Nano particle with core-shell structure as well as preparation method and application of nano particle

Abstract

The invention provides core-shell structure nanoparticles as well as a preparation method and application thereof, and belongs to the field of bone repair materials. The core-shell structure nanoparticle provided by the invention comprises a drug-loaded mesoporous silica nanoparticle and a polyphosphate layer coated on the surface of the drug-loaded mesoporous silica nanoparticle. According to the core-shell structure nanoparticle provided by the invention, the polyphosphate is adopted to coat the drug-loaded mesoporous silica nanoparticle, and the polyphosphate on the outer layer can be gradually depolymerized to release energy and phosphate groups under the action of alkaline phosphatase, so that explosive release of the drug in the nanoparticle can be avoided; the core-shell structure nano-particles are used as a core-shell structure, gradual release of drugs is achieved, a better bone repair curative effect is achieved, released energy and phosphate groups can be used for bone regeneration outside cells, and the bone repair effect of the core-shell structure nano-particles is further improved. The core-shell structure nanoparticle provided by the invention can enable the drug to be slowly released and achieve a better bone repair effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bone repair materials, in particular to a core-shell structure nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] Bone defect is caused by trauma, infection or tumor resection, etc., and is one of the most common orthopedic diseases in the world. Although many bone repair materials have been used at present, the repair effect is still not ideal, which brings great pain and economic burden to patients, and the mismatch between the bone repair material and the bone healing microenvironment is one of the key factors affecting the repair effect.

[0003] Bone repair is a complex and phased dynamic process, including inflammation stage and osteogenesis stage. Bone injury is accompanied by excessive ROS production, and the rupture of blood vessels after injury leads to insufficient oxygen supply, causing continuous increase of ROS, which in turn causes secondary damage to the tissue. At the same time, the pH value of the injury site also decreases, thereby exacerbating the inflammatory response and inhibiting the repair of bone tissue. The behavior and differentiation of bone cells are greatly influenced by the pH value of the microenvironment. At present, researchers mainly focus on the bone repair inflammation stage in the study of bone tissue repair materials, and the study of bone tissue repair materials in the osteogenesis stage is less.

[0004] However, the osteogenesis stage is crucial in the process of bone tissue repair, and it is the key period of bone tissue repair, which plays a decisive role in restoring the structure and function of bone. When the existing drugs are used for treatment in the osteogenesis stage, the release of the drugs is usually too fast, and the rapid loss of biological activity of the drugs causes poor treatment effect. SUMMARY

[0005] The purpose of the present application is to provide a core-shell structure nanoparticle and a preparation method and application thereof. The core-shell structure nanoparticle provided by the present application can slowly release the drug and achieve better bone repair effect.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The present application provides a core-shell structure nanoparticle, which comprises a drug-loaded mesoporous silica nanoparticle and a polyphosphate layer coated on the surface of the drug-loaded mesoporous silica nanoparticle.

[0007] Preferably, the particle size of the drug-loaded mesoporous silica nanoparticle is 80-120 nm. The specific surface area of the mesoporous silica nanoparticle in the drug-loaded mesoporous silica nanoparticle is 400-500 m 2 / g.

[0008] Preferably, the polyphosphate in the polyphosphate layer comprises calcium polyphosphate, zinc polyphosphate, strontium polyphosphate or iron polyphosphate.

[0009] Preferably, the drug-loaded mesoporous silica nanoparticles have a drug loading amount of 1-8 wt%.

[0010] The present application also provides a preparation method of the core-shell structure nanoparticles as described in the above technical solutions, comprising the following steps: The mesoporous silica nanoparticles are mixed with a drug solution, and then centrifuged, left standing and dried in sequence to obtain drug-loaded mesoporous silica nanoparticles. The drug-loaded mesoporous silica nanoparticles, a soluble inorganic salt and water are first mixed, and then added into a sodium polyphosphate solution to perform an ion cross-linking-precipitation reaction to obtain the core-shell structure nanoparticles; the soluble inorganic salt includes a soluble calcium salt, a soluble zinc salt, a soluble strontium salt or a soluble iron salt.

[0011] Preferably, the mass ratio of the drug-loaded mesoporous silica nanoparticles to the soluble inorganic salt is (20-30):1.

[0012] Preferably, the drug-loaded mesoporous silica nanoparticles, the soluble inorganic salt and water are first mixed, and then added into the sodium polyphosphate solution at an addition rate of 0.1-3 mL / min.

[0013] Preferably, the mass-volume concentration of the sodium polyphosphate solution is 1-5%.

[0014] Preferably, the pH of the ion cross-linking-precipitation reaction is 9.5-10.5.

[0015] The present application also provides an application of the core-shell structure nanoparticles as described in the above technical solutions or the core-shell structure nanoparticles prepared by the preparation method as described in the above technical solutions in bone repair materials in an osteogenic stage.

[0016] The present application provides a kind of core-shell structure nanoparticles, including drug-loaded mesoporous silica nanoparticles and the polyphosphate layer coated on the surface of drug-loaded mesoporous silica nanoparticles.The present application uses polyphosphate to coat drug-loaded mesoporous silica nanoparticles, wherein the outer polyphosphate can be gradually depolymerized under the action of alkaline phosphatase (osteogenic stage alkaline phosphatase significantly increases) to release energy and phosphate groups, which not only can avoid the explosive release of the drug therein, achieve gradual release of the drug, achieve better bone repair efficacy, but also can use the released energy and phosphate groups for bone regeneration outside the cell, further improve the effect of core-shell structure nanoparticles bone repair.The results of examples show that the core-shell structure nanoparticles provided by the present application will not leak early in the slow-release experiment, and 100% of the drug is released within 15h, the in-vitro osteogenic differentiation capacity experiment shows that the core-shell structure nanoparticles have better osteogenic differentiation capacity, which shows that the core-shell structure nanoparticles provided by the present application can slowly release the drug and achieve better bone repair effect. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 SEM image of mSiO2 in Example 1 of the present application; Figure 2 SEM image of Cur@mSiO2 in Example 1 of the present application; Figure 3 SEM image of Cur@mSiO2-Capp in Example 1 of the present application; Figure 4 TEM image of mSiO2 in Example 1 of the present application; Figure 5 TEM image of Cur@mSiO2 in Example 1 of the present application; Figure 6 TEM image of Cur@mSiO2-Capp in Example 1 of the present application; Figure 7 EDS full spectrum of Cur@mSiO2-Capp in Example 1 of the present application; Figure 8 Surface distribution of each element in Cur@mSiO2-Capp in Example 1 of the present application; Figure 9 XRD pattern of mSiO2, Cur@mSiO2 and Cur@mSiO2-Capp in Example 1 of the present application; Figure 10 Infrared spectrum of mSiO2, Cur@mSiO2 and Cur@mSiO2-Capp in Example 1 of the present application; Figure 11Release profile of Cur in Cur@mSiO2, Cur@mSiO2-Capp (without enzyme) and Cur@mSiO2-Capp (with enzyme) in Example 1 of the present application; Figure 12 Cell viability column chart of rBMSCs co-incubated with mSiO2, Cur@mSiO2 and Cur@mSiO2-Capp at different concentrations for 1 day in Example 1 of the present application; Figure 13 Cell OD value column chart of rBMSCs co-incubated with mSiO2, Cur@mSiO2 and Cur@mSiO2-Capp at 100 µg / mL concentration for 1 day, 3 days and 5 days in Example 1 of the present application; Figure 14 Fluorescence distribution of cytoskeleton of rBMSCs co-incubated with mSiO2, Cur@mSiO2 and Cur@mSiO2-Capp for 3 days in Example 1 of the present application; Figure 15 Fluorescence distribution of alkaline phosphatase of rBMSCs co-incubated with mSiO2, Cur@mSiO2 and Cur@mSiO2-Capp for 5 days and 10 days in Example 1 of the present application; Figure 16 Relative activity column chart of alkaline phosphatase of rBMSCs co-incubated with mSiO2, Cur@mSiO2 and Cur@mSiO2-Capp for 5 days and 10 days in Example 1 of the present application; Figure 17 Fluorescence distribution of calcium nodules obtained after rBMSCs co-incubated with mSiO2, Cur@mSiO2 and Cur@mSiO2-Capp for 21 days in Example 1 of the present application; Figure 18 Solution obtained after staining calcium nodule degradation after rBMSCs co-incubated with mSiO2, Cur@mSiO2 and Cur@mSiO2-Capp for 21 days in Example 1 of the present application; Figure 19 OD value (562 nm) column chart of red calcium nodules degraded by 10% cetylpyridinium chloride solution after rBMSCs co-incubated with mSiO2, Cur@mSiO2 and Cur@mSiO2-Capp for 21 days in Example 1 of the present application. DETAILED DESCRIPTION

[0018] The present application provides a core-shell structure nanoparticle, comprising a drug-loaded mesoporous silica nanoparticle and a polyphosphate layer coated on the surface of the drug-loaded mesoporous silica nanoparticle.

[0019] In one embodiment of the present invention, the particle size of the drug-loaded mesoporous silica nanoparticles can be 80-120 nm or 90-110 nm; the specific surface area of ​​the drug-loaded mesoporous silica nanoparticles can be 400-500 m². 2 / g, or 420~480m 2 / g. By limiting the particle size and specific surface area of ​​the drug-loaded mesoporous silica nanoparticles to the above-mentioned range, the present invention can achieve better drug loading.

[0020] In one embodiment of the present invention, the drug loading of the drug-loaded mesoporous silica nanoparticles can be 1-8 wt%, 2-6 wt%, or 3-4 wt%; the drug in the drug-loaded mesoporous silica nanoparticles can be a small molecule chemical, a biological macromolecule, or an inorganic salt metal complex; the small molecule chemical can specifically include, but is not limited to, curcumin, simvastatin, or raloxifene; the biological macromolecule can specifically include, but is not limited to, BMP-2 or teriparatide; the inorganic salt metal complex can specifically include, but is not limited to, strontium ranelate or zoledronic acid. The present invention limits the drug loading and drug type of the drug-loaded mesoporous silica nanoparticles to the above-mentioned ranges to achieve better repair effects on bone damage during the osteogenic stage.

[0021] In one embodiment of the present invention, the thickness of the polyphosphate layer can be 3-8 nm, 4-6 nm, or 5 nm; the polyphosphate in the polyphosphate layer can include calcium polyphosphate, zinc polyphosphate, strontium polyphosphate, or iron polyphosphate; the chain length of the polyphosphate can be 3-100, 6-90, 15-70, or 30-50. In an embodiment of the present invention, the polyphosphate in the polyphosphate layer is calcium polyphosphate; the chain length of the calcium polyphosphate is 40. By limiting the thickness of the polyphosphate layer and the type of polyphosphate to the above ranges, the present invention can better achieve the sustained-release effect of drugs in core-shell structured nanoparticles and further improve the bone repair effect.

[0022] This invention uses polyphosphate to coat drug-loaded mesoporous silica nanoparticles. The outer polyphosphate layer can gradually depolymerize under the action of alkaline phosphatase, releasing energy and phosphate groups. This not only avoids the explosive release of the drug and achieves gradual drug release for better bone repair efficacy, but also utilizes the released energy and phosphate groups for extracellular bone regeneration, further improving the bone repair effect of core-shell structured nanoparticles.

[0023] This invention also provides a method for preparing the core-shell structured nanoparticles described in the above technical solution, comprising the following steps: Mesoporous silica nanoparticles were mixed with a drug solution and then centrifuged, allowed to stand, and dried sequentially to obtain drug-loaded mesoporous silica nanoparticles. The drug-loaded mesoporous silica nanoparticles, soluble inorganic salts, and water are first mixed and then added to a sodium polyphosphate solution for an ion crosslinking-precipitation reaction to obtain core-shell structured nanoparticles; the soluble inorganic salt solution includes soluble calcium salts, soluble zinc salts, soluble strontium salts, or soluble iron salts.

[0024] In this invention, mesoporous silica nanoparticles are mixed with a drug solution and then centrifuged, allowed to stand, and dried sequentially to obtain drug-loaded mesoporous silica nanoparticles.

[0025] As one embodiment of the present invention, the method for preparing the mesoporous silica nanoparticles may include the following steps: Hexadecyltrimethylammonium bromide, ammonium fluoride and water were mixed and then tetraethoxysilane was added to induce self-assembly. The mixture was then centrifuged, dried and calcined in sequence to obtain mesoporous silica nanoparticles.

[0026] In one embodiment of the present invention, the mixing of hexadecyltrimethylammonium bromide, ammonium fluoride, and water can be carried out by stirring; the mixing temperature can be 80-100°C, or 90-100°C; and the mixing time can be 1-2 hours. The present invention does not have a particular limitation on the stirring speed; stirring speeds commonly used by those skilled in the art can be used. Limiting the mixing temperature and time to the above-mentioned ranges ensures the complete dissolution of hexadecyltrimethylammonium bromide and ammonium fluoride.

[0027] The present invention does not have a particular limitation on the rate at which tetraethoxysilane is added, as long as the tetraethoxysilane is added in droplet form to the mixed solution of hexadecyltrimethylammonium bromide, ammonium fluoride and water.

[0028] In one embodiment of the present invention, the mass ratio of hexadecyltrimethylammonium bromide, ammonium fluoride, and tetraethoxysilane can be 1:(1~3):(4~6), 1:(1.5~2.5):(4.5~5.5), or 1:(1.5~2):(4.5~5). Limiting the mass ratio of hexadecyltrimethylammonium bromide, ammonium fluoride, and tetraethoxysilane to the above ranges ensures sufficient self-assembly.

[0029] In one embodiment of the present invention, the volume ratio of tetraethoxysilane to deionized water can be (0.5~0.8):11 or (0.8~1.2):11. Limiting the volume ratio of tetraethoxysilane to deionized water to the above range allows for better reaction performance.

[0030] In one embodiment of the present invention, the self-assembly temperature can be 60~90℃ or 70~80℃; the self-assembly time can be 1.5~2.5h or 2h. Limiting the self-assembly temperature and time to the above ranges ensures the reaction proceeds sufficiently.

[0031] As one embodiment of the present invention, after the self-assembly is completed, the reaction solution can be cooled to room temperature and allowed to stand, and then centrifuged, dried and calcined in sequence to obtain mesoporous silica nanoparticles.

[0032] In one embodiment of the present invention, the settling time can be 12-24 hours. The present invention utilizes settling to make the self-assembled silica precursor more stable.

[0033] The present invention does not have a special limitation on the centrifugation speed, as long as it can achieve solid-liquid separation.

[0034] In one embodiment of the present invention, the solid obtained by centrifugation can be washed after centrifugation.

[0035] The present invention does not have any special limitations on the cleaning agent or the number of cleaning cycles used for the cleaning process; cleaning agents and cleaning cycles commonly used by those skilled in the art can be used.

[0036] In one embodiment of the present invention, the drying can be freeze drying; the freeze drying temperature can be -50 to -70°C; and the freeze drying time can be 10 to 20 hours.

[0037] In one embodiment of the present invention, the heating rate to the calcination temperature can be 1~2℃ / min; the calcination temperature can be 550~600℃; and the calcination time can be 4~6h. Controlling the heating rate to 1~2℃ / min in this invention results in a small temperature difference between the inside and outside of the particles, low pore wall stress, and optimal morphology preservation. A calcination temperature of 550~600℃ allows for the formation of clear, open mesoporous channels, achieving a high specific surface area, and the original morphology of the particles is well preserved within this temperature range. A calcination time of 4~6h ensures the uniformity of the mesoporous silica particles and the complete removal of hexadecyltrimethylammonium bromide.

[0038] In one embodiment of the present invention, the concentration of the drug solution can be 1-10 mg / mL, 2-8 mg / mL, 4-6 mg / mL, or 5 mg / mL; the solvent of the drug solution can be ethanol or phosphate buffer. Limiting the concentration of the drug solution to the above range in the present invention facilitates the adsorption of the drug by mesoporous silica nanoparticles.

[0039] The present invention does not have any particular limitation on the mixing of the mesoporous silica nanoparticles and the drug solution; it is sufficient to perform preliminary mixing of the mesoporous silica nanoparticles and the drug solution.

[0040] As one embodiment of the present invention, the centrifugation speed can be 8000~12000 rpm, 9000~11000 rpm, or 10000 rpm; the centrifugation time can be 8~12 min or 10 min.

[0041] In one embodiment of the present invention, the settling time can be 20-30 hours, 22-26 hours, or 24 hours.

[0042] This invention enables the loading of drugs into mesoporous silica nanoparticles through centrifugation and settling.

[0043] In one embodiment of the present invention, after the settling period, a second centrifugation can be performed. The parameter settings for the second centrifugation are consistent with those for the centrifugation described above, and will not be repeated here. The present invention achieves solid-liquid separation through the second centrifugation.

[0044] As one embodiment of the present invention, the solid obtained after the second centrifugation can be washed.

[0045] In one embodiment of the present invention, the washing solution used for washing can be anhydrous ethanol, phosphate buffer, or deionized water; the number of washing cycles can be three.

[0046] In one embodiment of the present invention, the drying can be freeze-drying; the freeze-drying temperature can be -50 to -70°C; and the freeze-drying time can be 10 to 20 hours. Limiting the freeze-drying temperature and time to the above ranges allows for better drying of drug-loaded mesoporous silica nanoparticles.

[0047] After obtaining drug-loaded mesoporous silica nanoparticles, the present invention first mixes the drug-loaded mesoporous silica nanoparticles, soluble inorganic salts and water, and then adds them to sodium polyphosphate solution to carry out ion crosslinking-precipitation reaction to obtain core-shell structured nanoparticles.

[0048] In one embodiment of the present invention, the soluble inorganic salt may include soluble calcium salts, soluble zinc salts, soluble strontium salts, or soluble iron salts. The present invention limits the types of soluble inorganic salts to those within the above-mentioned range that can yield polyphosphate precipitation.

[0049] In one embodiment of the present invention, the mass ratio of the drug-loaded mesoporous silica nanoparticles to the soluble inorganic salt can be (20~30):1, (25~30):1, or (28~30):1. Limiting the mass ratio of the drug-loaded mesoporous silica nanoparticles to the soluble inorganic salt to the above range creates favorable conditions for subsequent polyphosphate coating.

[0050] In one embodiment of the present invention, ultrasound can be performed during the first mixing process; the frequency of the ultrasound can be 40-50 kHz; and the duration of the ultrasound can be 10-20 min. The present invention uses ultrasound to make the drug-loaded mesoporous silica nanoparticles and soluble inorganic salts mix more uniformly.

[0051] In one embodiment of the present invention, the molar ratio of the soluble inorganic salt to the sodium polyphosphate in the sodium polyphosphate solution can be 1:(0.006~0.084), 1:(0.006~0.02), 1:(0.0063~0.01), or 1:(0.0063~0.007). The present invention limits the molar ratio of the soluble inorganic salt to the sodium polyphosphate in the sodium polyphosphate solution to the above ranges to better form the polyphosphate layer.

[0052] In one embodiment of the present invention, the mass-volume concentration of the sodium polyphosphate solution can be 1-5%, 2-4%, or 2-3%. Limiting the mass-volume concentration of the sodium polyphosphate solution to the above range facilitates the subsequent ion crosslinking-precipitation reaction.

[0053] In one embodiment of the present invention, the addition rate of the drug-loaded mesoporous silica nanoparticles, soluble inorganic salts, and water to the sodium polyphosphate solution after the first mixture can be 0.1~3 mL / min, 0.5~2 mL / min, or 1~1.5 mL / min. By limiting the addition rate of the drug-loaded mesoporous silica nanoparticles, soluble inorganic salts, and water to the sodium polyphosphate solution within the above-mentioned range, the present invention ensures the complete progress of the reaction.

[0054] In one embodiment of the present invention, the pH of the ion crosslinking-precipitation reaction can be 9.5~10.5, or even 10. By limiting the pH of the ion crosslinking-precipitation reaction to the above range, the present invention can ensure the ion crosslinking-precipitation reaction proceeds and forms a polyphosphate layer of better quality.

[0055] In one embodiment of the present invention, the pH is adjusted to 9.5-10.5 during the ion crosslinking-precipitation reaction using an alkaline solution; the alkaline solution can be NaOH solution, KOH solution, or ammonia water; the concentration of the alkaline solution can be 0.1-1M.

[0056] This invention does not have a special limitation on the reaction time of the ion crosslinking-precipitation reaction. It can completely add the mixed solution of drug-loaded mesoporous silica nanoparticles, soluble inorganic salts and water into the sodium polyphosphate solution and allow the raw materials to react completely.

[0057] After the ion crosslinking-precipitation reaction is completed, the product of the ion crosslinking-precipitation reaction can be centrifuged, washed and dried in sequence to obtain core-shell structured nanoparticles.

[0058] In one embodiment of the present invention, the centrifugation speed of the ion crosslinking-precipitation reaction product can be 3000~5000 rpm, or even 4000 rpm; the centrifugation time can be 4~6 min, or even 5 min. By limiting the centrifugation speed and time to the above ranges, the present invention can achieve solid-liquid separation.

[0059] As one embodiment of the present invention, the solid obtained by centrifugation can be washed after centrifuging the ion crosslinking-precipitation reaction product.

[0060] In one embodiment of the present invention, the washing liquid used for washing can be deionized water; the washing can be performed three times.

[0061] In one embodiment of the present invention, the drying can be freeze-drying; the freeze-drying temperature can be -50 to -70°C; and the freeze-drying time can be 10 to 20 hours. By limiting the freeze-drying temperature and time to the above ranges, the present invention can better dry the obtained core-shell structured nanoparticles.

[0062] This invention also provides the application of the core-shell structured nanoparticles described in the above technical solutions or the core-shell structured nanoparticles prepared by the preparation methods described in the above technical solutions in bone repair materials during the osteogenic stage.

[0063] As one application method of the present invention, the core-shell structured nanoparticles can be used to inject alkaline phosphatase in vitro. As one embodiment of the present invention, the timing of the in vitro injection of alkaline phosphatase can be selected as needed. By injecting alkaline phosphatase in vitro, the core-shell structured nanoparticles of the present invention can achieve sudden drug release at a specific time point (the time point at which sudden drug release is required).

[0064] As one application method of this invention, the amount of alkaline phosphatase injected in vitro can be 10-100 units / mL. This invention accelerates the hydrolysis of polyphosphates through in vitro injection of alkaline phosphatase, thereby speeding up bone tissue repair.

[0065] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0066] Example 1 A core-shell structured nanoparticle consists of mesoporous silica nanoparticles loaded with curcumin and a layer of polyphosphate coating on the surface of the drug-loaded mesoporous silica nanoparticles. The curcumin-loaded mesoporous silica nanoparticles have a particle size of 80-120 nm; the specific surface area of ​​the mesoporous silica nanoparticles is 441 m². 2 / g; The drug loading of the curcumin-loaded mesoporous silica nanoparticles is 3.3 wt%. The thickness of the calcium polyphosphate layer is 5 nm; the average phosphate chain length in the calcium polyphosphate is 40. The method for preparing the core-shell structured nanoparticles comprises the following steps: Mesoporous silica nanoparticles (denoted as mSiO2) were mixed with curcumin solution (5 mg / mL), centrifuged at 10000 rpm for 10 min, and allowed to stand for 24 h. Then, the mixture was centrifuged at 8000 rpm for 5 min. The resulting solid was washed three times with anhydrous ethanol and then freeze-dried at -70 °C for 10 h to obtain curcumin-loaded mesoporous silica nanoparticles (denoted as Cur@mSiO2). The mesoporous silica nanoparticles loaded with curcumin, CaCl2·2H2O, and water were ultrasonically mixed at 60 kHz for 10 min and then added to a sodium polyphosphate solution (5% (w / v)) with an average phosphate chain length of 40. The pH was adjusted with 0.1 M NaOH solution to maintain the pH at 10.0 for ion crosslinking-precipitation reaction. The ion crosslinking-precipitation reaction product was then centrifuged at 4000 rpm for 5 min and washed three times with deionized water. Finally, it was freeze-dried at -70℃ for 10 h to obtain core-shell structured nanoparticles (denoted as Cur@mSiO2-Capp). The mass ratio of the curcumin-loaded mesoporous silica nanoparticles to CaCl2·2H2O is 28:1; The molar ratio of CaCl2·2H2O to sodium polyphosphate in the sodium polyphosphate solution is 1:0.0063; The method for preparing the mesoporous silica nanoparticles is as follows: Hexadecyltrimethylammonium bromide, ammonium fluoride, and water were stirred at 80°C for 1 hour, and then tetraethoxysilane was added to induce self-assembly for 2 hours. The mixture was then cooled to room temperature and left to stand for 12 hours. After centrifugation at 11000 rpm for 10 minutes, the solid was washed three times with anhydrous ethanol and then freeze-dried at -70°C for 10 hours. The solid was then heated to 600°C in air at a rate of 1 minute / °C and calcined for 6 hours to obtain mesoporous silica nanoparticles. The mass ratio of hexadecyltrimethylammonium bromide, ammonium fluoride, and tetraethoxysilane was 1:1.64:4.59, and the volume ratio of tetraethoxysilane to deionized water was 1:11.

[0067] Example 2 The only difference between Example 2 and Example 1 is that the drug used is BMP-2; otherwise, they are the same as in Example 1.

[0068] The morphology and structure of mSiO2, Cur@mSiO2 and Cur@mSiO2-Capp in Example 1 were observed using scanning electron microscopy (SEM, Nova Nano SEM450) and transmission electron microscopy (TEM).

[0069] The SEM image of mSiO2 in Example 1 is shown below. Figure 1 As shown, the SEM image of Cur@mSiO2 is as follows. Figure 2 As shown, the SEM image of Cur@mSiO2-Capp is as follows. Figure 3 As shown, the TEM image of mSiO2 is as follows. Figure 4 As shown, the TEM image of Cur@mSiO2 is as follows. Figure 5 As shown, the TEM image of Cur@mSiO2-Capp is as follows. Figure 6 As shown, the EDS full spectrum of Cur@mSiO2-Capp is as follows. Figure 7 As shown; the surface distribution diagram of each element in Cur@mSiO2-Capp is as follows. Figure 8 As shown.

[0070] from Figures 1 to 3As can be seen, the prepared mSiO2 is spherical and basically the same size. After adding curcumin (Cur) to mSiO2, the surface morphology did not change significantly. However, the surface of Cur@mSiO2-Capp nanoparticles became rough, which may be due to the encapsulation of Capp (calcium polyphosphate) on mesoporous silica. The diameters of mSiO2, Cur@mSiO2 and Cur@mSiO2-Capp are 92nm, 93nm and 97nm, respectively.

[0071] from Figures 4 to 6 As can be seen, both mSiO2 and Cur@mSiO2 exhibit an ordered mesoporous structure, while Cur@mSiO2-Capp shows a distinct core-shell structure with a shell thickness of 5 nm.

[0072] from Figure 7 and Figure 8 The distribution of elements in Cur@mSiO2-Capp can be seen, and it is found that Si, O, Ca and P (represented by red, yellow, green and blue regions, respectively) are uniformly distributed in the nanoparticles.

[0073] The morphology and structure of mSiO2, Cur@mSiO2 and Cur@mSiO2-Capp in Example 1 were characterized by X-ray diffraction (XRD, D8 Discover) and Fourier transform infrared spectroscopy (FTIR, ThermoScientific Nicolet 6700).

[0074] The XRD patterns of mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp in Example 1 are shown below. Figure 9 As shown, the infrared spectra of mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp in Example 1 are as follows: Figure 10 As shown.

[0075] from Figure 9 As can be seen, mSiO2 has an amorphous structure. Curcumin was not detected in the Cur@mSiO2 spectrum, which may be due to its low curcumin content. A broad peak of about 30° was detected in the Cur@mSiO2-Capp spectrum, which is a characteristic peak of Ca-polyP.

[0076] from Figure 10 As can be seen from the infrared spectrum of mSiO2, 3449 cm⁻¹ -1 The broad peak at 1635 cm⁻¹ is the asymmetric stretching vibration peak of structural water -OH. -1 The nearby peak is the HOH bending vibration peak of water, 810 cm⁻¹. -1 and 467cm-1 The peak at 958 cm⁻¹ is caused by the symmetrical stretching and bending vibrations of the Si-O bond. -1 The peak at 2923 cm⁻¹ belongs to the bending vibration absorption peak of Si-OH; in the Cur@mSiO₂ infrared spectrum, the peak at 2923 cm⁻¹ is... -1 The absorption peak at 3449 cm⁻¹ is the stretching vibration peak of -CH, while the absorption peak at 3449 cm⁻¹ is the stretching vibration peak of -CH. -1 and 1635cm -1 This is the characteristic peak of curcumin; in asymmetric vibration (915 cm⁻¹) -1 ) and symmetrical vibration (740cm) -1 The characteristic peak of polyphosphate was found at the wavelength of ).

[0077] Drug sustained-release experiments were conducted on Cur@mSiO2 and Cur@mSiO2-Capp from Example 1 using a UV spectrophotometer. Alkaline phosphatase was added to Cur@mSiO2-Capp to verify that Capp hydrolysis is accelerated in the presence of alkaline phosphatase. The group of Cur@mSiO2-Capp without added alkaline phosphatase was designated Cur@mSiO2-Capp (no enzyme), and the group with added alkaline phosphatase was designated Cur@mSiO2-Capp (with enzyme).

[0078] The release curves of Cur in Cur@mSiO2, Cur@mSiO2-Capp (enzyme-free), and Cur@mSiO2-Capp (enzyme-containing) in Example 1 are shown in the figure below. Figure 11 As shown, from Figure 11 It can be seen that Cur@mSiO2 exhibits a significant burst release in the first 5 hours of sustained release, and is almost completely released after 5 hours. In Cur@mSiO2-Capp (without enzyme), the amount of Cur released is relatively small in the first 20 hours. The small amount of Cur released is partly due to the core-shell structure not being completely wrapped with Cur@mSiO2, and partly due to the hydrolysis of calcium polyphosphate in the presence of water, which releases Cur. Cur@mSiO2-Capp (with enzyme) containing alkaline phosphatase released 100% of Cur within 15 hours, indicating that Capp hydrolysis is accelerated in the presence of alkaline phosphatase.

[0079] Cytotoxicity tests were performed on mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp from Example 1. The specific test method was as follows: Rat bone marrow mesenchymal stem cells (rBMSCs) were co-incubated with different concentrations (12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL) of mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp suspensions in α-MEM medium at 37°C and 5% CO2. To assess the biocompatibility of mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp, cells co-incubated for 1 day were collected and treated with CCK-8 reagent. The cell viability histograms for different concentrations of mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp co-incubated with rBMSCs for 1 day in Example 1 are shown below. Figure 12 As shown in Table 1, the cell viability data are as follows. Figure 12 In This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001.

[0080] Table 1. Cell viability data of different concentrations of mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp co-incubated with rBMSCs for 1 day in Example 1. unit(%)

[0081] From Table 1 and Figure 12 It can be seen that rat bone marrow mesenchymal stem cells (rBMSCs) were co-incubated with nanoparticles of different concentrations for 1 day. The nanoparticle concentration of 200 µg / mL was cytotoxic, while the concentration of 12.5~100 µg / mL was less cytotoxic. Finally, a concentration of 100 µg / mL was selected for subsequent cell experiments.

[0082] Based on the cytotoxicity assay results of rBMSCs co-incubated with different concentrations of nanoparticles, a concentration of 100 µg / mL was selected for subsequent cell proliferation experiments. The bar charts showing the OD values ​​of rBMSCs co-incubated with 100 µg / mL mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp at concentrations of 100 µg / mL for 1, 3, and 5 days are shown below. Figure 13 As shown in Table 2, the cell OD value data are as follows. Figure 13 In This indicates that P < 0.05. This indicates that P < 0.001. This means P < 0.0001.

[0083] Table 2. Cell OD values ​​of 100 µg / mL mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp co-incubated with rBMSCs for 1 day, 3 days, and 5 days in Example 1.

[0084] From Table 2 and Figure 13 It can be seen that the number of rBMSCs increases significantly with the extension of cell culture time. The cell proliferation rate of rBMSCs co-incubated with Cur@mSiO2-Capp nanoparticle suspension is higher than that of rBMSCs co-incubated with mSiO2 and Cur@mSiO2 nanoparticle suspension.

[0085] The cytoskeleton and cell nuclei of mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp in Example 1 were stained using the following methods: Rat bone marrow mesenchymal stem cells (rBMSCs) were co-incubated with mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp suspensions in α-MEM medium at 37°C and 5% CO2. To assess the effect of 100 µg / mL mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp suspensions on cell growth, cells co-incubated for 3 days were collected and treated with the FITC phalloidin kit.

[0086] The fluorescence distribution of the cytoskeleton of mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp co-incubated with rBMSCs for 3 days in Example 1 is shown in the figure below. Figure 14 As shown, the first row is a fluorescence image of the rBMSC cytoskeleton, the second row is a fluorescence image of the rBMSC nucleus, and the third row is a composite fluorescence image of the rBMSC cytoskeleton and nucleus. Figure 14 As can be seen, the rBMSCs have normal cell morphology, without any signs of curling, shrinkage or breakage, and can adhere well to the well plate.

[0087] The alkaline phosphatase and alizarin red of mSiO2, Cur@mSiO2 and Cur@mSiO2-Capp obtained in Example 1 were quantitatively and qualitatively analyzed to test the in vitro osteogenic differentiation ability of each nanoparticle.

[0088] The in vitro osteogenic differentiation capacity of mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp was detected using alkaline phosphatase and alizarin red assays: Rat bone marrow mesenchymal stem cells (rBMSCs) were co-incubated with suspensions of mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp in α-MEM medium at 37°C and 5% CO2 for 12 h. After 12 h, the basal cell culture medium was changed to osteogenic induction medium for mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp to induce rBMSC differentiation (osteogenic induction culture). The culture medium was prepared by adding ascorbic acid (50 nM), dexamethasone (0.1 μM), and sodium β-glycerophosphate (10 mM). rBMSCs were co-incubated with mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp suspensions for 5 and 10 days, respectively. The 24-well plates containing rBMSCs were then washed with PBS and fixed with 4% paraformaldehyde (FDA) at room temperature for 30 min. After fixation, the samples were stained with a BCIP / NBT alkaline phosphatase colorimetric kit, and then completely washed with PBS to terminate the staining. The samples were observed and photographed using an optical microscope. ALP (alkaline phosphatase) activity was quantitatively analyzed using p-nitrophenyl phosphate reagent and a BCA protein assay kit. Absorbance at 405 nm and 562 nm was recorded using a microplate reader, and the contents of alkaline phosphatase and protein were quantified using a standard curve. Finally, ALP activity was normalized to total protein content.

[0089] The fluorescence distribution of alkaline phosphatase obtained by co-incubating mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp with rBMSCs for 5 and 10 days in Example 1 is shown in the figure below. Figure 15 As shown in the bar chart of relative alkaline phosphatase activity, see below. Figure 16 As shown in Table 3, the relative activity data of alkaline phosphatase are as follows. Figure 16 In This indicates that P < 0.05.

[0090] Table 3. Relative alkaline phosphatase activities of mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp co-incubated with rBMSCs for 5 and 10 days in Example 1.

[0091] From Table 3 and Figures 15 to 16 As can be seen, ALP is an early marker of bone formation during osteoblast differentiation, reflecting both the differentiation state and functional activity of osteoblasts. Therefore, we measured ALP activity in rBMSCs co-cultured with different nanoparticle suspensions for 5 and 10 days. Figure 16As shown, there was no statistically significant difference in ALP activity among the nanoparticle groups after 5 days of co-culture. However, when the incubation time was extended to 10 days, rBMSCs co-cultured with Cur@mSiO2-Capp nanoparticle suspension exhibited the highest ALP activity, which may be attributed to the enhanced ALP activity of cellular ALP ions released by Cur@mSiO2-Capp nanoparticles. This indicates that Cur@mSiO2-Capp nanoparticles can effectively promote early osteogenic differentiation of rBMSCs. ALP staining results were consistent with the obtained ALP activity data.

[0092] After co-incubating rBMSCs with the mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp suspensions from Example 1 for 21 days, the 6-well plates inoculated with rBMSCs were washed with PBS and then fixed with 4% paraformaldehyde at room temperature for 30 minutes. Alizarin Red solution was added at predetermined time points to stain calcium nodules for 30 minutes at room temperature. The samples were then thoroughly washed with distilled water, and the cells were observed using an optical microscope. Finally, 10% hexadecylpyridine chloride was added to degrade the calcium nodules, and the resulting solution was collected and its absorbance (OD value) was measured at 562 nm for quantitative analysis.

[0093] The fluorescence distribution of calcium nodules obtained after co-incubating mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp with rBMSCs for 21 days in Example 1 is shown in the figure below. Figure 17 As shown, the solution obtained after the degradation of stained calcium nodules is as follows: Figure 18 As shown in the bar chart, the OD values ​​(562nm) of the red calcium nodules after degradation by 10% hexadecylpyridine chloride solution are as follows. Figure 19 As shown, the OD value (562nm) data are shown in Table 4. Figure 19 In This means P < 0.0001.

[0094] Table 4. OD values ​​of red calcium nodules after 21 days of co-incubation of mSiO2, Cur@mSiO2, and Cur@mSiO2-Capp with rBMSCs in Example 1, followed by degradation by 10% hexadecylpyridine chloride solution.

[0095] From Table 4 and Figures 17 to 19 It can be seen that a small number of red mineralized nodules are scattered at the bottom of the mSiO2 and Cur@mSiO2 well plates, and cell membranes formed by rBMSCs are visible in some areas. Compared with mSiO2 and Cur@mSiO2, a large number of dark red nodules appeared at the bottom of the Cur@mSiO2-Capp well plates. Compared with mSiO2 and Cur@mSiO2, Cur@mSiO2-Capp NPs have the strongest osteogenic differentiation ability.

[0096] The core-shell nanoparticles provided by this invention do not leak drugs in the early stages of sustained-release experiments. With the extension of release time, 100% of the drug is released within 15 hours. In vitro osteogenic differentiation capacity experiments show that the core-shell nanoparticles have better osteogenic differentiation capacity, indicating that the core-shell nanoparticles provided by this invention can enable slow drug release and achieve better bone repair effects.

[0097] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A core-shell structured nanoparticle, characterized by, The mesoporous silica nanoparticles loaded with drugs and a polyphosphate layer coated on the surface of the mesoporous silica nanoparticles loaded with drugs.

2. The core-shell structured nanoparticle according to claim 1, wherein The particle size of the mesoporous silica nanoparticles loaded with drugs is 80-120 nm. The specific surface area of the mesoporous silica nanoparticles in the drug-loaded mesoporous silica nanoparticles is 400-500 m 2 / g.

3. The core-shell structured nanoparticle according to claim 1, wherein The polyphosphate in the polyphosphate layer includes calcium polyphosphate, zinc polyphosphate, strontium polyphosphate or iron polyphosphate.

4. The core-shell structured nanoparticle according to claim 1, wherein The drug loading amount of the mesoporous silica nanoparticles loaded with drugs is 1-8 wt%.

5. A preparation method of the core-shell structure nanoparticles according to any one of claims 1-4, comprising the following steps: The mesoporous silica nanoparticles loaded with drugs are obtained by mixing the mesoporous silica nanoparticles with a drug solution and then sequentially performing centrifugation, standing and drying; The core-shell structure nanoparticles are obtained by adding a sodium polyphosphate solution to the mixture of the mesoporous silica nanoparticles loaded with drugs, a soluble inorganic salt and water for ion cross-linking-precipitation reaction; the soluble inorganic salt includes a soluble calcium salt, a soluble zinc salt, a soluble strontium salt or a soluble iron salt.

6. The production method according to claim 5, characterized by, The mass ratio of the mesoporous silica nanoparticles loaded with drugs to the soluble inorganic salt is (20-30):

1.

7. The preparation method according to claim 5, characterized in that, The sodium polyphosphate solution is added at a rate of 0.1-3 mL / min after the first mixing of the mesoporous silica nanoparticles loaded with drugs, the soluble inorganic salt and water.

8. The preparation method according to claim 7, characterized in that, The mass-volume concentration of the sodium polyphosphate solution is 1-5%.

9. The preparation method according to claim 5, characterized in that, The pH of the ion cross-linking-precipitation reaction is 9.5-10.

5.

10. The use of the core-shell structure nanoparticles according to any one of claims 1-4 or prepared by the preparation method according to any one of claims 5-9 in bone repair materials in the osteogenic stage.

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