification pathway. Furthermore, this invention uses the fluorescent small molecule AF647 NHS activated ester and BSA protein as drug models to verify the feasibility of hydrophilic magnetic iron oxide nanoparticles as delivery carriers for small molecules and biomacromolecules. In summary, this invention achieves the controllable synthesis of hydrophilic, size-tunable, and functionally functionalized hydrophilic magnetic iron oxide nanoparticles by controlling the reaction system and heating conditions. These iron oxide nanoparticles can serve as a magnetic nanomedicine platform and have wide applications in drug delivery, magnetic resonance imaging, and magnetothermal therapy. This invention solves the problems of complex preparation steps, single particle size, and difficulty in functional group modification in the existing technology of water-dispersible iron oxide nanoparticles, and has good technical promotion value and application prospects. Attached Figure Description

[0031] Figure 1 The image shows the powder X-ray diffraction characterization of the iron oxide nanoparticles obtained in Example 1.

[0032] Figure 2 This is a transmission electron microscope image of the iron oxide nanoparticles obtained in Example 1;

[0033] Figure 3 The graph shows the hydration particle size characterization and stability test results of the iron oxide nanoparticles obtained in Example 1.

[0034] Figure 4 The infrared spectra of the iron oxide nanoparticles and mPEG2000-Mal obtained in Example 1 are shown below.

[0035] Figure 5 For Fe 3+ Concentration standard curve;

[0036] Figure 6 Transmission electron microscopy and hydration particle size characterization of the iron oxide nanoparticles obtained in Example 3;

[0037] Figure 7Transmission electron microscopy and hydration particle size characterization of the iron oxide nanoparticles obtained in Example 4;

[0038] Figure 8 Transmission electron microscopy and hydration particle size characterization images of the iron oxide nanoparticles obtained in Example 5;

[0039] Figure 9 The diagram shows the structure of the iron oxide nanoparticles obtained in Examples 1 and 6.

[0040] Figure 10 The image shows the powder X-ray diffraction characterization of the iron oxide nanoparticles obtained in Example 6.

[0041] Figure 11 This is a transmission electron microscope image of the iron oxide nanoparticles obtained in Example 6;

[0042] Figure 12 The hydration particle size characterization diagram of the iron oxide nanoparticles obtained in Example 6 is shown.

[0043] Figure 13 The figures show the results of polypropylene gel electrophoresis (SDS-PAGE) and non-denaturing polypropylene gel electrophoresis (Native-PAGE) analysis of the iron oxide nanoparticles coupled with proteins obtained in Examples 1 and 6.

[0044] Figure 14 The images show fluorescence imaging of the iron oxide nanoparticles coupled with fluorescent small molecules obtained in Examples 1 and 6. Detailed Implementation

[0045] This invention provides a method for preparing hydrophilic magnetic iron oxide nanoparticles with controllable size and surface functional groups, comprising the following steps:

[0046] A mixture of acetylacetone iron, benzyl ether, and polyethylene glycol modifier was preheated to obtain a preheated mixture.

[0047] The preheated mixture is heated to the thermal decomposition temperature of acetylacetone iron to carry out a high-temperature thermal decomposition reaction, and the high-temperature thermal decomposition reaction product is obtained.

[0048] The high-temperature thermal decomposition reaction products were subjected to centrifugation, magnetic separation, and membrane filtration to obtain hydrophilic magnetic iron oxide nanoparticles.

[0049] Unless otherwise specified, all raw materials used in this invention are commercially available.

[0050] This invention involves mixing acetylacetone iron, benzyl ether, and a polyethylene glycol (PEG) modifier, followed by preheating to obtain a preheated mixture. In this invention, the PEG modifier comprises a PEG molecular structure and modifying groups located at one or both ends of the PEG molecular structure. Specifically, the PEG modifier includes one or more of the following: PEG modifiers containing a single modifying group, PEG modifiers containing identical dual modifying groups, and PEG modifiers containing dissimilar dual modifying groups. That is, when the modifying group is located at one end of the PEG molecular structure, the PEG modifier is a PEG modifier containing a single modifying group; when the modifying group is located at both ends of the PEG molecular structure, if the modifying groups are identical, the PEG modifier is a PEG modifier containing identical dual modifying groups; if the modifying groups are different, the PEG modifier is a PEG modifier containing dissimilar dual modifying groups.

[0051] In this invention, the polyethylene glycol modifier containing a single modifying group preferably includes one or a combination of methoxy-PEG-maleimide (mPEG-Mal), methoxy-PEG-amino (mPEG-NH2), methoxy-PEG-carboxyl (mPEG-COOH), methoxy-PEG-hydroxy (mPEG-OH), methoxy-PEG-azide (mPEG-N3), and methoxy-PEG-mercapto (mPEG-SH).

[0052] In this invention, the polyethylene glycol modifier containing the heterodimodified groups preferably includes one or a combination of carboxyl-PEG-maleimide (COOH-PEG-Mal), amino-PEG-maleimide (NH2-PEG-Mal), carboxyl-PEG-amino (COOH-PEG-NH2), hydroxy-PEG-maleimide (HO-PEG-Mal), carboxyl-PEG-hydroxy (COOH-PEG-OH), and amino-PEG-hydroxy (NH2-PEG-OH).

[0053] In this invention, the polyethylene glycol modifier containing the same dual modification groups preferably includes one or a combination of carboxyl-PEG-carboxyl (COOH-PEG-COOH), amino-PEG-amino (NH2-PEG-NH2), hydroxy-PEG-hydroxy (HO-PEG-OH), and maleimide-PEG-maleimide (Mal-PEG-Mal).

[0054] In this invention, the molecular weight of the PEG unit in the polyethylene glycol modifier is preferably 1 to 5 kDa, specifically 1 kDa, 2 kDa, 2.5 kDa, 3 kDa, 3.5 kDa, 4 kDa, 4.5 kDa or 5 kDa, and more preferably 2 kDa.

[0055] In this invention, the polyethylene glycol modifier is preferably one or more of methoxy-PEG2000-maleimide (mPEG2000-Mal), methoxy-PEG2000-carboxyl (mPEG2000-COOH), methoxy-PEG2000-amino (mPEG2000-NH2), and carboxyl-PEG2000-maleimide (COOH-PEG2000-Mal).

[0056] In this invention, the molar ratio of iron acetylacetone to polyethylene glycol modifier is preferably 1~4:0.5~3, more preferably 2~3:1~2; the molar ratio of iron acetylacetone to benzyl ether is 0.5~1 mmol:2~4 mL, more preferably 0.6~0.7 mmol:2~2.5 mL.

[0057] In this invention, the mixing method preferably includes the following steps: heating and melting the polyethylene glycol modifier, sequentially adding benzyl ether and ferric acetylacetone, and stirring and mixing. In this invention, the stirring and mixing rate is preferably 500-800 rpm, more preferably 600-700 rpm, and the mixing time is preferably 5-10 min, more preferably 6-8 min.

[0058] In this invention, the preheating temperature is preferably 80~150℃, more preferably 110℃, and the preheating time is preferably 30~100 min, more preferably 60 min; the heating rate to the preheating temperature is preferably 1~10℃ / min, more preferably 1~6℃ / min, specifically 1℃ / min, 3℃ / min, 6℃ / min, or 10℃ / min. In this invention, the preheating serves to remove moisture from the reaction system. In this invention, the preheating is preferably performed during magnetic stirring.

[0059] After obtaining the preheated mixture, the present invention heats the preheated mixture to the thermal decomposition temperature of acetylacetone iron to carry out a high-temperature thermal decomposition reaction, thereby obtaining the high-temperature thermal decomposition reaction product. In the present invention, the temperature of the high-temperature thermal decomposition reaction is preferably 200~300 ℃, more preferably 240~260 ℃, and the time is preferably 1~4 h, more preferably 2~3 h. In the present invention, the heating rate from the preheating temperature to the thermal decomposition temperature is preferably 1~10 ℃ / min, more preferably 1~6 ℃ / min, specifically 1 ℃ / min, 3 ℃ / min, 5 ℃ / min, 6 ℃ / min, 8 ℃ / min, or 10 ℃ / min. In the present invention, the high-temperature thermal decomposition is preferably carried out under magnetic stirring conditions, and after the high-temperature thermal decomposition reaction is completed, the present invention preferably cools to room temperature under magnetic stirring conditions. This invention employs low heating rates (e.g., 1~5 ℃ / min) and medium heating rates (6~10 ℃ / min), which is beneficial for obtaining hydrophilic magnetic iron oxide nanoparticles with relatively uniform and dispersed sizes. By controlling the composition of the raw materials and heating conditions of the reaction system, this invention achieves the controllable synthesis of hydrophilic, tunable-size magnetic iron oxide nanoparticles with functional groups on their surface.

[0060] Following the high-temperature thermal decomposition reaction, the present invention performs centrifugal separation, magnetic separation, and membrane filtration on the products of the high-temperature thermal decomposition reaction to obtain hydrophilic magnetic iron oxide nanoparticles. In the present invention, the centrifugal separation preferably includes the following steps:

[0061] The high-temperature thermal decomposition reaction product was mixed with n-hexane, shaken, and the lower black product was collected.

[0062] The lower black product is mixed with water, centrifuged, and the upper solution is collected as the centrifugation product.

[0063] In this invention, the volume of the high-temperature thermal decomposition reaction product is preferably the same as the volume of n-hexane. In this invention, the water is preferably deionized water, the centrifugation rate is preferably 4000 rpm, and the centrifugation time is preferably 30 min.

[0064] In this invention, the magnetic separation preferably includes the following steps:

[0065] The centrifuged product was added to an LS magnetic separation column and eluted with water.

[0066] Remove the LS magnetic separation column from the magnetic pole, add water to the magnetic separation column, and use a plunger to squeeze out the hydrophilic magnetic iron oxide nanoparticle solution.

[0067] In this invention, the water is preferably deionized water.

[0068] In this invention, the filter membrane used for membrane filtration is preferably an aqueous polycarbonate filter membrane, and the pore size of the filter membrane is preferably 0.1 μm.

[0069] This invention provides hydrophilic magnetic iron oxide nanoparticles prepared by the above-described method. The preferred particle size of the hydrophilic magnetic iron oxide nanoparticles obtained by this invention is 10-100 nm, more preferably 10-50 nm, and even more preferably 10-20 nm.

[0070] The following detailed description, in conjunction with embodiments, illustrates the size- and surface-group-controllable hydrophilic magnetic iron oxide nanoparticles and their preparation method provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0071] Example 1: Preparation of Fe3O4 nanoparticles with hydrophilic maleimide groups of different particle sizes by controlling the heating rate

[0072] (1) 0.46 mmol mPEG2000-Mal was placed in a 25 mL two-necked flask and heated to 60 °C until PEG melted; 2.3 mL benzyl ether was added and the mixture was magnetically stirred at 600 rpm until the solution was fully mixed, followed by the addition of 0.6 mmol ferric acetylacetone. The resulting mixture was heated to 110 °C and kept at that temperature for 1 h. Under reflux, the temperature was increased to 245 °C at 1 °C / min (slow heating), 6 °C / min (medium heating), and 25 °C / min (rapid heating) for 2.3 h, respectively. The mixture was then cooled to room temperature with stirring to obtain the high-temperature thermal decomposition reaction product.

[0073] (2) Transfer the high-temperature thermal decomposition reaction product obtained in step (1) to a 50 mL centrifuge tube, add an equal amount of n-hexane, shake vigorously, and then remove the lower black product. Repeat the washing process three times. Add 20 mL of deionized water to resuspend the product, centrifuge at 4000 rpm for 30 min, remove the lower precipitate, and the upper layer is a solution of iron oxide nanoparticles. Take 3 mL of the product solution and add it to an LS magnetic separation column, and wash the column with 6 mL of deionized water. Remove the magnetic separation column from the magnetic pole, add 1 mL of deionized water, and use the plunger to squeeze out the iron oxide nanoparticle solution. Filter the collected iron oxide nanoparticle solution through a 0.1 μm aqueous polycarbonate filter membrane and store it in a 4 ℃ refrigerator for later use. Different sizes of hydrophilic magnetic iron oxide nanoparticles with maleimide-modified surfaces were obtained and labeled as Fe3O4-Mal-1, Fe3O4-Mal-6, and Fe3O4-Mal-25, respectively.

[0074] Example 2: Characterization of the iron oxide nanoparticles prepared in Example 1

[0075] 1) The structure of the iron oxide nanoparticles prepared in Example 1 was characterized by X-ray diffraction (XRD), and the results are as follows: Figure 1 As shown, the diffraction peak positions of the three samples are consistent with the characteristic peaks of iron(III) oxide reported in the literature, indicating that the nanoparticles prepared under different heating reaction conditions are all iron(III) oxide.

[0076] 2) The particle size and morphology of the iron oxide nanoparticles prepared in Example 1 were characterized using transmission electron microscopy (TEM), and the results are as follows: Figure 2 As shown. Figure 2 In the images, (a) is a TEM image of Fe3O4-Mal-1; (b) is a TEM image of Fe3O4-Mal-6; (c) is a TEM image of Fe3O4-Mal-25; and (d) is a particle size distribution chart of the nanoparticles. It can be seen that the Fe3O4-Mal-1 and Fe3O4-Mal-6 nanoparticles prepared at the three different heating rates are all spherical. The Fe3O4-Mal-1 and Fe3O4-Mal-6 nanoparticles are relatively uniform in size and dispersed, while the Fe3O4-Mal-25 nanoparticle sample shows obvious aggregation. Figure 2 The red arrow in (c) indicates its aggregate, named Fe3O4-Mal-25-AGG. Statistical analysis of nanoparticle sizes shows that Fe3O4-Mal-1 has a size of 10.6 ± 1.77 nm, Fe3O4-Mal-6 has a size of 14.4 ± 1.94 nm, Fe3O4-Mal-25 monomers have a size of 17.0 ± 2.59 nm, while Fe3O4-Mal-25-AGG has a diameter of 101.4 ± 23.47 nm. Figure 2 The yellow double arrow in (c) indicates the method for measuring the aggregate diameter. These results show that during the high-temperature thermal decomposition stage, the size of the iron(III) oxide nanoparticles gradually increases with increasing heating rate. However, excessively rapid heating rates (>25 °C / min) lead to severe aggregation of the nanoparticles, forming larger aggregates.

[0077] 3) The hydration diameters of the Fe3O4-Mal-1, Fe3O4-Mal-6, and Fe3O4-Mal-25 nanoparticles prepared in Example 1 were measured using dynamic light scattering (DLS). The results are as follows: Figure 3As shown in (a), the hydrated diameters of the three nanoparticles were 26.2±1.86 nm, 31.7±0.53 nm, and 71.4±9.03 nm, respectively, with PDI coefficients of 0.191±0.0153, 0.198±0.0085, and 0.228±0.005, respectively. DLS data further confirmed the aggregation phenomenon in the Fe3O4-Mal-25 nanoparticle sample. The particle size change of the nanoparticles in water over 40 days was further investigated, and the results are as follows... Figure 3 As shown in (b), compared to Fe3O4-Mal-25 nanoparticles, the particle size of Fe3O4-Mal-1 and Fe3O4-Mal-6 nanoparticles only fluctuated slightly during the test, exhibiting higher stability.

[0078] 4) The functional groups of the iron oxide nanoparticles obtained in Example 1 were characterized using Fourier transform infrared spectroscopy, and the results are as follows: Figure 4 As shown. Figure 4 In the image, (a) shows the infrared spectrum of mPEG2000-Mal; (b) shows the infrared spectra of three types of iron oxide nanoparticles. Figure 4 As shown by the red arrow in (b) of the image, the three nanoparticles are at 690 cm⁻¹. -1 The presence of out-of-plane bending vibration characteristic peaks of the =CH group in the vicinity confirms that the surface of the nanoparticles has been successfully modified with maleimide groups.

[0079] 5) The Prussian blue colorimetric method for determining iron ion concentration is as follows:

[0080] An equal volume of 3 mol / L hydrochloric acid was added to a solution of iron(III) oxide nanoparticles, and the mixture was heated at 60 °C for 30 min to allow for complete dissociation. Subsequently, excess potassium ferrocyanide was added to react with the released Fe... 3+ The reaction produces Prussian blue, and the absorbance is measured at 710 nm using a microplate reader. Different concentrations of Fe are then introduced. 3+ The calculations are performed using a standard curve plotted after the reaction with potassium ferrocyanide. The standard curve is shown below. Figure 5 As shown, this method exhibits good linearity and high quantitative accuracy.

[0081] Example 3: Preparation of surface-modified amino-based hydrophilic iron oxide nanoparticles

[0082] The difference from Example 1 is that mPEG2000-Mal was replaced with mPEG2000-NH2, and a heating rate of 6 °C / min was used to obtain hydrophilic magnetic iron oxide nanoparticles with amino-modified surface, denoted as Fe3O4-NH2-6. Figure 6The images show the transmission electron microscopy (TEM) and hydration particle size characterization of the obtained Fe3O4-NH2-6 nanoparticles. (a) is a TEM image of the Fe3O4-NH2-6 nanoparticles, which shows that the Fe3O4-NH2-6 nanoparticles are spherical, uniform in size and dispersed, with a particle size of 8.1 ± 1.23 nm. Figure 6 (b) in the figure is the characterization diagram of hydrated particle size, which is 17.6±1.08 nm and the PDI coefficient is 0.313±0.0132.

[0083] Example 4: Preparation of surface-modified carboxyl-modified hydrophilic iron oxide nanoparticles

[0084] The difference from Example 1 is that mPEG2000-Mal was replaced with mPEG2000-COOH, and a heating rate of 6 °C / min was used to obtain hydrophilic magnetic iron oxide nanoparticles with carboxyl groups modified on the surface, denoted as Fe3O4-COOH-6. Figure 7 The images show the transmission electron microscopy (TEM) and hydration particle size characterization of the obtained Fe3O4-COOH-6 nanoparticles. (a) is a TEM image of the Fe3O4-COOH-6 nanoparticles. The Fe3O4-COOH-6 nanoparticles are spherical, uniform in size and dispersed, with a particle size of 11.3 ± 2.55 nm. Figure 7 (b) in the figure is the characterization diagram of hydrated particle size, which is 20.4±1.99 nm and the PDI coefficient is 0.308±0.0181.

[0085] Example 5: Preparation of iron oxide nanoparticles using HO-PEG2000-OH

[0086] The difference from Example 1 is that the polyethylene glycol modifier HO-PEG2000-OH containing the same dual modification groups was used instead of mPEG2000-Mal, and the heating rate was 6 °C / min. The prepared iron oxide nanoparticles were named Fe3O4-OH-6. Figure 8 The images show the transmission electron microscopy (TEM) and hydration particle size characterization of the obtained iron oxide nanoparticles. Figure 8 (a) is a TEM image of Fe3O4-OH-6 nanoparticles. The Fe3O4-OH-6 nanoparticles are regular spheres with uniform and dispersed size, and the particle size is 9.1±0.96 nm. Figure 8 (b) in the figure is the characterization diagram of hydrated particle size, which is 24.8±5.01 nm and the PDI coefficient is 0.188±0.0195.

[0087] Example 6: Preparation of iron oxide nanoparticles using COOH-PEG2000-Mal

[0088] The difference from Example 1 is that mPEG2000-Mal was replaced with COOH-PEG2000-Mal, and the heating rate was 6 °C / min. The prepared iron oxide nanoparticles were named Fe3O4-C-Mal.

[0089] Schematic diagrams of the iron oxide nanoparticle structures obtained in Examples 1 and 5 are shown below. Figure 9 As shown. Example 1 uses mPEG2000-Mal to prepare iron oxide nanoparticles. This is achieved by weakly coordinating the oxygen in the methoxy group (-OCH3) with iron ions to couple mPEG2000-Mal to the surface of iron oxide, exposing maleimide groups. Figure 9 As shown in (a) above. When COOH-PEG2000-Mal is used as a raw material, the carboxyl group (-COOH) forms bidentate or monodentate coordination complexes with iron ions, such as... Figure 9 As shown in (b) above, the carboxyl group has a stronger coordination effect with iron ions compared to the methoxy group. This strong coordination enhances the stability of the surface ligands, reduces the risk of ligand dissociation from the nanoparticle surface, and improves the dispersibility and stability of the nanoparticles in solution. In in vivo applications, this coordination structure endows the nanoparticles with greater environmental adaptability, helping them to remain stable and function in complex in vivo environments.

[0090] Example 7: Characterization of Fe3O4-C-Mal nanoparticles prepared in Example 6

[0091] 1) The structure of the Fe3O4-C-Mal nanoparticles prepared in Example 6 was characterized by X-ray diffraction (XRD), and the results are as follows: Figure 10 As shown, the diffraction peak positions of the sample are consistent with the characteristic peaks of iron(III) oxide reported in the literature, indicating that Fe3O4-C-Mal is iron(III) oxide.

[0092] 2) TEM results of the Fe3O4-C-Mal nanoparticles prepared in Example 6 are as follows: Figure 11 As shown, the nanoparticles are spherical in shape, uniform in size, and have a statistically significant particle size of 17.8 ± 2.34 nm.

[0093] 3) The DLS determination results of the hydrated particle size of Fe3O4-C-Mal nanoparticles in Example 6 are as follows: Figure 12 As shown, Figure 12 (a) shows the hydration particle size characterization results of Fe3O4-C-Mal nanoparticles, and (b) is a comparison of the PDI of Fe3O4-C-Mal nanoparticles and Fe3O4-Mal-6 nanoparticles. Figure 12 As can be seen from (a), the hydrated particle size of the Fe3O4-C-Mal nanoparticles is 32.0 ± 1.11 nm, and the PDI is 0.109 ± 0.0133. (From...) Figure 12 As can be seen from (b), the PDI of Fe3O4-C-Mal nanoparticles is significantly reduced compared to that of Fe3O4-Mal-6 nanoparticles, indicating that Fe3O4-C-Mal nanoparticles have a narrower particle size distribution in water, approaching a monodisperse state.

[0094] Example 8: Calculation of the number of PEG-coupled particles on the surface of individual iron oxide nanoparticles prepared in Examples 1 and 6

[0095] Fe3O4-Mal-1, Fe3O4-Mal-6, Fe3O4-Mal-25, and Fe3O4-C-Mal nanoparticle solutions were prepared into solid powders using vacuum freeze-drying technology, and the mass of the solid powders was weighed. A certain mass of Fe3O4-Mal-1, Fe3O4-Mal-6, Fe3O4-Mal-25, and Fe3O4-C-Mal nanoparticle solutions were then added to a certain volume of 6 N (V) solution. HCl The iron ion concentration was completely dissolved in hydrochloric acid (mFe3O4 = 10:1). The Prussian blue colorimetric method from Example 2 was used to quantify the iron ion concentration. The mass of iron oxide (Fe3O4) and PEG in the powder was calculated based on the iron ion concentration. The mass of a single nanoparticle was calculated based on the diameter and density of the iron oxide nanoparticles; combined with the total mass of the iron oxide nanoparticles and PEG, the number of iron oxide nanoparticles and PEG molecules was further calculated. Finally, the number of PEG molecules on the surface of a single nanoparticle was calculated, and the results are shown in Table 1. It can be seen that using COOH-PEG2000-Mal as the raw material, under the same heating rate (6 °C / min), the prepared surface maleimide-functionalized iron oxide nanoparticles (Fe3O4-C-Mal) have a higher PEG coupling efficiency, providing more favorable conditions for subsequent drug loading or ligand modification.

[0096] Table 1. Number of PEG particles coupled to the surface of different iron oxide nanoparticles

[0097]

[0098] Example 9: Performance Testing of Ferric Oxide Nanoparticles

[0099] 1) To evaluate the ability of iron oxide nanoparticles with maleimide-modified groups to couple with biomolecules. BSA protein (molecular weight 66.4 kDa) was selected as the model macromolecule for this study. 200 μL of 0.1 M NaHCO3 (pH=8.0) solution was added to 2 mL of 2 mg / mL BSA solution, followed by the addition of the thiolation reagent 2-iminothione hydrochloride. The reaction was carried out at room temperature for 2 h, followed by ultrafiltration to remove the free thiolation reagent. The resulting thiol-modified BSA protein (BSA-SH) was redissolved in PBS (pH=7.4) and quantified using a BCA protein quantification kit. One mg of BSA-SH was reacted with two mg of Fe3O4-Mal-1, Fe3O4-Mal-6, Fe3O4-Mal-25, and Fe3O4-C-Mal nanoparticles in PBS (pH=6.2) solution at room temperature with shaking for 12 h. Unreacted free proteins were then removed by LS magnetic column separation. The BSA-bound iron oxide nanoparticles were resuspended in PBS (pH=7.4) buffer to a final volume of 1 mL, and the solution was filtered through a 0.22 μm sterile aqueous polycarbonate membrane. These were named Fe3O4-1-BSA, Fe3O4-6-BSA, Fe3O4-25-BSA, and Fe3O4-C-BSA, respectively.

[0100] The coupling efficiency between iron oxide nanoparticles and BSA protein was investigated using SDS-PAGE gel electrophoresis, and the results are as follows: Figure 13 As shown in (a) above. ImageJ software was used to analyze the grayscale values ​​of the protein bands and to plot the protein quantification curve, as shown below. Figure 13 As shown in (b) of the figure. According to the analysis results, the coupling binding rates of Fe3O4-Mal-1, Fe3O4-Mal-6, Fe3O4-Mal-25, and Fe3O4-C-Mal to BSA protein were 4.9%, 6.4%, 3.5%, and 8.9%, respectively. The results indicate that when using mPEG2000-Mal to prepare Fe3O4 nanoparticles, the Fe3O4 nanoparticles prepared at a moderate heating rate (5~10 ℃ / min) showed the strongest protein coupling ability; under the same conditions, the Fe3O4 nanoparticles prepared by replacing mPEG2000-Mal with COOH-PEG2000-Mal showed even stronger protein coupling ability. The Native-PAGE gel electrophoresis results are shown in the figure. Figure 13 As shown in (c), the protein bands in the nanoparticle group are significantly weaker than the free BSA bands. Since the BSA coupled to the iron oxide nanoparticles cannot enter the protein lanes, it indicates that the BSA and the iron oxide nanoparticles are covalently bound, and there is almost no interference from free BSA in the sample.

[0101] 2) Evaluation of the ability of iron(III) oxide nanoparticles modified with maleimide groups to couple small molecules. AF647 NHS activated ester (molecular weight 1070.36) fluorescent small molecule was selected as a model for the study. First, the nanoparticles and cysteine ​​were reacted overnight at room temperature in PBS buffer (pH=7.4). The thiol groups in cysteine ​​reacted with the maleimide groups on the nanoparticle surface via a Michael addition reaction. Excess free cysteine ​​was removed using an LS magnetic column. Subsequently, the cysteine-modified nanoparticles and AF647 NHS were reacted for 3 h at room temperature in 10 mM NaHCO3 buffer (pH=8.0). Fluorescent labeling was completed by the reaction of the amino group of cysteine ​​with the NHS ester group. Unbound fluorescent small molecules were removed using an LS column, finally obtaining nanoparticles labeled with fluorescent molecules, named Fe3O4-1-647, Fe3O4-6-647, Fe3O4-25-647, and Fe3O4-C-647, respectively. The nanoparticle solution was filtered through a 0.22 μm filter membrane, brought to a final volume, and stored at 4 °C. The efficiency of nanoparticle-coupled fluorescent small molecules was characterized using a fluorescence module in a small animal in vivo imaging instrument, and the results are as follows: Figure 14 As shown. Figure 14 (a) is a fluorescence imaging image of the iron(III) oxide nanoparticles coupled with fluorescent small molecules; (b) is a quantitative fluorescence image. Figure 14 As shown in (a), the iron(III) oxide nanoparticles were successfully coupled with fluorescent small molecules. The quantitative analysis results of the fluorescence intensity are as follows: Figure 14 As shown in (b), the fluorescence intensities of Fe3O4-1-647, Fe3O4-6-647, and Fe3O4-C-647 are similar and all higher than those of Fe3O4-25-647. This indicates that the coupling ability of Fe3O4-Mal-1, Fe3O4-Mal-6, and Fe3O4-C-Mal nanoparticles to small molecules is similar and superior to that of Fe3O4-Mal-25 nanoparticles.

[0102] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing hydrophilic magnetic iron oxide nanoparticles with controllable size and surface functional groups, characterized in that, Includes the following steps: A mixture of acetylacetone iron, benzyl ether, and polyethylene glycol modifier was preheated to obtain a preheated mixture. The preheated mixture is heated to the thermal decomposition temperature of acetylacetone iron to carry out a high-temperature thermal decomposition reaction, and the high-temperature thermal decomposition reaction product is obtained. The high-temperature thermal decomposition reaction products were subjected to centrifugation, magnetic separation and membrane filtration to obtain hydrophilic magnetic iron oxide nanoparticles. The polyethylene glycol modifier includes a polyethylene glycol molecular structure and a modifying group located at one or both ends of the polyethylene glycol molecular structure. The preheating temperature is 80~150℃; The high-temperature thermal decomposition reaction temperature is 200~245℃; The heating rate from the preheating temperature to the thermal decomposition temperature is 6 °C / min; The preheating holding time is 30~100 min; The high-temperature thermal decomposition reaction takes 1-4 hours; The polyethylene glycol modifier includes carboxyl-PEG-maleimide.

2. The preparation method according to claim 1, characterized in that, The PEG unit in the polyethylene glycol modifier has a molecular weight of 1~5 kDa.

3. The preparation method according to claim 1, characterized in that, The molar ratio of iron acetylacetone to polyethylene glycol modifier is 1~4: 0.5~3; The volume ratio of the iron in the acetylacetone to the benzyl ether is 0.5~1 mmol: 2~4 mL.

4. The preparation method according to claim 1, characterized in that, The magnetic separation includes the following steps: The centrifuged product was added to an LS magnetic separation column and eluted with water. Remove the LS magnetic separation column from the magnetic pole, add water to the magnetic separation column, and use a plunger to squeeze out the hydrophilic magnetic iron oxide nanoparticle solution. The filter membrane used for the membrane filtration has a pore size of 0.1 μm.

5. Hydrophilic magnetic iron tetroxide nanoparticles prepared by the preparation method according to any one of claims 1 to 4.

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