Application of diphosphonic acid group-containing small molecules in preparation of hydrophilic inorganic nanoparticles

Abstract

The invention discloses application of small molecules containing diphosphonic acid groups in preparation of hydrophilic inorganic nanoparticles, and relates to the technical field of preparation of inorganic nanoparticles. The method comprises the following steps: dispersing inorganic nanoparticles coated with a hydrophobic organic compound on the surface into tetrahydrofuran, dropwise adding into a small molecule solution containing a diphosphonic acid group, uniformly mixing, and adding diethyl ether to prepare the hydrophilic inorganic nanoparticles. The method is simple and convenient to operate, mild in condition, rapid in conversion, high in conversion rate, energy-saving and efficient, and the converted nano-particle freeze-dried powder is stable in property and easy to store and transport and has certain popularization value. The problems that in the prior art, an inorganic nanoparticle hydrophilic modification method is complex, and conversion efficiency is low are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of inorganic nanoparticle preparation, and specifically relates to the application of small molecules containing bisphosphonic acid groups in the preparation of hydrophilic inorganic nanoparticles. Background Art

[0002] Inorganic nanoparticles generally include materials such as ferrite nanoparticles, gold nanoparticles, or quantum dots. Among them, ferrite nanoparticles are a large class of materials including nanoparticles such as iron(III) oxide, iron(II,III) oxide, manganese ferrite, cobalt ferrite, zinc ferrite, and manganese-zinc ferrite. Such materials have great application potential in the biomedical field, such as magnetic resonance imaging, targeted drug delivery, gene therapy, DNA purification, and magnetic hyperthermia (Meng et al., Journal of Nanobiotechnology, 2024, 22, 24).

[0003] The existing common methods for preparing ferrite nanoparticles mainly include chemical coprecipitation method, hydrothermal synthesis method, high-temperature thermal decomposition method, etc. Among them, the high-temperature thermal decomposition method is increasingly used in the preparation of nanomaterials due to the advantages of controllable size, uniform particle size distribution and good crystallinity of the prepared nanoparticles. However, most of the ferrite nanoparticles prepared by the high-temperature thermal decomposition method are coated with hydrophobic organic compounds such as oleic acid or oleylamine on the outer layer, and it is necessary to convert their surface from hydrophobic to hydrophilic to realize their application in the biomedical field. The existing common methods for converting nanoparticles from hydrophobic to hydrophilic can be mainly divided into three types: one is to achieve the conversion of its hydrophilicity by coating hydrophobic nanoparticles with amphiphilic polymers (Bronstein et al., Journal of Physical Chemistry C, 2010, 114, 21908). Usually, the particle size of the nanoparticles obtained by this method will increase due to the accumulation of the surface polymer layer, and due to the existence of the polymer layer with a large molecular weight, the direct contact between the surface of the magnetic nanoparticles and water is also affected, thereby affecting their application as a contrast agent in magnetic resonance imaging; the second is through ligand exchange, using other hydrophilic molecules to replace the oleic acid or oleylamine on the surface of the nanoparticles to perform hydrophilic-hydrophobic conversion on the surface of the nanoparticles. For example, the Fan research group realized the hydrophilic conversion of manganese ferrite nanoparticles using phosphorylated polyethylene glycol (Fan et al., ACS Nano, 2017, 11, 3614). However, the amount of phosphorylated polyethylene glycol consumed by this method is much more than the amount of nanoparticles (the mass ratio of the two is 10:1), and it causes certain pollution to the environment (the organic solvents used in the experiment need to be volatilized into the atmosphere in an argon atmosphere), and the preparation process takes more than two days; the third is to directly oxidize the fatty acids on the surface of the nanoparticles to convert them from hydrophobic to hydrophilic (Ruiz-Cabello et al., Chemistry-European Journal, 2008, 14, 9126). However, this method has harsh reaction conditions and is time-consuming. It is necessary to first treat the nanoparticles with a strong oxidant potassium permanganate (heating under reflux for 4 h), and then under strong acid (pH = 2.9) or strong base conditions, heat under reflux and stir the nanoparticles for a long time (20 h or 32 h). Therefore, there is an urgent need for a new hydrophilic modification method. Summary of the Invention

[0004] In order to solve the above technical problems, the object of the present invention is to provide the application of small molecules containing bisphosphonic acid groups in the preparation of hydrophilic inorganic nanoparticles to solve the problems of complex hydrophilic modification methods and low conversion efficiency of inorganic nanoparticles in the prior art.

[0005] The technical solution of the present invention for solving the above technical problems is as follows: providing the application of a small molecule containing a bisphosphonic acid group in the preparation of hydrophilic inorganic nanoparticles, and the structure of the small molecule containing a bisphosphonic acid group contains two phosphorous acid groups -PO 3 H 2 and the two phosphorous acid groups -PO 3 H 2 are connected to the same carbon atom.

[0006] The beneficial effects of the present invention are as follows: The present invention uses a small molecule containing a bisphosphonic acid group, which is simple and easy to prepare, as a ligand to modify the surface of metal nanoparticles through metal-ligand interaction. By ligand exchange, the transformation of metal nanoparticles from hydrophobic to hydrophilic is successfully achieved, and good results are obtained. This method is simple to operate, mild in conditions, fast in transformation, high in conversion rate, energy-saving and efficient. The freeze-dried powder of the transformed nanoparticles has stable properties, is easy to store and transport, and has certain popularization value.

[0007] In the present invention, the bisphosphonic acid group in the small molecule containing a bisphosphonic acid group is a functional group that can coordinate with the constituent metals (such as manganese, iron, cobalt, nickel, magnesium, calcium, strontium, barium, etc.) of inorganic nanoparticles. Different from the common diphosphoric acid group structure (P-O-P), the phosphorus atoms in the bisphosphonic acid group skeleton are connected by a carbon atom (P-C-P), so its coordination with metals (ions or nanoparticles) is stronger, and the formed metal-ligand structure is more stable.

[0008] On the basis of the above technical solution, the present invention can also be improved as follows:

[0009] Further, the structural formula of the small molecule containing a bisphosphonic acid group is

[0010] Further, through the coordination of the bisphosphonic acid group and inorganic nanoparticles, the hydrophobic organic compound on the surface of the inorganic nanoparticles is replaced with a small molecule containing a bisphosphonic acid group to obtain hydrophilic inorganic nanoparticles.

[0011] Further, the inorganic nanoparticles are at least one of iron oxide nanoparticles, magnetite nanoparticles, manganese ferrite nanoparticles, cobalt ferrite nanoparticles, zinc ferrite nanoparticles, and manganese-zinc ferrite nanoparticles.

[0012] Further, the hydrophobic organic compound is oleic acid or oleylamine.

[0013] Further, the structure of the small molecule containing a bisphosphonic acid group further contains at least one of a mercapto group, a carboxyl group, an amino group, an amine group, and a maleimide group.

[0014] Further, the structure of the small molecule containing a bisphosphonic acid group contains two phosphorous acid groups -PO3 H 2 and two phosphite groups -PO 3 H 2 are connected to the same carbon atom; the small molecule structure containing a bisphosphonic acid group also contains at least one of a mercapto group, a carboxyl group, an amino group, an amine group, and a maleimide group.

[0015] Furthermore, the small molecule containing a bisphosphonic acid group is prepared by the following method: acryloylated pamidronic acid is dissolved in water, a mercapto source is added, stirred, concentrated, acetone is added to obtain a white precipitate, impurities are removed, and vacuum drying is performed to obtain the small molecule containing a bisphosphonic acid group.

[0016] Furthermore, acryloylated pamidronic acid is prepared by the following method:

[0017] (1) Dissolve β-alanine in a solvent, drop in phosphorus trichloride, stir at 80 - 85 °C for 4 - 6 h, cool to room temperature, then add deionized water, hydrolyze at 100 - 110 °C for 3 - 5 h, cool to room temperature, add sodium hydroxide solution, add methanol, collect the precipitate, and obtain pamidronic acid.

[0018] (2) Dissolve the pamidronic acid prepared in step (1) in sodium hydroxide solution, drop in acryloyl chloride at 0 °C, then adjust the pH value to 9 - 11, repeat the operations of dropping acryloyl chloride and adjusting the pH value 3 times, then return to room temperature, stir for 1 - 2 h, extract and collect the aqueous phase, remove water to obtain a solid-phase product, wash and perform vacuum drying to obtain acryloylated pamidronic acid.

[0019] Furthermore, in step (1), the solvent is methanesulfonic acid.

[0020] Furthermore, in step (1), phosphorus trichloride is dropped in under stirring.

[0021] Furthermore, in step (1), phosphorus trichloride is dropped in and stirred at 82 °C for 5 h.

[0022] Furthermore, in step (1), the mass-to-volume ratio of β-alanine, solvent, phosphorus trichloride, deionized water, sodium hydroxide solution, and methanol is 2 - 3 g : 8 - 12 mL : 5 - 10 mL : 10 - 15 mL : 10 - 15 mL : 38 - 42 mL.

[0023] Furthermore, in step (1), the mass-to-volume ratio of β-alanine, solvent, phosphorus trichloride, deionized water, sodium hydroxide solution, and methanol is 2.23 g : 10 mL : 7 mL : 12 mL : 12 mL : 40 mL.

[0024] Furthermore, in step (1), hydrolysis is performed at 105 °C for 4 h.

[0025] Further, in step (1), the concentration of the sodium hydroxide solution is 45-55 wt%.

[0026] Further, in step (1), the concentration of the sodium hydroxide solution is 50 wt%.

[0027] Further, in step (2), the mass-volume ratio of pamidronic acid, the sodium hydroxide solution, and acryloyl chloride is 0.4-0.5 g: 18-22 mL: 150-180 μL, and the volume of the acryloyl chloride is the volume of a single dropwise addition.

[0028] Further, in step (2), the mass-volume ratio of pamidronic acid, the sodium hydroxide solution, and acryloyl chloride is 0.47 g: 20 mL: 160 μL, and the volume of the acryloyl chloride is the volume of a single dropwise addition.

[0029] Further, in step (2), the concentration of the sodium hydroxide solution is 3-5 wt%.

[0030] Further, in step (2), the concentration of the sodium hydroxide solution is 4 wt%.

[0031] Further, in step (2), the pH value is adjusted to 10.

[0032] Further, in step (2), ethyl acetate extraction is used.

[0033] Further, in step (2), water is removed by rotary evaporation.

[0034] Further, in step (2), methanol washing is used.

[0035] Further, the thiol source is 1,4-dithiothreitol, cysteine, glutathione, or mercaptoethylamine.

[0036] Further, the mass-volume ratio of acryloylated pamidronic acid, the thiol source, water, and acetone is 240-260 mg: 1300-1350 mg: 20-30 mL: 8-12 mL.

[0037] Further, the mass-volume ratio of acryloylated pamidronic acid, the thiol source, water, and acetone is 250 mg: 1335 mg: 25 mL: 10 mL.

[0038] Further, the white precipitate is dissolved in deionized water, and then methanol is added to precipitate a white solid, completing the impurity removal process.

[0039] The present invention also provides a method for preparing hydrophilic inorganic nanoparticles, comprising the following steps: dispersing inorganic nanoparticles coated with a hydrophobic organic compound in tetrahydrofuran to obtain a mixed solution, dropping the mixed solution into a small molecule solution containing a bisphosphonic acid group, mixing evenly, adding ether, centrifuging, filtering the aqueous phase, and freeze-drying to obtain hydrophilic inorganic nanoparticles.

[0040] Further, the inorganic nanoparticles coated with a hydrophobic organic compound are manganese ferrite nanoparticles coated with oleic acid.

[0041] Further, the inorganic nanoparticles coated with a hydrophobic organic compound are prepared by the following method: dissolving manganese oleate, iron erucate, oleyl alcohol and oleic acid in benzyl ether at room temperature, heating and reacting under an argon atmosphere, then cooling to room temperature, centrifuging, and separating the precipitate to obtain inorganic nanoparticles coated with a hydrophobic organic compound.

[0042] Further, the inorganic nanoparticles coated with a hydrophobic organic compound are prepared by the following method: dissolving manganese oleate, iron erucate, oleyl alcohol and oleic acid in benzyl ether at room temperature, heating to 110 °C and maintaining for 30 min under an argon atmosphere, when the temperature rises to 130 °C, carrying out condensation reflux, when the temperature reaches 265 °C, maintaining for 30 min, then cooling to room temperature, using ethanol as a precipitant and n-hexane as a dispersant, centrifuging, and separating the precipitate to obtain inorganic nanoparticles coated with a hydrophobic organic compound.

[0043] Further, the particle size of the inorganic nanoparticles coated with a hydrophobic organic compound is 1-100 nm.

[0044] Further, the mass-volume ratio of the inorganic nanoparticles coated with a hydrophobic organic compound to tetrahydrofuran is 20-25 mg: 1-2 mL.

[0045] Further, the mass-volume ratio of the inorganic nanoparticles coated with a hydrophobic organic compound to tetrahydrofuran is 23.8 mg: 1.5 mL.

[0046] Further, the inorganic nanoparticles coated with a hydrophobic organic compound are dispersed in tetrahydrofuran by ultrasonic treatment.

[0047] Further, the ultrasonic treatment is carried out for 0.8-3 min.

[0048] Further, the mass-volume ratio of the small molecule containing a bisphosphonic acid group to water in the small molecule solution containing a bisphosphonic acid group is 45-55 mg: 2-5 mL.

[0049] Further, the mass-volume ratio of the small molecule containing a bisphosphonic acid group to water in the small molecule solution containing a bisphosphonic acid group is 50 mg: 3 mL.

[0050] Furthermore, the volume ratio of tetrahydrofuran to diethyl ether is 1:8 - 12.

[0051] Furthermore, the volume ratio of tetrahydrofuran to diethyl ether is 1:10.

[0052] Furthermore, centrifuge at 8000 - 12000 rpm / min for 10 - 20 min.

[0053] Furthermore, filter using a 0.2 - 0.3 μm membrane.

[0054] Furthermore, filter using a 0.22 μm membrane.

[0055] Furthermore, lyophilize for 15 - 20 h.

[0056] Furthermore, lyophilize for 18 h.

[0057] Furthermore, the mass ratio of the inorganic nanoparticles surface - coated with a hydrophobic organic compound to the small molecule containing a bisphosphonic acid group is 1:1.8 - 2.2.

[0058] Furthermore, the mass ratio of the inorganic nanoparticles surface - coated with a hydrophobic organic compound to the small molecule containing a bisphosphonic acid group is 1:2.

[0059] The beneficial effects of adopting the above - mentioned further technical solutions are as follows: At this ratio, the complete conversion of the inorganic nanoparticles from hydrophobic to hydrophilic can be achieved, and the dosage of the small molecule is reasonable, eliminating the need to remove the small molecules not connected to the surface of the inorganic nanoparticles subsequently.

[0060] The present invention also provides the hydrophilic inorganic nanoparticles prepared by the above - mentioned method.

[0061] The present invention also provides the applications of the above - mentioned hydrophilic inorganic nanoparticles in magnetic resonance imaging, preparation of magnetic hyperthermia nanoparticles, hydrogel labeling, preparation of gene therapy drugs, and preparation of functionalized hydrophilic inorganic nanoparticles.

[0062] Furthermore, label the hydrophilic inorganic nanoparticles with growth factors, glycosaminoglycans, polyethylene glycol, or fluorescent markers to achieve the functionalization of the hydrophilic inorganic nanoparticles.

[0063] Furthermore, the growth factor is bone morphogenetic protein 2.

[0064] Furthermore, the glycosaminoglycan is hyaluronic acid.

[0065] Furthermore, label the hydrophilic inorganic nanoparticles with a fluorescent marker to achieve the functionalization of the hydrophilic inorganic nanoparticles.

[0066] Furthermore, the fluorescent marker is DyLight TM800 maleimide or Alexa Fluor TM 647.

[0067] Furthermore, the method for labeling hydrophilic inorganic nanoparticles with a fluorescent marker is as follows: disperse the hydrophilic inorganic nanoparticles in deionized water, add the fluorescent marker, shake evenly to obtain a dispersion system, dialyze, and freeze-dry to prepare.

[0068] Furthermore, the mass-volume ratio of the hydrophilic inorganic nanoparticles, the fluorescent marker, and deionized water is 2 mg: 20 mg: 1 mL.

[0069] Furthermore, place the dispersion system in a dialysis bag, place the dialysis bag in deionized water, adjust the pH value of the deionized water to 4 with hydrochloric acid, perform dialysis in the dark, and repeat the dialysis in the dark 2 times.

[0070] The present invention has the following beneficial effects:

[0071] 1. The modification of the hydrophilicity of the nanoparticle surface in the present invention is simple, efficient, and mild in conditions. Only by dissolving the small molecule containing a bisphosphonic acid group in water at room temperature and mixing and shaking it with the tetrahydrofuran dispersion system of the nanoparticles, the modification of the nanoparticles from hydrophobic to hydrophilic can be immediately achieved (less than 1 min), avoiding the need in the prior art to use a large excess of high-purity hydrophilic polymers and taking several hours to modify the nanoparticles. Among them, in the prior art, the large excess means that the mass of the polymer is ten times or more the mass of the nanoparticles, and several hours means 20 hours or more.

[0072] 2. The purification of the hydrophilic inorganic nanoparticles in the present invention is simple. Only by using ether and removing the organic phase and oleic acid impurities after centrifugation, a high-purity aqueous dispersion system of hydrophilic inorganic nanoparticles can be obtained.

[0073] 3. The hydrophilic inorganic nanoparticles obtained in the present invention are in powder form after freeze-drying, can be stored at room temperature for a long time without particle aggregation; and when further redispersed in water, good dispersibility is still achieved, and the particle size distribution of the nanoparticles does not change significantly.

[0074] 4. The small molecule containing a bisphosphonic acid group used in the present invention is easy to prepare, and its structure can further contain functional groups such as a mercapto group, a carboxyl group, an amino group, an amine group, and a maleimide group, and molecules such as growth factors such as bone morphogenetic protein 2, glycosaminoglycans such as hyaluronic acid, polyethylene glycol, and fluorescent markers can be further linked to the surface of the hydrophilic inorganic nanoparticles, thereby realizing the functionalization of the hydrophilic inorganic nanoparticles.

[0075] 5. The hydrophilic inorganic nanoparticles prepared in the present invention can label biomaterials such as hydrogels to achieve magnetic resonance imaging (MRI) of the hydrogel material. Description of the Drawings

[0076] Figure 1 It is a schematic diagram of the hydrophilic modification of the present invention;

[0077] Figure 2 It is a schematic diagram of labeling hydrophilic inorganic nanoparticles with a fluorescent marker according to the present invention;

[0078] Figure 3 It shows inorganic nanoparticles coated with oleic acid prepared in Comparative Example 1 uniformly dispersed in n-hexane;

[0079] Figure 4 It shows hydrophilic inorganic nanoparticles prepared in Example 1 uniformly dispersed in deionized water;

[0080] Figure 5 It is a TEM image of inorganic nanoparticles coated with oleic acid prepared in Comparative Example 1;

[0081] Figure 6 It is a TEM image of hydrophilic inorganic nanoparticles prepared in Example 1;

[0082] Figure 7 It is a TEM image of inorganic nanoparticles coated with oleic acid prepared in Comparative Example 2;

[0083] Figure 8 It is the particle size statistics of inorganic nanoparticles coated with oleic acid prepared in Comparative Example 2;

[0084] Figure 9 It is a TEM image of hydrophilic inorganic nanoparticles prepared in Example 2;

[0085] Figure 10 It is the particle size statistics of hydrophilic inorganic nanoparticles prepared in Example 2;

[0086] Figure 11 It shows inorganic nanoparticles coated with oleic acid prepared in Comparative Example 3 aggregated in water;

[0087] Figure 12 It shows hydrophilic inorganic nanoparticles prepared in Example 3 uniformly dispersed in water. Detailed Embodiments

[0088] The principles and features of the present invention will be described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0089] Example 1:

[0090] A hydrophilic inorganic nanoparticle (a hydrophilic manganese ferrite nanoparticle modified by a mercapto-diphosphonic acid small molecule), and a preparation method thereof comprises the following steps:

[0091] (1) Preparation of pamidronic acid: Dissolve β-alanine in a methanesulfonic acid solvent, stir vigorously, dropwise add phosphorus trichloride, stir at 82 °C for 5 h, cool to room temperature, then add deionized water, hydrolyze at 105 °C for 4 h, cool to room temperature, add a sodium hydroxide solution (concentration 50 wt%), add methanol, a white precipitate appears, collect the precipitate by suction filtration to obtain pamidronic acid; wherein, the mass-volume ratio of β-alanine, solvent, phosphorus trichloride, deionized water, sodium hydroxide solution and methanol is 2.23 g: 10 mL: 7 mL: 12 mL: 12 mL: 40 mL;

[0092] The reaction equation is as follows:

[0093]

[0094] (2) Preparation of acryloylated pamidronic acid: Dissolve the pamidronic acid prepared in step (1) in a sodium hydroxide solution (concentration 4 wt%), at 0 °C, dropwise add acryloyl chloride, then adjust the pH value from 2 to 10, repeat the operations of dropping acryloyl chloride and adjusting the pH value 3 times, then return to room temperature, stir for 1.5 h, extract with ethyl acetate to collect the aqueous phase, remove water by rotary evaporation to obtain a solid-phase product, wash with methanol, collect the solid insoluble in methanol, and dry in vacuum to obtain acryloylated pamidronic acid; wherein, the mass-volume ratio of pamidronic acid, sodium hydroxide solution and acryloyl chloride is 0.47 g: 20 mL: 160 μL, and the volume of the acryloyl chloride is the volume of a single dropwise addition;

[0095] The reaction equation is as follows:

[0096]

[0097] (3) Preparation of a small molecule containing a diphosphonic acid group: Dissolve the acryloylated pamidronic acid prepared in step (2) in water, add a mercapto source (1,4-dithiothreitol), stir overnight at room temperature, concentrate by rotary evaporation, add acetone, filter to collect the white precipitate, dissolve the white precipitate in deionized water, then add methanol, and a white solid precipitates to complete the impurity removal process, and dry in vacuum to obtain a small molecule containing a diphosphonic acid group with one end being a mercapto; wherein, the mass-volume ratio of acryloylated pamidronic acid, mercapto source, water and acetone is 250 mg: 1335 mg: 25 mL: 10 mL;

[0098] The reaction equation is as follows:

[0099]

[0100] (4) Preparation of inorganic nanoparticles (inorganic manganese ferrite nanoparticles) with a hydrophobic organic compound-coated surface: Synthesized according to the reference ACS Nano 2017, 11, 3614 - 3631;

[0101] At room temperature, 0.62 g of manganese oleate, 1.07 g of iron erucate, 1.61 g of oleyl alcohol, and 0.57 g of oleic acid were dissolved in 10 g of benzyl ether. The mixture was transferred to a three-necked flask. Under an argon atmosphere, it was heated to 110 °C and maintained for 30 min. Using a programmable temperature-controlled magnetic stirrer heater, the temperature of the reaction system was raised to 265 °C at a rate of 5 °C / min. When the temperature reached 130 °C, an air condenser was placed on the three-necked flask to achieve solvent condensation and reflux. When the temperature reached 265 °C, it was maintained for 30 min. Then, the solution in the flask was transferred to an ice bath and quickly cooled to room temperature. Ethanol was used as the precipitant and n-hexane as the dispersant. The mixture was centrifuged three times with a high-speed centrifuge (8000 rpm / min, 10 min) to separate the precipitate, and inorganic nanoparticles with a hydrophobic organic compound-coated surface (particle size: 3 nm) were obtained;

[0102] (5) Hydrophilic modification: The inorganic nanoparticles with a hydrophobic organic compound-coated surface prepared in step (4) were dispersed in tetrahydrofuran (mass-to-volume ratio: 23.8 mg: 1.5 mL) and sonicated for 1 min to uniformly disperse them in tetrahydrofuran, obtaining a mixed solution. The mixed solution was dropped into the small molecule solution containing bisphosphonic acid groups prepared in step (3) (mass-to-volume ratio: 50 mg: 3 mL), and the mixture was shaken with an oscillator for 1 min. The whole system showed uniform mixing without precipitation. Ether was added, and the oleic acid in the system was removed by centrifugation with a high-speed centrifuge (8000 rpm / min, 10 min). The upper organic phase was poured off, and the lower aqueous phase was filtered through a 0.22 μm membrane and freeze-dried for 18 h to obtain hydrophilic inorganic nanoparticles (see Figure 1 ); wherein, the mass ratio of the inorganic nanoparticles with a hydrophobic organic compound-coated surface to the small molecule containing bisphosphonic acid groups is 1:2.

[0103] The hydrophilic inorganic nanoparticles were labeled with a fluorescent marker. The method includes the following steps (see Figure 2 ):

[0104] The hydrophilic inorganic nanoparticles were dispersed in deionized water, and a fluorescent marker (DyLight TM 800 maleimide) was added and shaken for 1 min to obtain a dispersion system. The dispersion system was placed in a dialysis bag (MW3500), and the dialysis bag was placed in deionized water. The pH value of the deionized water was adjusted to 4 with hydrochloric acid, and dark dialysis was carried out. Dark dialysis was repeated twice, and then freeze-dried for 4 h to obtain the product.

[0105] Example 2:

[0106] A hydrophilic inorganic nanoparticle (a hydrophilic cobalt ferrite particle modified by a mercapto-bisphosphonic acid small molecule), and a preparation method thereof comprises the following steps:

[0107] (1) Preparation of pamidronic acid: Dissolve β-alanine in a methanesulfonic acid solvent, stir vigorously, dropwise add phosphorus trichloride, stir at 80 °C for 6 h, cool to room temperature, then add deionized water, hydrolyze at 100 °C for 5 h, cool to room temperature, add a sodium hydroxide solution (concentration 45 wt%), add methanol, a white precipitate appears, collect the precipitate by suction filtration to obtain pamidronic acid; wherein, the mass-volume ratio of β-alanine, solvent, phosphorus trichloride, deionized water, sodium hydroxide solution and methanol is 2 g: 8 mL: 5 mL: 10 mL: 10 mL: 38 mL;

[0108] (2) Preparation of acryloylated pamidronic acid: Dissolve the pamidronic acid prepared in step (1) in a sodium hydroxide solution (concentration 3 wt%), at 0 °C, dropwise add acryloyl chloride, then adjust the pH value from 2 to 9, repeat the operations of dropping acryloyl chloride and adjusting the pH value 3 times, then return to room temperature, stir for 1 h, extract with ethyl acetate to collect the aqueous phase, remove water by rotary evaporation to obtain a solid-phase product, wash with methanol, collect the solid insoluble in methanol, and dry in vacuum to obtain acryloylated pamidronic acid; wherein, the mass-volume ratio of pamidronic acid, sodium hydroxide solution and acryloyl chloride is 0.4 g: 18 mL: 150 μL, and the volume of the acryloyl chloride is the volume of a single dropwise addition;

[0109] (3) Preparation of a small molecule containing a bisphosphonic acid group: Dissolve the acryloylated pamidronic acid prepared in step (2) in water, add a mercapto source (1,4-dithiothreitol), stir overnight at room temperature, concentrate by rotary evaporation, add acetone, filter to collect the white precipitate, dissolve the white precipitate in deionized water, then add methanol, a white solid precipitates, complete the impurity removal process, and dry in vacuum to obtain a small molecule containing a bisphosphonic acid group with a mercapto group at one end; wherein, the mass-volume ratio of acryloylated pamidronic acid, mercapto source, water and acetone is 240 mg: 1300 mg: 20 mL: 8 mL;

[0110] (4) Preparation of an inorganic nanoparticle (cobalt ferrite nanoparticle) with a hydrophobic organic compound coated on the surface: Synthesized with reference to ACS Nano 2017, 11, 3614-3631;

[0111] At room temperature, 0.62 g of cobalt oleate, 1.07 g of iron erucate, 1.61 g of oleyl alcohol and 0.57 g of oleic acid were dissolved in 10 g of benzyl ether. The mixture was transferred to a three-necked flask. Under an argon atmosphere, it was heated to 110 °C and maintained for 30 min. Using a programmable temperature-controlled magnetic stirrer heater, the temperature of the reaction system was raised to 265 °C at a rate of 5 °C / min. When the temperature reached 130 °C, an air condenser was placed on the three-necked flask to achieve solvent condensation reflux. When the temperature reached 265 °C, it was maintained for 30 min. Then the solution in the flask was transferred to an ice bath and quickly cooled to room temperature. Ethanol was used as the precipitant and n-hexane was used as the dispersant. It was centrifuged 3 times with a high-speed centrifuge (8000 rpm / min, 10 min) to separate the precipitate, and inorganic nanoparticles (particle size 4.3 nm) coated with hydrophobic organic compounds were obtained;

[0112] (5) Hydrophilic modification: The inorganic nanoparticles coated with hydrophobic organic compounds prepared in step (4) were dispersed in tetrahydrofuran and sonicated for 0.8 min to make them uniformly dispersed in tetrahydrofuran to obtain a mixed solution (mass-volume ratio 20 mg: 1 mL). The mixed solution was dropped into the small molecule solution containing bisphosphonic acid groups prepared in step (3) (mass-volume ratio 45 mg: 2 mL), and shaken with an oscillator for 1 min. The whole system was uniformly mixed and no precipitation occurred. Ether was added, and the oleic acid in the system was removed by centrifugation with a high-speed centrifuge (9000 rpm / min, 15 min). The upper organic phase was poured off, and the lower aqueous phase was filtered through a 0.2 μm membrane and freeze-dried for 15 h to obtain hydrophilic inorganic nanoparticles; among them, the mass ratio of the inorganic nanoparticles coated with hydrophobic organic compounds to the small molecule containing bisphosphonic acid groups was 1:1.8.

[0113] The hydrophilic inorganic nanoparticles were labeled with a fluorescent label, and the method included the following steps:

[0114] The hydrophilic inorganic nanoparticles were dispersed in deionized water, and a fluorescent label (DyLight TM 800 maleimide) was added and shaken for 1 min to obtain a dispersion system. The dispersion system was placed in a dialysis bag (MW3500), and the dialysis bag was placed in deionized water. The pH value of the deionized water was adjusted to 4 with hydrochloric acid, and dark dialysis was carried out. Dark dialysis was repeated 2 times and freeze-dried for 4 h to obtain.

[0115] Example 3:

[0116] A kind of hydrophilic inorganic nanoparticles (hydrophilic manganese ferrite nanoparticles modified by cysteine-bisphosphonic acid small molecules), and its preparation method includes the following steps:

[0117] (1) Preparation of pamidronic acid: Dissolve β-alanine in methanesulfonic acid solvent, stir vigorously, dropwise add phosphorus trichloride, stir at 85 °C for 4 h, cool to room temperature, then add deionized water, hydrolyze at 110 °C for 3 h, cool to room temperature, add sodium hydroxide solution (concentration 55 wt%), add methanol, white precipitate appears, collect the precipitate by suction filtration to obtain pamidronic acid; wherein, the mass-volume ratio of β-alanine, solvent, phosphorus trichloride, deionized water, sodium hydroxide solution and methanol is 3 g: 12 mL: 10 mL: 15 mL: 15 mL: 42 mL;

[0118] (2) Preparation of acrylated pamidronic acid: Dissolve the pamidronic acid prepared in step (1) in sodium hydroxide solution (concentration 4 wt%), at 0 °C, dropwise add acryloyl chloride, then adjust the pH value from 2 to 11, repeat the operations of dropping acryloyl chloride and adjusting the pH value 3 times, then return to room temperature, stir for 2 h, use ethyl acetate to extract and collect the aqueous phase, remove water by rotary evaporation to obtain a solid-phase product, wash with methanol, collect the solid insoluble in methanol, and dry in vacuum to obtain acrylated pamidronic acid; wherein, the mass-volume ratio of pamidronic acid, sodium hydroxide solution and acryloyl chloride is 0.5 g: 22 mL: 180 μL, and the volume of the acryloyl chloride is the volume of a single dropwise addition;

[0119] (3) Preparation of small molecule containing bisphosphonate group: Dissolve the acrylated pamidronic acid prepared in step (2) in water, add a thiol source (cysteine), stir overnight at room temperature, concentrate by rotary evaporation, add acetone, filter to collect the white precipitate, dissolve the white precipitate in deionized water, then add methanol, white solid precipitates, complete the impurity removal process, and dry in vacuum to obtain a small molecule containing bisphosphonate group with a thiol group at one end; wherein, the mass-volume ratio of acrylated pamidronic acid, thiol source, water and acetone is 260 mg: 1350 mg: 30 mL: 12 mL;

[0120] The reaction equations are as follows:

[0121]

[0122] (4) Preparation of inorganic nanoparticles (inorganic manganese ferrite nanoparticles) coated with hydrophobic organic compounds: Synthesized with reference to ACS Nano 2017, 11, 3614 - 3631;

[0123] At room temperature, 0.62 g of manganese oleate, 1.07 g of iron erucate, 1.61 g of oleyl alcohol and 0.57 g of oleic acid were dissolved in 10 g of benzyl ether. The mixture was transferred to a three-necked flask. Under an argon atmosphere, it was heated to 110 °C and maintained for 30 min. Using a programmable temperature-controlled stirring heater, the temperature of the reaction system was raised to 265 °C at a rate of 5 °C / min. When the temperature reached 130 °C, an air condenser was placed on the three-necked flask to achieve solvent condensation and reflux. When the temperature reached 265 °C, it was maintained for 30 min. Then the solution in the flask was transferred to an ice bath and quickly cooled to room temperature. Ethanol was used as the precipitant and n-hexane was used as the dispersant. It was centrifuged 3 times with a high-speed centrifuge (8000 rpm / min, 10 min) to separate the precipitate, and inorganic nanoparticles (particle size 20 nm) coated with a hydrophobic organic compound on the surface were prepared;

[0124] (5) Hydrophilic modification: The inorganic nanoparticles coated with a hydrophobic organic compound prepared in step (4) were dispersed in tetrahydrofuran (mass-to-volume ratio of 25 mg: 2 mL), and ultrasonicated for 3 min to make it uniformly dispersed in tetrahydrofuran to obtain a mixed solution. The mixed solution was dropped into the small molecule solution containing a bisphosphonic acid group prepared in step (3) (mass-to-volume ratio of 55 mg: 5 mL), and shaken with an oscillator for 1 min. The whole system was uniformly mixed and no precipitate appeared. Ether was added, and the oleic acid in the system was removed by centrifugation with a high-speed centrifuge (12000 rpm / min, 20 min). The upper organic phase was poured off, and the lower aqueous phase was filtered through a 0.3 μm membrane and freeze-dried for 20 h to obtain hydrophilic inorganic nanoparticles; among them, the mass ratio of the inorganic nanoparticles coated with a hydrophobic organic compound to the small molecule containing a bisphosphonic acid group was 1:2.

[0125] The hydrophilic inorganic nanoparticles were labeled with a fluorescent label, and the method included the following steps:

[0126] The hydrophilic inorganic nanoparticles were dispersed in deionized water, and a fluorescent label (Alexa Fluor TM 647) was added and shaken for 1 min to obtain a dispersion system. The dispersion system was placed in a dialysis bag (MW3500), and the dialysis bag was placed in deionized water. The pH value of the deionized water was adjusted to 4 with hydrochloric acid, and dark dialysis was carried out. Dark dialysis was repeated 2 times, and then freeze-dried for 4 h to obtain the product.

[0127] Example 4:

[0128] A hydrophilic inorganic nanoparticle, and its preparation method includes the following steps:

[0129] Steps (2)-(3) were not included, and direct hydrophilic modification was carried out with pamidronic acid, and the rest was the same as in Example 1.

[0130] Comparative Example 1:

[0131] A manganese ferrite nanoparticle with oleic acid coated on its surface, and its preparation method includes the following steps:

[0132] It only includes step (4) of Example 1.

[0133] Comparative Example 2:

[0134] A manganese ferrite nanoparticle with oleic acid coated on its surface, and its preparation method includes the following steps:

[0135] It only includes step (4) of Example 2.

[0136] Comparative Example 3:

[0137] A manganese ferrite nanoparticle with oleic acid coated on its surface, and its preparation method includes the following steps:

[0138] It only includes step (4) of Example 3.

[0139] Test Example

[0140] I. Mix the hydrophilic inorganic nanoparticles prepared in Example 1 and the inorganic nanoparticles with oleic acid coated on their surface prepared in Comparative Example 1 with n - hexane solution respectively, as shown in Figures 3 - 4 .

[0141] It can be seen from Figure 3 that the inorganic nanoparticles with oleic acid coated on their surface are uniformly dispersed in n - hexane, proving that they are oil - soluble.

[0142] It can be seen from Figure 4 that the hydrophilic inorganic nanoparticles are uniformly dispersed in deionized water, proving that they are water - soluble.

[0143] II. Characterize the hydrophilic inorganic nanoparticles prepared in Example 1 and the inorganic nanoparticles with oleic acid coated on their surface prepared in Comparative Example 1 by dynamic light scattering (DLS) and transmission electron microscopy (TEM) respectively, and measure their particle sizes. The results are shown in Figures 5 - 6 .

[0144] It can be seen from Figure 5 that the particle size of the inorganic nanoparticles with oleic acid coated on their surface is measured and statistically analyzed to be 3.1 nm.

[0145] It can be seen from Figure 6 that the particle size of the hydrophilic inorganic nanoparticles is measured and statistically analyzed to be 3.0 nm.

[0146] III. Characterize the hydrophilic inorganic nanoparticles prepared in Example 2 and the inorganic nanoparticles with oleic acid coated on their surface prepared in Comparative Example 2 by transmission electron microscopy (TEM) respectively, and measure their particle sizes. The results are shown in Figures 7 - 10 .

[0147] It can be seen from Figures 7 - 8 that the particle size of the inorganic nanoparticles coated with oleic acid is measured and statistically analyzed to be 4.3 nm.

[0148] It can be seen from Figures 9 - 10 that the particle size of the hydrophilic inorganic nanoparticles is measured and statistically analyzed to be 4.3 nm.

[0149] IV. The hydrophilic inorganic nanoparticles prepared in Example 3 and the inorganic nanoparticles coated with oleic acid prepared in Comparative Example 3 are respectively dispersed in water, and their dispersibility in water is observed. The results are shown in Figures 11 - 12 .

[0150] It can be seen from Figure 11 that the manganese ferrite nanoparticles coated with oleic acid in Comparative Example 3 cannot be dispersed in water and agglomerate.

[0151] It can be seen from Figure 12 that the manganese ferrite nanoparticles modified with cysteine-diphosphonic acid group small molecules in Example 3 are uniformly dispersed in water ( Figure 12 ).

[0152] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of small molecules containing bisphosphonic acid groups in the preparation of hydrophilic inorganic nanoparticles, characterized in that: The structure of the small molecule containing a diphosphonic acid group contains two phosphite groups -PO3H2, and the two phosphite groups -PO3H2 are connected to the same carbon atom.

2. The use of the small molecule containing bisphosphonic acid groups according to claim 1 in the preparation of hydrophilic inorganic nanoparticles, characterized in that: Through the coordination effect between the bisphosphonic acid group and the inorganic nanoparticles, the hydrophobic organic compound on the surface of the inorganic nanoparticles is replaced by a small molecule containing the bisphosphonic acid group to obtain the hydrophilic inorganic nanoparticles.

3. The use of the small molecule containing bisphosphonic acid groups according to claim 2 in the preparation of hydrophilic inorganic nanoparticles, characterized in that: The inorganic nanoparticles are at least one of ferric oxide nanoparticles, ferroferric oxide nanoparticles, manganese ferrite nanoparticles, cobalt ferrite nanoparticles, zinc ferrite nanoparticles and manganese-zinc ferrite nanoparticles.

4. The use of the small molecule containing bisphosphonic acid groups according to claim 1 in the preparation of hydrophilic inorganic nanoparticles, characterized in that: The small molecule structure containing the bisphosphonic acid group also contains at least one of a thiol group, a carboxyl group, an amino group, an amine group and a maleimide group.

5. Use of the small molecule containing bisphosphonic acid groups according to claim 4 in the preparation of hydrophilic inorganic nanoparticles, characterized in that: The bisphosphonic acid group-containing small molecule is prepared by the following method: dissolving acryloyl pamidronate in water, adding a thiol source, stirring, concentrating, adding acetone to obtain a white precipitate, removing impurities, and vacuum drying to obtain the bisphosphonic acid group-containing small molecule.

6. Use of the small molecule containing bisphosphonic acid groups according to claim 5 in the preparation of hydrophilic inorganic nanoparticles, characterized in that: The sulfhydryl source is 1,4-dithiothreitol, cysteine, glutathione or mercaptoethylamine.

7. A method for preparing hydrophilic inorganic nanoparticles, characterized in that: The method comprises the following steps: dispersing inorganic nanoparticles coated with a hydrophobic organic compound on the surface in tetrahydrofuran to obtain a mixed solution, dropping the mixed solution into a small molecule solution containing a bisphosphonic acid group according to any one of claims 1 to 6, mixing evenly, adding ether, centrifuging, filtering the aqueous phase, and freeze-drying to obtain hydrophilic inorganic nanoparticles.

8. A hydrophilic inorganic nanoparticle, characterized in that: The method according to claim 7 is adopted to prepare the product.

9. Use of the hydrophilic inorganic nanoparticles according to claim 8 in magnetic resonance imaging, preparation of magnetic hyperthermia nanoparticles, hydrogel labeling, preparation of gene therapy drugs and preparation of functionalized hydrophilic inorganic nanoparticles.

10. The use of the hydrophilic inorganic nanoparticles according to claim 9 in magnetic resonance imaging, preparation of magnetic hyperthermia nanoparticles, hydrogel labeling, preparation of gene therapy drugs and preparation of functionalized hydrophilic inorganic nanoparticles, characterized in that: The hydrophilic inorganic nanoparticles are labeled with growth factors, glycosaminoglycans, polyethylene glycol or fluorescent markers to achieve functionalization of the hydrophilic inorganic nanoparticles.

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