A magnetic nanocomposite with adjustable magnetic properties and a preparation method thereof

Abstract

The present invention belongs to the technical field of nanomaterial science and specifically relates to a magnetic nanocomposite with adjustable magnetic properties and a preparation method thereof. First, an iron oleate complex precursor is prepared, then oleic acid-coated iron oxide nanoparticles are prepared, and then citric acid-coated iron oxide nanoparticles (NPs) are prepared. By collecting cell microvesicles and combining with the electroporation technology, the magnetic nanomaterials are transferred into the interior of the vesicles. By adjusting the electroporation voltage, the loading amount of the magnetic nanoparticles is regulated to construct a magnetic nanocomposite with adjustable magnetic properties. Moreover, the preparation method is simple to operate, does not require large-scale equipment during the process, has low requirements for the professionalism of the operators, and the prepared magnetic nanocomposite has good biological safety and good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterial science, and particularly relates to a magnetic nanocomposite with adjustable magnetic properties and a preparation method thereof. Background Art

[0002] Magnetic nanocomposites are multi-component materials, usually formed by combining nanoscale magnetic materials with polymer polymers, silica, carbon or metal-organic framework materials, etc., and are widely used in magnetic separation, catalysis, medical diagnosis, treatment, etc. Magnetic nanocomposites exhibit the property of responding to an external magnetic field and exhibit special properties when exposed to a static or alternating magnetic field, and can be used for brakes, magnetic separation or drug delivery.

[0003] The polymer coating method usually grinds ferromagnetic metal materials, such as cobalt powder or iron powder, into ferromagnetic materials with a size of nanometers to micrometers, and then coats the magnetic material surface with a polymer through a solvent evaporation technique to form a magnetic nanocomposite. In this process, parameters such as the stirring speed and the viscosity of the polymer will directly affect the size and shape of the composite material, and the magnetization intensity will also decrease with the increase of the coating thickness. The magnetic properties of the magnetic nanocomposite are regulated by changing the grinding time of the magnetic material and the stirring time of the material and the polymer. This method has great interference, the size of the prepared microspheres is uncontrollable, and the effective regulation of the magnetic properties of the magnetic nanocomposite cannot be achieved.

[0004] In addition, some researchers have proposed to construct magnetic nanocomposites with different magnetic response speeds by changing the feed ratio. For example, after swelling micron-sized polystyrene particles in an aqueous solution of N-methyl-2-pyrrolidone (NMP), they are mixed with superparamagnetic iron oxide nanoparticles, and the magnetic nanoparticles can diffuse into the polymer microspheres. By regulating the swelling ratio of the polymer and the concentration of the magnetic nanoparticles, the magnetic properties of the magnetic polymer microspheres can be changed. However, with the increase of the swelling rate of the polymer solution, polymer loss will occur, and moreover, due to the acute toxicity of NMP and the fact that it cannot be discharged arbitrarily, the environmental protection and biological safety of the synthesis process need to be investigated, which affects its application range.

[0005] In recent years, the bionic technology developed can combine cell membranes with functional nanoparticles, playing a huge role in biomedical research. Currently, some researchers use cell membranes to coat magnetic nanoparticles. By a top-down approach, cell membranes from different sources such as cancer cells, immune cells, red blood cells, and bacteria are collected and coated on the surface of magnetic nanoparticles through ultrasonic or co-extrusion methods, constructing magnetic nanocomposites with natural membrane structures. This method cannot directly regulate the magnetic properties of the magnetic nanocomposites, but the idea of using biologically natural components to construct magnetic nanocomposites with good biosafety in this method is worth learning from. Based on this, we hope to design a magnetically tunable magnetic nanocomposite that not only has good biosafety but also can regulate magnetism. Summary of the Invention

[0006] Aiming at the problems and disadvantages of the existing technology, the present invention provides a preparation method of a magnetically tunable magnetic nanocomposite, including the following steps:

[0007] S1: Dissolve FeCl3·6H2O and sodium oleate in a solvent, place them in a container, introduce nitrogen, perform oil bath heating and reaction stirring to obtain a reactant, wash and rotary evaporate the reactant, and vacuum dry to obtain an iron oleate complex precursor;

[0008] S2: Dissolve the iron oleate complex precursor obtained in S1 and oleic acid in n-octyl ether, then place it in a container, introduce nitrogen and heat, then cool, sediment the nanoparticles, and wash through a filter element to obtain oleic acid-coated iron oxide nanoparticles;

[0009] S3: Disperse the oleic acid-coated iron oxide nanoparticles obtained in S2 in a mixed solution of 1,2-dichlorobenzene and DMF, add anhydrous citric acid, introduce nitrogen and heat and stir to react, sediment the nanoparticles, and obtain citric acid-coated iron oxide nanoparticles by magnetic adsorption;

[0010] S4: Culture mouse fibroblasts, collect cell microvesicles by centrifugation, mix the cell microvesicles and the citric acid-coated iron oxide nanoparticles with a volume ratio of 1:1, add them to an electroporation dish, and perform electroporation to obtain a magnetic nanocomposite.

[0011] Further, the time for introducing nitrogen in S1 is 15 - 20 min to remove excess air in the reaction container, and the continuous reaction time after heating to 70 °C is 3.5 - 4 h.

[0012] Further, the mass ratio of FeCl3·6H2O to sodium oleate in S1 is 3.6:12.17.

[0013] Further, the drying time in S1 is 10 - 12 h.

[0014] Further, the heating temperature in S2 is 290 °C, the centrifugation rate is 12,000 rpm, and the centrifugation time is 5 min.

[0015] Further, the mass ratio of the iron oleate complex precursor to oleic acid in S2 is 1.57:1.

[0016] Further, the mass ratio of oleic acid to anhydrous citric acid in S3 is 1:2, and the stirring time is 24 h.

[0017] Further, the centrifugation time in S4 is 2 h, and the voltages during the electroporation process are 100 V, 250 V, and 500 V.

[0018] A magnetic property - adjustable magnetic nanocomposite, the nanocomposite is in a spherical structure and internally loaded with magnetic nanomaterials.

[0019] Beneficial effects

[0020] (1) Through the preparation method of a magnetic property - adjustable magnetic nanocomposite provided by the present invention, iron oxide nanoparticles with good biosecurity, chemical catalytic properties, magnetism, and low price are used. And cell microvesicles are phospholipid bilayer membrane vesicles secreted by various types of cells. As an important medium for inter - cellular communication, they can be found in biological fluids such as cell culture media, blood, saliva, urine, and breast milk. According to the differences in their size and generation mechanism, they are usually divided into three different groups: cell microvesicles, apoptotic bodies, and exosomes. Cell microvesicles are directly germinated from the cell membrane and have a particle size of 100 - 1000 nanometers. Cell microvesicles carry biological information such as proteins, nucleic acids, and lipids related to their parent cells. More prominently, they have the characteristic of a natural cavity structure, which provides great advantages for the construction of magnetic nanocomposites.

[0021] (2) Through a magnetic property - adjustable magnetic nanocomposite provided by the present invention, based on the natural cavity structure of cell microvesicles and the excellent magnetism of iron oxide nanoparticles, iron oxide nanoparticles are loaded into the vesicles by using electroporation technology. By changing the voltage, magnetic nanocomposites with different magnetic response speeds are constructed and applied in fields such as catalysis, magnetic heat transfer, magnetic storage, batteries, and biomedicine. Especially in the field of biomedicine, it has been widely used in magnetic separation, imaging contrast agents, magnetic hyperthermia, magnetic targeting, biological antibacterial, and immunotherapy. Description of the drawings

[0022] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings. In the drawings:

[0023] Figure 1 It is a diagram of hydrophilic and hydrophobic magnetic nanoparticles of Embodiment 1 of the present invention;

[0024] Figure 2 It is a magnetic adsorption effect diagram of magnetic nanocomposites constructed with different electroporation parameters of Embodiment 1 of the present invention;

[0025] Figure 3 It is a transmission electron microscope diagram of the magnetic nanocomposite of Embodiment 1 of the present invention. Specific Embodiments

[0026] The following will combine Embodiment 1 of the present invention and the attached Figures 1 to 3 , to clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention

[0027] Embodiment 1

[0028] S1: First, dissolve 3.6 g of FeCl3·6H2O and 12.17 g of sodium oleate in 27 mL of ethanol, 20 mL of deionized water, and 46.7 mL of n-hexane to obtain a mixed solution. Then, place the mixed solution in a three-necked flask, and introduce nitrogen into it for 20 min. Then, place the three-necked flask in an oil bath, heat it to 70 °C at room temperature, and stir and reflux at this temperature for 4 h. After the reaction is completed, transfer the reactant to a separatory funnel, and wash the organic layer part containing iron oleate complex in the reactant with 50 mL of deionized water multiple times. After washing, further remove the residual n-hexane using a rotary evaporator, and place it in a vacuum drying oven for 10 h to obtain an iron oleate complex precursor in the form of an oily wax-like solid;

[0029] S2: Weigh 0.45 g of the iron oleate complex precursor obtained in S1 and dissolve it with 0.286 g of oleic acid in 12.346 mL of n-octyl ether to obtain a reactant. Place it in a three-necked flask, introduce nitrogen into it to remove the excess air in the device, then heat it to 290 °C at a constant heating rate and maintain it at this temperature for 30 min. After the reaction, place the three-necked flask containing the nanocrystal reactant in ice water and quickly cool it to room temperature. Then add 50 mL of ethanol to precipitate the nanoparticles, and wash them by centrifugation multiple times. The centrifugation rate is 12,000 rpm and the centrifugation time is 5 min to obtain oleic acid-coated iron oxide nanoparticles;

[0030] S3: Disperse 50 mg of oleic acid-coated iron oxide nanoparticles in 15 mL of a mixed solution of 1,2-dichlorobenzene and DMF (1:1, V / V) in a three-necked flask, add 100 mg of anhydrous citric acid, introduce nitrogen into the device to remove oxygen, raise the temperature to 100 °C at room temperature, and continuously reflux and stir for 24 h; after the reaction, add ether to the three-necked flask to precipitate the nanoparticles, collect and sediment the citric acid-coated iron oxide nanoparticles by magnetic adsorption, and let them stand in a fume hood for a period of time to remove the excess ether; weigh the dried iron oxide nanoparticles and add a certain amount of deionized water to obtain a citric acid-coated iron oxide nanoparticle solution with a fixed concentration and good dispersibility in water, seal it and store it in a 4 °C refrigerator to obtain citric acid-coated iron oxide nanoparticles (NPs);

[0031] S4: Culture mouse fibroblasts (L929) in an incubator at 37 °C with 5% CO2 and 95% air; when the cells can almost cover the entire culture flask, they can be cultured with serum-free DMEM high-glucose medium for 24 h to stimulate the cells to secrete extracellular vesicles by starvation culture; collect the culture supernatant, and remove the excess cell debris by pre-centrifugation (centrifugation rate is 2000 rpm, centrifugation rate is 10 min); centrifuge the supernatant at 4 °C, 5000 g in an ultra-high-speed centrifuge for 2 h to collect and concentrate the extracellular vesicles. The collected microvesicles are resuspended in a small amount of PBS, and the protein content is measured by the BCA protein assay method. The vesicles collected from every 200 mL of culture supernatant are resuspended in 4 mL of PBS, and the protein content is about 2.47 mg / mL; store them in an -80 °C refrigerator for later use to obtain cell microvesicles (MVs);

[0032] Mix 500 μL of MVs at 2.47 mg / mL and 500 μL of NPs at 10 mg / mL evenly, add them to an electroporation dish, and conduct an electroporation experiment under the conditions of 100 V and 350 μF; prepare magnetic nanocomposites under the conditions of 250 V and 500 V respectively in the same way; after electroporation, put them into a centrifuge tube and incubate at 37 °C for 30 min to restore the membrane structure of the vesicles; then collect MVs@NPs prepared under different voltages (100 V, 250 V, 500 V) by magnetic separation; store them in a refrigerator at 4 °C for standby, and thus obtain a magnetic nanocomposite with adjustable magnetic properties.

[0033] Combined with Figure 1 , it can be seen that the water-phase iron oxide nanoparticles coated with citric acid are evenly dispersed in water, and the oil-phase iron oxide nanoparticles coated with oleic acid are insoluble in water and only soluble in chloroform. The prepared iron oxide nanoparticles need to have good dispersibility in water. The final material synthesized by the three-step method - the iron oxide nanoparticles coated with citric acid - uses the ligand exchange method to enable them to be evenly dispersed in water without obvious precipitation.

[0034] Combined with Figure 2 , it can be known that after the same magnetic adsorption time of 15 min, the magnetic adsorption amounts of the magnetic nanocomposites constructed by different electroporation voltages are significantly different. The reason is that the loading amounts of the magnetic nanomaterials inside the vesicles in the constructed magnetic nanocomposites are different. Combined with Figure 3 , it can be seen from the transmission image of the magnetic nanocomposite that the small image in the upper right corner corresponds to the (311) crystal plane of γ-Fe2O3.

[0035] Disperse the input magnetic nanoparticles NPs and the prepared magnetic nanocomposite MVs@NPs in pure water respectively, transfer them to a glass tube, and digest them with nitric acid. Determine the iron element content by atomic absorption spectrometry, and then calculate the encapsulation efficiency through the following formula to obtain Table 1.

[0036]

[0037]

[0038] Table 1 Encapsulation efficiency of NPs and MVs@NPs under different electroporation voltages

[0039] Through a preparation method of a magnetic nanocomposite with adjustable magnetic properties provided by the present invention, a cell microvesicle with a natural cavity structure is used in combination with iron oxide nanoparticles to obtain excellent magnetism. Then, the iron oxide nanoparticles are loaded into the interior of the vesicles by using electroporation technology, and magnetic nanocomposites with different magnetic response speeds are constructed by changing the voltage. This method is simple to operate, does not require large-scale equipment during the process, has low requirements for the professionalism of operators, and the prepared magnetic nanocomposites have good biosafety and good application prospects.

[0040] Through a magnetic nanocomposite with adjustable magnetic properties provided by the present invention, it is spherical under a transmission electron microscope and is a magnetic nanocomposite loaded with magnetic nanomaterials inside. Moreover, due to the small particle size of the synthesized magnetic nanoparticles and their good dispersibility in water, a single nanoparticle cannot be manipulated by a magnet. After being transferred into the interior of the vesicles, the particles aggregate, and the synthesized magnetic nanocomposite can be manipulated by a magnet.

Claims

1. A method for preparing a magnetic nanocomposite material with adjustable magnetic properties, characterized in that: The following steps are involved: S1: dissolving FeCl3·6H2O and sodium oleate in a solvent, placing the mixture in a container, introducing nitrogen, heating the mixture in an oil bath, reacting and stirring the mixture to obtain a reactant, washing the reactant with rotary evaporation, and vacuum drying the mixture to obtain an iron oleate complex precursor; S2: dissolving the oleic acid iron complex precursor obtained in S1 and oleic acid in n-octyl ether, then placing in a container, introducing nitrogen gas to heat, then cooling, precipitating nanoparticles, and washing by centrifugation to obtain oleic acid-coated iron oxide nanoparticles; S3: dispersing the oleic acid-coated iron oxide nanoparticles obtained in S2 in a mixture of 1,2-dichlorobenzene and DMF, adding anhydrous citric acid, introducing nitrogen gas to heat and stir the reaction, precipitating the nanoparticles, and obtaining citric acid-coated iron oxide nanoparticles by magnetic adsorption. The dried citric acid-coated iron oxide nanoparticles are weighed and a certain amount of deionized water is added to obtain a citric acid-coated iron oxide nanoparticle solution with a fixed concentration and good dispersibility in water, and the solution is sealed and stored in a refrigerator at 4°C to obtain a citric acid-coated iron oxide nanoparticle solution; S4: Culture mouse fibroblasts, collect cell microvesicles by centrifugation, resuspend the collected microvesicles in a small amount of PBS, and determine the protein content by BCA protein assay. The vesicles collected from every 200 mL of culture medium supernatant are resuspended in 4 mL of PBS, and the protein content is 2.47 mg / mL; store in a -80°C refrigerator for later use to obtain a cell microvesicle suspension, mix the cell microvesicle suspension and the citric acid-coated iron oxide nanoparticle solution in a volume ratio of 1:1, add to an electroporation dish, and perform electroporation to obtain a magnetic nanocomposite material; The method uses electroporation technology to load iron oxide nanoparticles into vesicles, and constructs magnetic nanocomposites with different magnetic response speeds by changing the voltage.

2. The method for preparing a magnetic nanocomposite material with controllable magnetic properties according to claim 1, characterized in that: The nitrogen gas is introduced for 15-20 minutes in S1 to remove excess air in the reaction vessel, and the reaction is continued for 3.5-4 hours after the temperature is raised to 70°C.

3. The method for preparing a magnetic nanocomposite material with controllable magnetic properties according to claim 1, characterized in that: The mass ratio of FeCl3·6H2O to sodium oleate described in S1 is 3.6:12.

17.

4. The method for preparing a magnetic nanocomposite material with controllable magnetic properties according to claim 1, characterized in that: The drying time described in S1 is 10-12 hours.

5. The method for preparing a magnetic nanocomposite material with controllable magnetic properties according to claim 1, characterized in that: The heating temperature described in S2 is 290° C., the centrifugal speed is 12000 rpm, and the centrifugal time is 5 min.

6. The method for preparing a magnetic nanocomposite material with controllable magnetic properties according to claim 1, characterized in that: The mass ratio of the iron oleate complex precursor to oleic acid described in S2 is 1.57:

1.

7. The method for preparing a magnetic nanocomposite material with controllable magnetic properties according to claim 1, characterized in that: The mass ratio of oleic acid to anhydrous citric acid in S3 is 1:2, and the stirring time is 24 hours.

8. The method for preparing a magnetic nanocomposite material with controllable magnetic properties according to claim 1, characterized in that: The centrifugation time described in S4 is 2 h, and the voltage during the electroporation process is 100 V, 250 V, and 500 V.

9. A magnetic nanocomposite material with adjustable magnetic properties obtained by the preparation method according to any one of claims 1 to 8, characterized in that: The magnetic nano-composite material is a spherical structure, and the magnetic nano-material is loaded inside.

Images