Modified magnetic nanoparticles, methods of making and using the same

Abstract

The application relates to the technical field of biological medicine, and provides modified magnetic nanoparticles as well as a preparation method and application thereof. The core of the modified magnetic nanoparticles is a magnetic metal oxide, the surface of the magnetic metal oxide is at least partially wrapped with a silica coating, and the surface of the silica coating is modified with fluorosilane. The modified magnetic nanoparticles have high magnetism and fluorine compatibility, have wide application prospects, and are particularly suitable for use in magnetic ultrasonic contrast agents, so that the obtained magnetic ultrasonic contrast agents can better meet the requirements of external stimulation targeted delivery, thereby realizing local high-concentration targeted delivery of drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to modified magnetic nanoparticles, their preparation methods, the magnetic nanoparticles prepared by the method, and the applications of the magnetic nanoparticles. Background Technology

[0002] Currently, pH-sensitive and microenvironment-sensitive drug carriers have emerged in the field of targeted drug delivery. Although these passive targeting strategies based on specific targets have made significant progress, their targeting accuracy remains poor. This is because targets sensitive to microenvironments such as pH are not only present at the lesion site. This "off-target" result often leads to uncontrollable systemic toxicity. Therefore, researching a nanocarrier that can be delivered in a controllable manner through external stimulation will be of epoch-making significance for disease treatment, especially cancer treatment.

[0003] Nanodroplets are droplets in which liquid is encapsulated primarily by membrane materials such as phospholipids, proteins, and polymers. In recent years, ultrasonic nanodroplets, due to their favorable particle size distribution, have become capable of penetrating the intercellular spaces of blood vessels in tumor tissues. Combined with ultrasound-targeted delivery (UTMD) technology, this has made intra-tissue drug delivery a reality. However, in practical clinical applications, magnetic nanodroplets often suffer from low loading efficiency and insufficient magnetism, failing to adequately meet the requirements for targeted delivery of external stimuli and making it difficult to achieve localized high-concentration targeted drug delivery.

[0004] Therefore, it is of great significance to study a nanodroplet with high loading efficiency and high magnetic properties. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing magnetic nanodroplets, such as low loading efficiency and insufficient magnetism, and to provide a modified magnetic nanoparticle, its preparation method, the magnetic nanoparticles prepared by this method, and the applications of the magnetic nanoparticles. The modified magnetic nanoparticles of this invention possess high magnetic properties and fluorine compatibility, and have broad application prospects. They are particularly suitable for use in magnetic ultrasound contrast agents, enabling the resulting magnetic ultrasound contrast agents to better meet the requirements of targeted delivery of external stimuli, thereby achieving localized high-concentration targeted drug delivery.

[0006] The inventors of this invention discovered that magnetic nanoparticles are first modified with silica to form a silica coating on the surface of the nanoparticles, and then the surface of the silica coating is modified with fluorosilane. This allows the magnetic ultrasonic contrast agent obtained by assembling the modified magnetic nanoparticles with a fluorine phase liquid and a specific surfactant to load more magnetic nanoparticles, thus significantly improving the magnetism of the magnetic nanodroplets (or contrast agent).

[0007] To achieve the above objectives, the first aspect of the present invention provides a modified magnetic nanoparticle, wherein the core of the magnetic nanoparticle is a magnetic metal oxide, and the surface of the magnetic metal oxide is at least partially coated with a silicon dioxide coating, and the surface of the silicon dioxide coating is modified with fluorosilane.

[0008] In this invention, the content of the silica coating is 50-500 parts by weight and the content of fluorosilane is 50-400 parts by weight relative to 100 parts by weight of magnetic nanoparticles.

[0009] To further enhance the surface modification effect of the modified magnetic nanoparticles, the content of the silica coating is 60-300 parts by weight and the content of fluorosilane is 150-380 parts by weight relative to 100 parts by weight of magnetic nanoparticles; more preferably, the content of the silica coating is 70-90 parts by weight and the content of fluorosilane is 330-350 parts by weight relative to 100 parts by weight of magnetic nanoparticles.

[0010] In this invention, "at least partially" means that the surface of the magnetic nanoparticles is partially or completely coated with a silicon dioxide coating. In general, the surface of the magnetic nanoparticles is completely coated with a silicon dioxide coating.

[0011] In this invention, the microstructure of the modified magnetic nanoparticles is preferably such that the surface of the magnetic nanoparticles is completely coated with a silica coating, and the surface of the silica coating is modified with a fluorosilane film. The thickness of the silica coating is preferably 2-5 nm, and the thickness of the fluorosilane film is preferably 1-2 nm.

[0012] Preferably, the fluorosilane refers to a substance having the following molecular formula: (CH3-(CH2)) a O)3Si-(CH2) b -(CF2) c A class of substances containing -CF3; the structural formula (I) of fluorosilanes is shown below:

[0013]

[0014] Where a is a positive integer ≥ 0, b is a positive integer ≥ 2, and c is a positive integer between 3 and 20.

[0015] In one specific embodiment of the present invention, the value of a can be, but is not limited to, a positive integer from 0 to 30, preferably a positive integer from 0 to 10, and more preferably 0, 1, 2, or 3.

[0016] In one specific embodiment of the present invention, the value of b can be, but is not limited to, a positive integer from 2 to 20, preferably a positive integer from 2 to 10, and more preferably 2, 3, 4, or 5.

[0017] In one specific embodiment of the present invention, the value of c can be, but is not limited to, a positive integer from 3 to 20, preferably a positive integer from 3 to 15, and more preferably 4, 5, 6, 7, 8, 9, or 10.

[0018] In one specific embodiment of the present invention, the fluorosilane has the following molecular formula: (CH3-(CH2) a O)3Si-(CH2) b -(CF2) c -CF3, where a is 0, 1, 2, 3; b is 2, 3, 4, 5; and c is 5, 6, 7, 8.

[0019] In a preferred embodiment of the present invention, the fluorosilane is heptadecafluorodecyltrimethoxysilane (CAS: 83048-65-1).

[0020] In this invention, the magnetic nanoparticles can be selected from those commonly used in magnetic ultrasound contrast agents in the art. To further enhance the modification effect of the magnetic nanoparticles, preferably, the magnetic metal oxide is selected from one or more of Fe3O4, Fe2O3, CoFe2O4, NiFe2O4, and MnFe2O4.

[0021] In a preferred embodiment of the present invention, the magnetic metal oxide is Fe3O4. Fe3O4 nanoparticles can be obtained commercially or prepared by a co-precipitation method.

[0022] According to a specific embodiment of the present invention, the preparation method of Fe3O4 nanoparticles includes the following steps: mixing FeCl2 and FeCl3 in a 1:1 molar ratio to obtain a mixed solution; refluxing the above mixed solution under nitrogen at a constant temperature of 50-90°C; adding alkaline solution dropwise until pH=6-8, and observing the formation of a black viscous substance; separating the solid and liquid phases to obtain solid Fe3O4 nanoparticles.

[0023] In this invention, the magnetic nanoparticles have a particle size of 5nm-20nm, preferably 7nm-15nm.

[0024] In this invention, the term "particle size" refers to the geometric diameter of a single particle, not an average value. When a range is specified, it means that the particle sizes of all particles of that type in the same material fall within that range. This invention also allows for a certain margin of error; that is, particles whose diameters are outside the required range, representing less than 5% of the total number, are still considered to meet the requirements. In this invention, the particle size of the magnetic nanoparticles is measured using a transmission electron microscope.

[0025] A second aspect of the present invention provides a method for preparing the magnetic nanoparticles described in the first aspect, the method comprising the following steps:

[0026] (1) In a first alkaline environment, the alcohol-water system of silicate ester and magnetic nanoparticles are brought into first contact;

[0027] (2) In the second alkaline environment, the nanoparticles obtained in step (1) are brought into a second contact with the fluorosilane liquid.

[0028] In step (1), the conditions for the first contact include: a temperature of 30-70°C, preferably 40-60°C; and a time of 3-10 hours, preferably 5-8 hours.

[0029] Preferably, the effective amount of silicate ester, calculated as silicon dioxide, is 50-100 parts by weight relative to 100 parts by weight of magnetic nanoparticles. "Calculated as silicon dioxide" refers to converting the weight of the silicate ester into the weight of the resulting silicon dioxide coating. For example, if a silicate ester has two silicon atoms in its molecular formula and reacts to produce two SiO2 molecules, then when 1 mol of this silicate ester is used, it is equivalent to the weight of 2 mol of silicon dioxide, i.e., 120 g.

[0030] In this invention, the term "effective dosage" refers to the amount of raw material used that, ignoring losses during the preparation process, ultimately forms the product (e.g., a silica coating). Those skilled in the art can select and set the actual amount of raw material added based on the requirements of the effective dosage. For example, the range of raw material dosage can be between 200% and 500% of the effective dosage.

[0031] In this invention, "alcohol-water system" refers to a mixed liquid system of low carbon alcohol and water, wherein the low carbon alcohol is a C2-C6 alcohol, preferably a mixed liquid of ethanol and water, and more preferably the volume ratio of ethanol to water is 1:(0.2-0.3).

[0032] In this invention, the volume ratio of silicate ester to alcohol-water system is 1:(400-700). To further improve the coating effect of silica coating, the preferred volume ratio of silicate ester to alcohol-water system is 1:(450-550).

[0033] In this invention, the pH range of the first alkaline environment is 11-12. The first alkaline solution providing the first alkaline environment is selected from at least one of ammonia, sodium hydroxide solution, sodium carbonate solution, and sodium bicarbonate solution, preferably ammonia, and preferably the concentration of ammonia is 25%-28%.

[0034] To further accelerate the hydrolysis and condensation reaction of silicate and promote the coating of magnetic nanoparticles with silica coating, the volume ratio of silicate to ammonia is preferably 1:(10-40), more preferably 1:(15-25).

[0035] In step (2), the conditions for the second contact include: a temperature of 10-40°C, preferably 20-30°C; and a time of 5-20 hours, preferably 8-15 hours.

[0036] Preferably, the effective amount of fluorosilane liquid relative to 100 parts by weight of magnetic nanoparticles, calculated as fluorosilane film, is 50-400 parts by weight, more preferably 60-300 parts by weight, and more preferably 330-350 parts by weight.

[0037] In this invention, the pH range of the second alkaline environment is 11-12. The second alkaline solution providing the second alkaline environment is selected from at least one of ammonia, sodium hydroxide solution, sodium carbonate solution, and sodium bicarbonate solution, preferably ammonia, and preferably the concentration of ammonia is 25%-28%.

[0038] To further enhance the modification effect of fluorosilane, the preferred volume ratio of fluorosilane liquid to ammonia is 1:(5-15), and more preferably 1:(8-10).

[0039] It should be noted that between steps (1) and (2) above, there is also a step of separating the nanoparticles after the first contact, so that the individual nanoparticles can be used for the second contact in step (2).

[0040] In this invention, the amount of material (e.g., alcohol-water system, alkaline solution) that is compatible with silicate ester can preferably be calculated based on the actual amount of silicate ester added.

[0041] In one specific embodiment of the present invention, the method for preparing magnetic nanoparticles includes the following steps:

[0042] (I) The magnetic nanoparticles were uniformly dispersed in a mixed solution of deionized water and anhydrous ethanol and stirred at room temperature until homogeneous.

[0043] (II) Add alkali and ethyl silicate to the solution in step (I), stir and react at 30-70℃ for 3-10h, remove the supernatant by magnetic adsorption, and obtain magnetic nanoparticles with surface modified SiO2.

[0044] (III) Disperse the magnetic nanoparticles obtained in step (II) into anhydrous ethanol;

[0045] (IV) Add fluorosilane liquid and alkali to the solution in step (III) and react at 10-40℃ for 5-20h to obtain a black organic phase suspended in the upper layer of anhydrous ethanol solution.

[0046] (V) Separate the solid and liquid phases of the solution from step (IV), and dry the solid phase to obtain fluorosilane-modified magnetic nanoparticles.

[0047] The third aspect of the present invention provides modified magnetic nanoparticles prepared by the method described in the second aspect.

[0048] The modified magnetic nanoparticles prepared by the method described in the second aspect of the present invention have all the same advantages as the modified magnetic nanoparticles described in the first aspect, which will not be repeated here.

[0049] The fourth aspect of the present invention provides the application of the modified magnetic nanoparticles described in the first and third aspects of the present invention in magnetic ultrasound contrast agents.

[0050] The modified magnetic nanoparticles of this invention are particularly suitable for use in magnetic ultrasound contrast agents due to their excellent magnetic properties and fluorine compatibility. This enables the resulting magnetic ultrasound contrast agents to better meet the requirements of targeted delivery of external stimuli and achieve local high-concentration targeted delivery of drugs.

[0051] The preparation of magnetic ultrasound contrast agents using the magnetic nanoparticles can be carried out in accordance with conventional methods in the art.

[0052] According to a preferred application of the present invention, the magnetic nanoparticles of the present invention are applied in an ultrasound contrast agent in the following manner.

[0053] The magnetic nanoparticles of the present invention can be used to form a "raw material package" of magnetic nanoparticles (each component can be stored independently for ease of production, transportation and sales), that is, a composition of magnetic nanodroplets.

[0054] The composition of the magnetic nanodroplet includes surfactants and magnetic nanoparticles stored independently of each other, wherein the magnetic nanoparticles are the magnetic nanoparticles described in the first aspect of the present invention and / or the magnetic nanoparticles described in the third aspect of the present invention.

[0055] The surfactant is composed of surfactant A and surfactant B, both with HLB values ​​> 8. Surfactant A encapsulates magnetic nanoparticles, while surfactant B is in a free state.

[0056] Wherein, surfactant A is a carboxylated phospholipid; surfactant B is a nonionic surfactant.

[0057] Preferably, the amount of surfactant used is 50-500 parts by weight relative to 100 parts by weight of magnetic nanoparticles; more preferably, the amount of surfactant used is 100-300 parts by weight, and most preferably, the amount of surfactant used is 150-250 parts by weight.

[0058] Preferably, the HLB value of the surfactant is >10. In this invention, the HLB value is determined by reversed-phase gas chromatography, referring to Jiang Chaoxue et al., "Determination of the hydrophilic-lipophilic balance of nonionic emulsifiers by reversed-phase gas chromatography", Synthetic Rubber Industry, 1991, 14(6):399-401.

[0059] The formulation of surfactant A, surfactant B, and modified magnetic nanoparticles described above in this invention achieves good results. To further improve stability, preferably, based on the total weight of the surfactants, the content of surfactant A is 88-98% by weight, and the content of surfactant B is 2-12% by weight. Preferably, based on the total weight of the surfactants, the content of surfactant A is 90-96% by weight, and the content of surfactant B is 4-10% by weight. More preferably, based on the total weight of the surfactants, the content of surfactant A is 94-96% by weight, and the content of surfactant B is 4-6% by weight.

[0060] Preferably, the surfactant A is selected from one or more of distearylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG2000), 1,2-distearate-sn-glycerol phosphatidylcholine (DSPC), distearylphosphatidylethanolamine, and dipalmitoylphosphatidylcholine.

[0061] In a preferred embodiment of the present invention, the surfactant A is a combination of distearylphosphatidylethanolamine-polyethylene glycol and 1,2-distearate-sn-glycerophosphatidylcholine, more preferably the weight ratio of the two is 1:(0.8-1.2).

[0062] Preferably, the surfactant B agent is selected from one or more of poloxamer, poly(isobutylene-maleic anhydride), and poly(maleic anhydride-alt-1-octadecene).

[0063] In a preferred embodiment of the present invention, in order to achieve better synergistic effects with other components in the magnetic nanodroplet composition of the present invention, surfactant A may be a combination of distearylphosphatidylethanolamine-polyethylene glycol and 1,2-distearate-sn-glycerophosphatidylcholine, and surfactant B may be poloxamer. The content of surfactant A is 94-96% by weight, and the content of surfactant B is 4-6% by weight.

[0064] Furthermore, the composition further includes a fluorine phase liquid, wherein the amount of the fluorine phase liquid is 5-50 parts by weight relative to 100 parts by weight of magnetic nanoparticles; preferably, the content of the fluorine phase liquid is 10-30 parts by weight.

[0065] Preferably, the fluorinated liquid is an organic fluorine that can undergo phase change, and more preferably perfluorinated carbon with a carbon chain length greater than 4.

[0066] More preferably, the perfluorocarbon is selected from one or more of perfluorobutane, perfluoropentane and perfluorohexane, with perfluorohexane being the most preferred.

[0067] Furthermore, the composition further includes a base liquid, the content of which is 1000-5000 parts by weight relative to 100 parts by weight of magnetic nanoparticles; preferably, the content of the base liquid is 2000-3000 parts by weight relative to 100 parts by weight of magnetic nanoparticles; more preferably, the content of the base liquid is 2500-2550 parts by weight relative to 100 parts by weight of magnetic nanoparticles.

[0068] The base liquid is a continuous phase, and the magnetic nanodroplets are dispersed phases therein. In this invention, the base liquid is an aqueous liquid. In this invention, the term "aqueous liquid" refers to a liquid or solution that is hydrophilic, such as water, aqueous solutions of salts (e.g., physiological saline), hydrophilic organic liquids (e.g., ethanol, methanol, isopropanol), and mixtures of one or more of these.

[0069] Furthermore, the magnetic nanodroplet composition further includes a drug, the amount of which is 1-20 parts by weight relative to the sum of the weights of the surfactant and the magnetic nanoparticles (100 parts by weight).

[0070] It is understood that although the magnetic ultrasound contrast agent of the present invention can be used as a drug delivery agent, according to a specific embodiment, the ultrasound magnetic contrast agent composition of the present invention may not include a drug due to the needs of production, transportation, etc.

[0071] In this invention, the type of drug is not specifically limited, and commonly used drugs in the field of ultrasound contrast agents can be selected as needed. For example, the drugs include, but are not limited to, doxorubicin, bleomycin, irinotecan hydrochloride, oxaliplatin, paclitaxel, etc.

[0072] The magnetic nanodroplet composition of the present invention may also contain other conventional additives in the art. As long as they do not adversely affect the performance of other components, those skilled in the art can make such selections. The content of these other additives can refer to the conventional content in the art.

[0073] The magnetic nanoparticles and / or magnetic nanodroplets of the present invention can also be used to prepare magnetic ultrasound contrast agents.

[0074] The magnetic ultrasound contrast agent comprises a base liquid and magnetic nanodroplets dispersed in the base liquid, wherein the magnetic nanodroplets contain a composition of the magnetic nanodroplets.

[0075] In this invention, the magnetic ultrasound contrast agent comprises a continuous phase and a dispersed phase, wherein the continuous phase refers to the base liquid, and the dispersed phase refers to magnetic nanodroplets dispersed in the base liquid. The continuous phase (base liquid) can be a conventional continuous phase used in the art for preparing ultrasound contrast agents, such as a phosphate-buffered saline (PBS) buffer solution. The dispersed phase (magnetic nanodroplets) contains the composition of the magnetic nanodroplets described in this invention.

[0076] In this invention, the magnetic ultrasound contrast agent contains a large number of magnetic nanodroplets, wherein the magnetic nanodroplets contain at least the following components in the composition of the magnetic nanodroplets: modified magnetic nanoparticles, fluorine phase liquid, and surfactant.

[0077] Preferably, the magnetic nanodroplets have a particle size of 0.1 μm-1 μm, more preferably 0.1 μm-0.5 μm.

[0078] The present invention also provides a method for preparing the magnetic ultrasound contrast agent of the present invention, the method comprising the following steps:

[0079] (a) A third contact is made between surfactant A and surfactant B;

[0080] (b) Mix the drug with the material obtained in step (a);

[0081] (c) The material obtained in step (b) is subjected to a fourth contact with a fluorine phase liquid and magnetic nanoparticles, and ultrasonic cavitation is performed.

[0082] In the method for preparing the magnetic ultrasound contrast agent, the specific selection and proportion of the raw materials used are all in accordance with the limitations of the modified magnetic nanoparticles and the magnetic nanodroplets composition described in the first aspect of the present invention, and will not be repeated here.

[0083] Further, in step (a), the third contact involves dissolving surfactant A and surfactant B in a solvent and mixing them evenly. To ensure a more thorough mixing of surfactant A and surfactant B, thereby making the core-shell structure of the magnetic nanodroplets more stable, the conditions for the third contact preferably include: a temperature of 60-100℃, more preferably 80-90℃; and a time of 5-50 min, preferably 10-30 min.

[0084] Furthermore, in step (b), the mixing is carried out at a temperature of 5-30°C, preferably 10-20°C. For example, the material obtained in step (a) can be cooled to the range of 10-20°C before being mixed with the drug.

[0085] Furthermore, in step (c), the conditions for ultrasonic cavitation include: ultrasonic power of 5-20kW and time of 1-50min; preferably 8-12kW and time of 5-30min.

[0086] In a preferred embodiment of the present invention, the method for preparing the magnetic ultrasound contrast agent includes the following steps:

[0087] (i) Dissolve surfactant A and surfactant B in anhydrous ethanol at 80-100℃ and mix for 10-30 minutes until homogeneous;

[0088] (ii) Evaporate the solution obtained in step (i) and dissolve the evaporated product in a glycerol solution;

[0089] (iii) Dissolve the drug in the solution obtained in step (ii) at a temperature of 10-20°C;

[0090] (iv) Fluorine phase liquid and magnetic nanoparticles are added dropwise at a uniform rate to the solution obtained in step (iii) and subjected to ultrasonic cavitation treatment with an ultrasonic power of 5-20 kW and a time of 1-50 min.

[0091] In this invention, the redissolving of the surfactant in glycerol in step (ii) can give the final magnetic nanodroplets a more stable core-shell structure.

[0092] The present invention also provides a magnetic ultrasound contrast agent prepared according to the method for preparing a magnetic ultrasound contrast agent described herein.

[0093] The magnetic ultrasound contrast agent prepared by the method for preparing magnetoacoustic ultrasound contrast agents described in this invention has all the same advantages as the magnetic ultrasound agent described above, which will not be elaborated here.

[0094] The present invention, employing the above-mentioned technical solution, has the following beneficial effects: The modified magnetic nanoparticles of the present invention possess high magnetic properties and fluorine compatibility, exhibiting broad application prospects, and are particularly suitable for use in magnetic ultrasound contrast agents. Magnetic ultrasound contrast agents containing modified magnetic nanoparticles can load more magnetic nanoparticles, significantly improving the magnetism of magnetic nanodroplets (or contrast agents), and better meeting the requirements for targeted delivery of external stimuli, thereby achieving localized high-concentration targeted delivery of drugs.

[0095] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein. Attached Figure Description

[0096] Figure 1 The image shown is an electron microscope image of the magnetic nanoparticles A1 obtained in Example A1.

[0097] Figure 2 The image shown is an electron micrograph of the magnetic ultrasound contrast agent B1 obtained in Example B1.

[0098] Figure 3 The absorbance standard curve of doxorubicin in test example 4 is shown.

[0099] Figure 4 The standard curve of doxorubicin obtained by liquid chromatography-mass spectrometry in test example 5 is shown. Detailed Implementation

[0100] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0101] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0102] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0103] Preparation Example

[0104] FeCl2 and FeCl3 were mixed in a 1:1 molar ratio to obtain a mixed solution A. The mixed solution A was transferred to a four-necked flask and refluxed under nitrogen at 70°C. Ammonia was added dropwise until pH=7, and a black viscous substance was formed. The mixture was centrifuged (5000 rpm, 10 minutes), and the supernatant was separated by magnetic adsorption to obtain bare iron(III) oxide nanoparticles, which were denoted as nanoparticles.

[0105] Modified magnetic nanoparticles in Example A

[0106] Example A1

[0107] A modified magnetic nanoparticle, the preparation method of which includes the following steps:

[0108] (1) Disperse 5 mg of nanoparticles B uniformly into an alcohol-water system (a mixed solution of anhydrous ethanol and deionized water with a volume ratio of 1:0.25) and stir at room temperature until homogeneous.

[0109] (2) Add ammonia and ethyl orthosilicate to the solution in step (1) (the amount added makes the effective amount calculated as silicon dioxide 4 mg), stir at 50°C for 6.5 h, remove the supernatant by magnetic adsorption, and obtain magnetic nanoparticles with surface modified SiO2 coating, denoted as magnetic nanoparticle C.

[0110] The volume ratio of tetraethyl orthosilicate to alcohol-water system is 1:500; the volume ratio of tetraethyl orthosilicate to ammonia is 1:20.

[0111] (3) Disperse the magnetic nanoparticles C obtained in step (2) into anhydrous ethanol;

[0112] (4) Add heptadecafluorodecyltrimethoxysilane (the amount added is such that the effective amount is 17 mg based on the fluorosilane film layer) and ammonia to the solution in step (3), react at room temperature for 10 h, and obtain a black organic phase suspended in the upper layer of anhydrous ethanol solution.

[0113] The volume ratio of fluorosilane liquid to ammonia is 1:9;

[0114] (5) Centrifuge the solution from step (4) (5000 rpm, 10 minutes), remove the supernatant by magnetic separation, and vacuum dry for 24 hours to obtain modified magnetic nanoparticles, denoted as A1.

[0115] Example A2

[0116] A modified magnetic nanoparticle, the preparation method of which includes the following steps:

[0117] (1) Disperse 5 mg of nanoparticles B uniformly into an alcohol-water system (a mixed solution of anhydrous ethanol and deionized water with a volume ratio of 1:0.2) and stir at room temperature until homogeneous.

[0118] (2) Add ammonia and tetraethyl orthosilicate to the solution in step (1) (the amount added makes the effective amount calculated as silicon dioxide 3.5 mg), stir at 55°C for 6 h, remove the supernatant by magnetic adsorption, and obtain magnetic nanoparticles with surface modified SiO2, denoted as magnetic nanoparticle C.

[0119] The volume ratio of tetraethyl orthosilicate to alcohol-water system is 1:450; the volume ratio of tetraethyl orthosilicate to ammonia is 1:15.

[0120] (3) Disperse the magnetic nanoparticles C obtained in step (2) into anhydrous ethanol;

[0121] (4) Add heptadecafluorodecyltrimethoxysilane (the amount added is such that the effective amount is 16.5 mg based on the fluorosilane film layer) and ammonia to the solution in step (3), react at room temperature for 12 h, and obtain a black organic phase suspended in the upper layer of anhydrous ethanol solution.

[0122] The volume ratio of fluorosilane liquid to ammonia is 1:8;

[0123] (5) Centrifuge the solution from step (4) (5000 rpm, 10 minutes), remove the supernatant by magnetic separation, and vacuum dry for 24 hours to obtain modified magnetic nanoparticles, denoted as A2.

[0124] Example A3

[0125] A modified magnetic nanoparticle, the preparation method of which includes the following steps:

[0126] (1) Disperse 5 mg of nanoparticles B uniformly into an alcohol-water system (a mixed solution of anhydrous ethanol and deionized water with a volume ratio of 1:0.3) and stir at room temperature until homogeneous.

[0127] (2) Add ammonia and ethyl orthosilicate to the solution in step (1) (the amount added makes the effective amount based on silicon dioxide 4.5 mg), stir at 45°C for 8 h, remove the supernatant by magnetic adsorption, and obtain magnetic nanoparticles with surface modified SiO2, denoted as magnetic nanoparticle C.

[0128] The volume ratio of tetraethyl orthosilicate to alcohol-water system is 1:550; the volume ratio of tetraethyl orthosilicate to ammonia is 1:25.

[0129] (3) Disperse the magnetic nanoparticles C obtained in step (2) into anhydrous ethanol;

[0130] (4) Add 1.1 ml of heptadecafluorodecyltrimethoxysilane (the amount added makes the effective amount based on the fluorosilane film layer 17.5 mg) and ammonia to the solution in step (3), react at room temperature for 10 h, and obtain a black organic phase suspended in the upper layer of anhydrous ethanol solution.

[0131] The volume ratio of fluorosilane liquid to ammonia is 1:10;

[0132] (5) Centrifuge the solution from step (4) (5000 rpm, 10 minutes), remove the supernatant by magnetic separation, and vacuum dry for 24 hours to obtain modified magnetic nanoparticles, denoted as A3.

[0133] Example A4

[0134] A modified magnetic nanoparticle was prepared according to the method of Example A1, except that the amount of silicate was changed so that the effective amount, calculated as silicon dioxide, was 15 mg, and the modified magnetic nanoparticle was obtained, denoted as A4.

[0135] Example A5

[0136] A modified magnetic nanoparticle was prepared according to the method of Example A1, except that the amount of silicate was changed so that the effective amount, calculated as silicon dioxide, was 3 mg, and the modified magnetic nanoparticle was obtained, denoted as A5.

[0137] Example A6

[0138] A modified magnetic nanoparticle was prepared according to the method of Example A1, except that the amount of heptadecafluorodecyltrimethoxysilane was changed so that the effective amount, calculated as a fluorosilane film layer, was 19 mg, and the modified magnetic nanoparticle was obtained, denoted as A6.

[0139] Example A7

[0140] A modified magnetic nanoparticle was prepared according to the method of Example A1, except that the amount of heptadecafluorodecyltrimethoxysilane was changed so that the effective amount, calculated as a fluorosilane film layer, was 7.5 mg, and the modified magnetic nanoparticle was obtained, denoted as A7.

[0141] Comparative Example DA1

[0142] A modified magnetic nanoparticle was prepared according to the method of Example A1, except that the experimental process of coating with silica in step (2) was not performed, and the modified magnetic nanoparticle was obtained, denoted as DA1.

[0143] Comparative example DA2

[0144] A modified magnetic nanoparticle was prepared according to the method of Example A1, except that the fluorosilane modification process in step (5) was not performed, and the modified magnetic nanoparticle was obtained, denoted as DA2.

[0145] Comparative Example DA3

[0146] A type of iron oxide nanoparticle, obtained by referring to step (1) of the method in Example A1, is denoted as DA3.

[0147] Morphological characterization of modified magnetic nanoparticles in Test Example 1

[0148] (1) The modified magnetic nanoparticles prepared in Examples A1-A7 and Comparative Examples DA1-DA3 above were dissolved in anhydrous ethanol;

[0149] (2) Take 1 mg / mL of nanoparticles A1-A7 and DA1-DA3 dissolved in anhydrous ethanol into a 50 mL centrifuge tube, dilute the above stock solution by 1000 times, and add it dropwise to the surface of the copper mesh. Dry under vacuum at room temperature for 24 h until all solvent evaporates.

[0150] (3) The copper mesh containing the nanoparticles was transferred to a transmission electron microscope (TEM), and the accelerating voltage was set to 200kV. The microstructure of the nanoparticles was observed. The particle size results of nanoparticles A1-A7 and DA1-DA3 are shown in Table 1.

[0151] The electron microscopy results of magnetic nanoparticle A1 are shown below. Figure 1 As shown.

[0152] Test Example 2: Magnetic Characterization of Modified Magnetic Nanoparticles

[0153] 20 mg of nanoparticles A1-A7 and DA1-DA3 were vacuum-dried into powder and placed into the sample chamber of a vibrating sample magnetometer for measurement. Measurement parameters were: electrode diameter: 5 cm, room temperature measurement sensitivity: 5 × 10⁻⁶. -7 emu, moment measurement range: 5×10 -7 emu to 10 3 emu, maximum magnetic field: 2.17T @ 16.2mm pole spacing. The final test results are shown in Table 1.

[0154] Table 1

[0155] A1 7-15 59 A2 6-18 58 A3 6-19 58 A4 8-20 55 A5 6-18 57 A6 5-20 56 A7 5-20 56 DA1 3-15 62 DA2 5-20 60 DA3 3-15 62

[0156] As can be seen from Table 1, the modified magnetic nanoparticles of the present invention have a suitable particle size, good uniformity, and high saturation magnetization.

[0157] Example B: Magnetic ultrasound contrast agent

[0158] Example B1

[0159] A magnetic ultrasound contrast agent, the preparation method of which includes the following steps:

[0160] (1) Dissolve 10 mg of surfactant A and 0.5 mg of surfactant B in 10 ml of anhydrous ethanol at 80 °C and stir for 20 min to obtain solution B;

[0161] Surfactant A is a combination of DSPE-PEG2000 and DSPC (in a weight ratio of 1:1), and surfactant B is poloxamer. The HLB values ​​of both surfactant A and surfactant B are >10.

[0162] (2) Transfer solution B to a rotary evaporator and evaporate at 50°C for 5 minutes. Dissolve the evaporated product in 10 ml of 90% glycerol solution to obtain solution C.

[0163] (3) Dissolve 2 mg of doxorubicin hydrochloride completely in solution C at 15°C;

[0164] (4) 1 ml of perfluorohexane liquid and 5 mg of nanoparticles A1 were added dropwise to solution C at a uniform rate. The mixture was then subjected to ultrasonic treatment using an ultrasonic cavitation device with an ultrasonic power of 10 kW and a time of 20 min to obtain a magnetic ultrasonic contrast agent with internally modified magnetic nanoparticles, denoted as B1.

[0165] An appropriate amount of B1 was added dropwise to the surface of a copper mesh, and the mesh was vacuum dried at room temperature for 24 hours until all the solvent evaporated. The copper mesh containing the magnetic nanodroplets was then transferred to a transmission electron microscope (TEM), and the microstructure of B1 was observed with an accelerating voltage of 200 kV. The results are as follows. Figure 2 As shown. From Figure 2 It can be seen that the magnetic contrast agent is densely covered with nanodroplets with a particle size of about 0.3μm-1μm. The narrow particle size distribution of the nanodroplets can meet the imaging requirements of ultrasound contrast agents.

[0166] Example B2

[0167] A magnetic ultrasound contrast agent, the preparation method of which includes the following steps:

[0168] (1) Dissolve 11 mg of surfactant A and 1.5 mg of surfactant B in 10 ml of anhydrous ethanol at 90 °C and stir for 10 min to obtain solution B;

[0169] Surfactant A is a combination of DSPE-PEG2000 and DSPC (the weight ratio of the two is 1:0.8), and surfactant B is poloxamer. The HLB values ​​of surfactant A and surfactant B are both >10.

[0170] (2) Transfer solution B to a rotary evaporator and evaporate at 50°C for 5 minutes. Dissolve the evaporated product in 10 ml of 80% glycerol solution to obtain solution C.

[0171] (3) Dissolve 2 mg of doxorubicin hydrochloride completely in solution C at 18°C;

[0172] (4) 1.5 ml of perfluorohexane liquid and 5 mg of nanoparticles A1 were added dropwise to solution C at a uniform rate. The mixture was then subjected to ultrasonic treatment using an ultrasonic cavitation device with an ultrasonic power of 8 kW and a time of 25 min to obtain a magnetic ultrasonic contrast agent with internally modified magnetic nanoparticles, denoted as B2.

[0173] Example B3

[0174] A magnetic ultrasound contrast agent, the preparation method of which includes the following steps:

[0175] (1) Dissolve 8.64 mg of surfactant A and 0.36 mg of surfactant B in 10 ml of anhydrous ethanol at 85 °C and stir for 15 min to obtain solution B;

[0176] Surfactant A is a combination of DSPE-PEG2000 and DSPC (in a weight ratio of 1:1.2), and surfactant B is poloxamer. The HLB values ​​of both surfactant A and surfactant B are >10.

[0177] (2) Transfer solution B to a rotary evaporator and evaporate at 50°C for 5 minutes. Dissolve the evaporated product in 10 ml of 90% glycerol solution to obtain solution C.

[0178] (3) Dissolve 2 mg of doxorubicin hydrochloride completely in solution C at 18°C;

[0179] (4) 0.75 ml of perfluorohexane liquid and 5 mg of nanoparticles A1 were added dropwise to solution C at a uniform rate. The mixture was then subjected to ultrasonic treatment using an ultrasonic cavitation device with an ultrasonic power of 12 kW and a time of 8 min to obtain a magnetic ultrasonic contrast agent with internally modified magnetic nanoparticles, denoted as B3.

[0180] Examples B4-B7

[0181] A magnetic ultrasound contrast agent was prepared according to the method of Example B1, except that the magnetic nanoparticles were replaced. In Examples B4-B7, nanoparticles A4-A7 were used to replace nanoparticles A1 in equal amounts to obtain magnetic ultrasound contrast agents, denoted as B4, B5, B6, and B7.

[0182] Example B8

[0183] A magnetic ultrasound contrast agent was prepared according to the method of Example B1, except that an equal amount of DSPE-PEG2000 was used to replace DSPC to obtain a magnetic ultrasound contrast agent, denoted as B8.

[0184] Example B9

[0185] A magnetic ultrasound contrast agent was prepared according to the method of Example B1, except that the total amount of surfactant remained the same, the content of surfactant A was 80% by weight, and the content of surfactant B was 20% by weight, resulting in a magnetic ultrasound contrast agent, denoted as B9.

[0186] Example B10

[0187] A magnetic ultrasound contrast agent was prepared according to the method of Example B1, except that surfactant B was not included, the total amount of surfactant remained the same, and the ratio of DSPE-PEG2000 and DSPC in surfactant A remained the same, resulting in a magnetic ultrasound contrast agent, denoted as B10.

[0188] Comparative examples DB1-DB3

[0189] A magnetic ultrasound contrast agent was prepared according to the method of Example B1, except that in comparative examples DB1-DB3, nanoparticle A1 was replaced by equal amounts of nanoparticles DA1-DA3 to obtain magnetic ultrasound contrast agents, which were denoted as DB1, DB2, and DB3, respectively.

[0190] Test Example 3: Magnetic Characterization of Magnetic Ultrasound Contrast Agent

[0191] 5 mg of the magnetic ultrasonic contrast agent provided in Examples B1-B10 and Comparative Examples DB1-DB3 were respectively placed in the liquid sample chamber of the vibrating sample magnetometer, and measurements were started. Measurement parameters were: electrode diameter: 5 cm, room temperature measurement sensitivity: 5 × 10⁻⁶. -7 emu, moment measurement range: 5×10 -7 emu to 10 3 emu, maximum magnetic field: 2.17T@16.2mm pole spacing; final test results are shown in Table 2.

[0192] Test Example 4: Characterization of Drug Loading of Magnetic Ultrasound Contrast Agent

[0193] (1) Dilute the 5mg magnetic ultrasound contrast agent provided in Examples B1-B10 and Comparative Examples DB1-DB3 to 2mL of PBS. Take 200μL of the magnetic ultrasound contrast agent solution to be tested and place it in a centrifuge tube. Then add 800μL of acetonitrile solution, blow and swirl 10 times, and let stand for 2min to completely dissolve the nanodroplets. Specifically, the solution changes from the initial turbid state to clear and transparent, indicating that the drug has been completely released into the solution.

[0194] (2) Add 600 μL of PBS solution to the solution in step (1) to make the volume ratio of PBS to acetonitrile in the solution 1:1. Observe the total volume of the liquid and centrifuge at 4000 rpm for 5 min. Take out 100 μL of the upper layer solution and place it in a 96-well plate. Use a spectrophotometer to measure the absorbance (Od) of the solution in the 96-well plate at a wavelength of 485 nm.

[0195] (3) By matching the absorbance standard curve of doxorubicin at a wavelength of 485 nm (see...) Figure 3 The concentration of doxorubicin in each sample was quantitatively analyzed, and the drug loading rate of doxorubicin was quantitatively calculated.

[0196] Drug-loading capacity = (Total drug - Remaining drug) / (Total drug - Remaining drug + Membrane material) × 100%.

[0197] The doxorubicin loading efficiency of magnetic ultrasound contrast agents is shown in Table 2.

[0198] Table 2

[0199] B1 7 25 B2 6.8 24 B3 6.7 25 B4 6.5 21 B5 6.3 20 B6 6.7 18 B7 6.5 19 B8 6.6 20 B9 6.7 19 B10 6.5 15 DB1 0.15 20 DB2 0.12 19 DB3 0.18 19

[0200] As can be seen from Table 2, the magnetic ultrasound contrast agent of the present invention has high saturation magnetization and high drug loading efficiency, thus enabling the magnetic ultrasound contrast agent to well meet the requirements of in vivo circulation targeted delivery.

[0201] Test Example 5: Validation of in vivo delivery of magnetic ultrasound contrast agents

[0202] (1) Dilute human breast cancer cells MCF-7 to 1*10 6 Cells / mL, 300 μL of the above cell solution was injected into the inner side of the right leg of BALB / c nude mice, and the mice were kept in a sterile environment for 10 days until the subcutaneous tumor grew.

[0203] (2) Dilute 5 mg / mL magnetic ultrasound contrast agent B1 4.5 times, take 100 μL of the above solution and inject it into the nude mouse through the tail vein, and magnetically enrich it for 30 minutes.

[0204] (3) Doxorubicin stock solution with the same drug concentration as magnetic ultrasound contrast agent was injected into nude mice via the caudal vein and magnetically enriched for 30 minutes.

[0205] (4) Apply a 0.5 MHz ultrasound probe with a 50% duty cycle to the surface of the tumor to perform targeted bursting of the drug-loaded magnetic ultrasound contrast agent for 5 minutes, thereby releasing doxorubicin.

[0206] (5) After treatment, the nude mice were immediately euthanized by dislocation of the neck, and the tumor tissue was immediately frozen with liquid nitrogen after dissection.

[0207] (6) Weigh the above samples and add 0.3 mL of methanol to each sample. Vortex for 1 min, grind for 5 min, and vortex for 5 min. Centrifuge at 13000 rpm for 10 min and take the supernatant for analysis.

[0208] (7) Accurately weigh doxorubicin standard and prepare a stock solution of 2.00 mg / mL with DMSO and methanol. Dilute the stock solution with pure methanol to a standard solution with a concentration of 200 ng / mL and perform high performance liquid chromatography (HPLC) measurement.

[0209] (8) The chromatograms of the compounds were acquired and integrated using Xcilabur 3.0 (Thermo) software. Linear regression was performed with weighted coefficients to obtain the standard curve, and the results are as follows: Figure 4 As shown.

[0210] The final results showed that the average concentration of doxorubicin delivered by magnetic ultrasound contrast agent carrying doxorubicin was 16.91 ng / g, which was about 50% more efficient than the treatment strategy of direct injection of doxorubicin (8.7 ng / g).

[0211] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A modified magnetic nanoparticle for use in the preparation of magnetic ultrasound contrast agents, characterized in that, The core of the magnetic nanoparticles is a magnetic metal oxide, and the surface of the magnetic metal oxide is at least partially coated with a silica coating. The silica coating is modified with fluorosilane. Relative to 100 parts by weight of magnetic nanoparticles, the silica coating content is 70-90 parts by weight, and the fluorosilane content is 330-350 parts by weight. The particle size of the magnetic nanoparticles is 5 nm-20 nm, the thickness of the silica coating is 2-5 nm, and the thickness of the fluorosilane film is 1-2 nm. The fluorosilane has the following molecular formula: (CH3-(CH2)). a O)3Si-(CH2) b -(CF2) c -CF3; where a is 0, 1, 2, 3; b is 2, 3, 4, 5; c is 5, 6, 7, 8; The magnetic ultrasound contrast agent contains at least a fluorine phase liquid, and the magnetic nanoparticles can be uniformly dispersed in the fluorine phase liquid. When used to prepare the magnetic ultrasound contrast agent, the saturation magnetization and drug loading efficiency of the magnetic ultrasound contrast agent can be improved.

2. The modified magnetic nanoparticles for preparing magnetic ultrasound contrast agents according to claim 1, characterized in that, The magnetic metal oxide is selected from one or more of Fe3O4, Fe2O3, CoFe2O4, NiFe2O4, and MnFe2O4.

3. The modified magnetic nanoparticles for preparing magnetic ultrasound contrast agents according to claim 2, characterized in that, The magnetic metal oxide is Fe3O4.

4. A method for preparing modified magnetic nanoparticles according to any one of claims 1-3 for use in preparing magnetic ultrasound contrast agents, characterized in that, The method includes the following steps: (1) In the first alkaline environment, the alcohol-water system of silicate ester and magnetic nanoparticles are brought into first contact; (2) In the second alkaline environment, the nanoparticles obtained in step (1) are brought into a second contact with the fluorosilane liquid; In step (1), the effective amount of silicate relative to 100 parts by weight of magnetic nanoparticles is 70-90 parts by weight of silica. In step (2), the effective amount of fluorosilane is 330-350 parts by weight relative to 100 parts by weight of magnetic nanoparticles.

5. The method according to claim 4, characterized in that, In step (1), the volume ratio of silicate ester to alcohol-water system is 1:(400-700).

6. The method according to claim 4 or 5, characterized in that, The conditions for the first contact include: a temperature of 30-70°C and a time of 3-10 hours; and / or, The conditions for the second contact include a temperature of 10-40°C and a time of 5-20 hours.

7. Modified magnetic nanoparticles prepared by the method according to any one of claims 4-6.

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