A primary osteoporosis diagnosis and treatment nanoprobe with in-situ catalysis-on-demand gas production and its preparation method and application

Abstract

The purpose of the present invention is to provide a primary osteoporosis diagnosis and treatment nanoprobe with in-situ catalysis and on-demand gas production, and its preparation method and application, which belongs to the field of life medicine and nanobiomedical material technology. The nanoprobe uses rare earth core-shell nanoparticles with bidirectional conversion as carriers, and on this basis, amorphous calcium carbonate and romumab are coated to synthesize RE@Ca-Ig nanoprobes with uniform dispersion and uniform particle size, which have integrated fluorescence imaging and treatment. The nanoprobe of the present invention can be guided by activatable near-infrared imaging to perform on-demand "gas-drug-drug-antibody" combined treatment on osteoporosis sites. The preparation method is simple and easy to implement, and can achieve the integration of primary osteoporosis diagnosis and treatment, with obvious effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of life medicine and nano-biomedical materials, and specifically relates to a primary osteoporosis diagnosis and treatment nano-probe with in-situ catalysis-on-demand gas production, and a preparation method and application thereof. Background Art

[0002] With the acceleration of the aging process of the social population, osteoporosis has become a major chronic disease that affects the health and quality of life of the elderly population, with a prevalence of 53.85%, second only to cardiovascular disease and diabetes. Epidemiological survey results show that the incidence of fractures caused by osteoporosis is increasing year by year. One osteoporotic fracture occurs every 3 seconds worldwide, and most of them are elderly people and postmenopausal women. In the past few decades, more and more effective drugs have been available for the treatment of osteoporosis, such as hormone replacement therapy (HRT) and antiresorptive drugs. However, long-term HRT increases the risk of reproductive organ malignancies, such as breast cancer, as well as other risk factors for cardiovascular disease and venous thromboembolism (VTE). Long-term use of antiresorptive drugs may lead to the inhibition of bone turnover, which will destroy the natural mechanism of bone repair and increase the risk of osteonecrosis of the jaw (ONJ) ​​and atypical fractures. Therefore, it is crucial to develop alternative treatments with fewer side effects to combat this common metabolic disorder. With the development of nanotechnology, it is of great significance to utilize the characteristics of nanomaterials to combine the functions of targeted drug transport, real-time monitoring and drug treatment, and develop new and efficient nanomedicines for the diagnosis and treatment of primary osteoporosis. With the development of nanotechnology, it is of great significance to utilize the characteristics of nanomaterials to combine the functions of targeted drug transport, real-time monitoring and drug treatment, and develop new and efficient nanomedicines for the diagnosis and treatment of primary osteoporosis.

[0003] By utilizing the inherent characteristics of nanobiomedical materials, integrating functions such as drug treatment, targeted delivery, and real-time monitoring, the development of new nanoprobes plays an important role in the occurrence, development, and prognosis observation of primary osteoporosis. Summary of the invention

[0004] The object of the present invention is to provide a primary osteoporosis diagnosis and treatment nanoprobe with in-situ catalysis-on-demand gas production and its preparation method and application. The nanoprobe is replaced by a two-way conversion rare earth core-shell structure surface ligand, amorphous calcium carbonate is loaded on it to form a heterogeneous structure, and romumomab is loaded on the surface to successfully synthesize the primary osteoporosis nanoprobe with in-situ catalysis-on-demand gas production. After intravenous injection, the nanoprobe is actively targeted and aggregated at the osteoporosis site by romumomab, neutralizes with the acidic microenvironment, responsively releases calcium ions, combines with in-situ hydrogen phosphate ions, and promotes bone mineralization in situ. The two-way conversion rare earth core-shell structure is excited by 808nm laser, and the emission is converted at 350nm in the ultraviolet region to excite the GSNO of the sinusoidal endothelial cells adjacent to the bone microenvironment to release NO. The release of NO can promote bone formation, and the in-situ catalysis-on-demand gas production is achieved to effectively improve the osteoporosis microenvironment. By switching the emission at NIR-Ⅱ 1300nm, the skeletal system of the osteoporosis mouse model can be clearly observed.

[0005] The present invention adopts the following technical solution: A primary osteoporosis diagnosis and treatment nanoprobe with in-situ catalysis and on-demand gas production, the nanoprobe is a spherical structure, denoted as RENP@CaCO3-Ig, including a rare earth core-shell structure at the core, CaCO3 constituting the shell layer, and romumab connected to the outermost layer; the diameter of the nanoprobe is 50-75 nm.

[0006] The probe uses rare earth core-shell nanoparticles with bidirectional conversion as carriers, replaces the original oleic acid ligand with PAA (Mw≈ 15000) polymer, and then coats amorphous calcium carbonate on the surface by gas diffusion method, changes the water solubility of the particles by coating DSPE-PEG-COOH, and couples romumab by amide reaction. Since the primary osteoporosis microenvironment has the characteristics of high expression of SOST, romumab can specifically and actively target accumulation, neutralize the acidic microenvironment, release Ca ions, and promote bone mineralization; and utilizes the bidirectional conversion characteristics of RENP under 808nm excitation to construct nanoparticles with good biocompatibility to achieve in situ catalysis-on-demand generation of NO gas, and real-time monitoring through NIR-Ⅱ fluorescence emission, thereby realizing a new and efficient integrated diagnosis and treatment of primary osteoporosis.

[0007] The nanoprobe uses rare earth core-shell nanoparticles with bidirectional conversion as carriers, and on the basis of the carriers, amorphous calcium carbonate and romumomab are coated to synthesize RE@Ca-Ig nanoprobes with uniform dispersion and uniform particle size, which can integrate fluorescence imaging and treatment. The nanoprobe can be locally treated by enhanced penetration and retention effects and active targeted accumulation of romumomab in primary osteoporosis, and the released romumomab, a common drug for osteoporosis, can play a therapeutic role locally. At the same time, the characteristics of the microenvironment acidic environment degrading CaCO3 nanoparticles lead to the responsive degradation of the nanoprobe, and the released fluorescent molecule RENP has the imaging characteristics of high tissue resolution and spatial resolution in the down-conversion near-infrared region, and up-converts emission in the ultraviolet region to stimulate the GSNO of the sinusoidal endothelial cells adjacent to the bone microenvironment to release NO. The release of NO can promote bone formation, realizing a new therapeutic strategy of in-situ catalysis-on-demand gas production to effectively improve the osteoporosis microenvironment. That is, the nanoprobe of the present invention can be guided by imaging in the activatable near-infrared region to perform on-demand "gas-drug dual-antibody" combined treatment of osteoporosis sites.

[0008] A method for preparing a primary osteoporosis diagnosis and treatment nanoprobe with in-situ catalysis and on-demand gas production, comprising the following steps: S1. Synthesis of NaYF4:20%Yb / 0.5%Tm nanoparticles: put acetate hydrate or chloride hydrate of Y, acetate hydrate or chloride hydrate of Yb and acetate hydrate or chloride hydrate of Tm into a container containing oleic acid and 1-octadecene, heat the mixture to 140°C and keep it for 1 hour under nitrogen protection, then cool it to room temperature, and then slowly add methanol solution containing NaOH and NH4F, after stirring for 1 hour, heat the mixture to 80°C and keep it for 1 hour To remove excess methanol, the temperature was then raised to 290°C at a rate of 15°C / min and maintained for 1 h under nitrogen protection. The final solution was cooled to room temperature and then centrifuged at 10,000 rpm for 5 min to collect the precipitate, which was washed twice with excess ethanol and centrifuged. Finally, the precipitate was redispersed in cyclohexane and centrifuged at 3,000 rpm to remove NaF. The supernatant, i.e., the cyclohexane solution containing the NaYF4:20%Yb / 0.5%Tm core, was stored for later use. S2. Synthesis of core-shell nanoparticles: using the epitaxial growth method, add acetate hydrate or chloride hydrate of Y, acetate hydrate or chloride hydrate of Nd, oleic acid and 1-octadecene in a three-necked flask, then heat the solution to 140°C under nitrogen protection and keep it for 1 hour until an optically transparent solution is formed. When the solution temperature drops to room temperature, add a methanol solution containing NaOH and NH4F and a cyclohexane solution containing the synthesized NaYF4:20%Yb / 0.5%Tm core. After stirring for 1 hour, the mixture is heated to 80°C to remove excess methanol, and then the solution is heated to 300°C and kept for 1 hour under nitrogen protection. After cooling to room temperature, pour excess ethanol into the solution to precipitate NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd. The resulting product is centrifuged and washed twice with ethanol. The obtained nanocrystals are redispersed in cyclohexane. S3. Add acetate hydrate or chloride hydrate of Y, oleic acid and 1-octadecene to a three-necked flask, and then heat the solution to 140°C under nitrogen protection and keep it for 1 hour until an optically transparent solution is formed. When the solution temperature drops to room temperature, add a methanol solution containing NaOH and NH4F, and a cyclohexane solution containing the synthesized NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd core. After stirring for 1 hour, heat the mixture to 80°C to remove excess methanol, and then heat the solution to 300°C and keep it for 1 hour under nitrogen protection. After cooling to room temperature, pour excess ethanol into the solution to precipitate NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd@NaYF4. Centrifuge the resulting product and wash it twice with ethanol. The obtained nanocrystals are redispersed in cyclohexane. S4, preparation of hydrophilic NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd@NaYF4: washing NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd@NaYF4 nanocrystals with hydrochloric acid to remove the oleic acid ligands on the surface, then adding the nanocrystals and PAA into H2O and stirring for 12 h, finally, centrifuging to collect PAA-RENPs; S5. Synthesis of RENP@CaCO3 particles: CaCl2 and PAA-RENPs were pre-dissolved in ethanol through gas diffusion reaction and placed in a glass bottle. The bottle mouth was covered with aluminum foil and pierced with fine holes. Then, the glass bottle was placed in a vacuum drying oven filled with dry ammonium bicarbonate. After the whole system was kept in a vacuum environment for 24 h, the temperature was set to 30-70 °C to obtain RENPs nanoparticles containing CaCO3. The particles were separated by centrifugation at 8000 rpm, redispersed in anhydrous ethanol, and further modified. The modification of the above nanoparticles was carried out in two steps: first, the ethanol solution of RENP@CaCO3 and the chloroform solution of DOPA were mixed under ultrasound for 20 min, the resulting turbid solution was centrifuged to remove unbound free DOPA, and then redispersed in chloroform to obtain RENP@CaCO3-DOPA; then, DSPE-PEG-COOH and the chloroform solution of RENP@CaCO3-DOPA were stirred vigorously overnight to PEGylate the nanoparticles, and then, the chloroform was evaporated and the obtained RENP@CaCO3-PEG nanoparticles were dissolved in aqueous solution for use; S6. The prepared RENP@CaCO3-PEG was washed with water for multiple times and purified by centrifugation. The purified RENP@CaCO3-PEG solution was added to an EDC / NHS mixture to activate the carboxyl group for 40 min, and romumab was added. The mixture was incubated at 37 °C overnight, and dispersed in water by centrifugation at 5000 rpm for 5 min to obtain RE@Ca-Ig.

[0009] Further, the acetate hydrate or chloride hydrate of Y, the acetate hydrate or chloride hydrate of Yb and the acetate hydrate or chloride hydrate of Tm in S1 are C6H9O6Y·6H2O or YCl3·6H2O, Yb(C2H3O2)3·6H2O or YbCl3·6H2O and Tm(OOCCH3)3·6H2O or TmCl3·6H2O, respectively.

[0010] Further, in S1, the molar ratio of the acetate hydrate or chloride hydrate of Y, the acetate hydrate or chloride hydrate of Yb and the acetate hydrate or chloride hydrate of Tm is 0.795-0.8:0.2:0.005; The mass concentration of oleic acid is 90%, the mass concentration of 1-octadecene is 90%, and the volume ratio of oleic acid to 1-octadecene is 6:15; The dosage ratio of NaOH, NH4F and methanol is 2.5mmol:4mmol:8mL; The amount of cyclohexane used is 5 mL.

[0011] Furthermore, the acetate hydrate or chloride hydrate of Y and the acetate hydrate or chloride hydrate of Nd in S2 are C6H9O6Y·6H2O or YCl3·6H2O, C6H9O6Nd·6H2O or NdCl3·6H2O, respectively.

[0012] Further, the usage ratio of the acetate hydrate or chloride hydrate of Y, the acetate hydrate or chloride hydrate of Nd, oleic acid and 1-octadecene in S2 is 0.795-0.8 mmol: 0.2 mmol: 6 mL: 15 mL; The dosage ratio of NaOH, NH4F and methanol is 2.5mmol:4mmol:8mL; The amount of cyclohexane solution containing NaYF4:20%Yb / 0.5%Tm core was 5mL; The amount of cyclohexane used is 5 mL.

[0013] Further, the acetate hydrate or chloride hydrate of Y in S3 is Y(CH3CO2)3 or YCl3; The usage ratio of the acetate hydrate or chloride hydrate of Y, oleic acid and 1-octadecene is 0.5 mmol: 6 mL: 15 mL; The dosage ratio of NaOH, NH4F and methanol is 2.5mmol:4mmol:8mL; The amount of cyclohexane solution containing NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd core used is 5mL.

[0014] The amount of cyclohexane used is 5 mL.

[0015] Furthermore, the concentration of the hydrochloric acid in S4 is 0.1 M, and an ultrasonic disperser is used for washing at a frequency of 50 kHz; the mass concentration of PAA is 10%, the amount used is 2 mL, and the amount of H2O used is 3 mL.

[0016] Further, the dosage ratio of CaCl2, PAA-RENPs and ethanol in S5 is 5 mg: 10 mg: 100 mL; the dosage of ammonium bicarbonate is 5 g; The dosage ratio of the RENP@CaCO3 ethanol solution and the DOPA chloroform solution is 20 mg:1 mL; wherein the DOPA chloroform solution contains 2 mg of DOPA per mL of chloroform; The dosage of the DSPE-PEG-COOH is 10-100 mg.

[0017] Furthermore, the dosage ratio of the purified RENP@CaCO3-PEG solution, EDC / NHS mixed solution, romumab, and water in S6 is 5 mg: 50 μL: 10 μL: 80 μL; among which, the concentration of romumab is 5 μM; and the mass ratio of EDS to NHS is 1 mg: 1-10 mg.

[0018] A primary osteoporosis diagnostic and therapeutic nanoprobe with in situ catalysis and on-demand gas production is used in the combined treatment of primary osteoporosis with "gas, drug and dual antibody" under the guidance of near-infrared second-zone fluorescence imaging.

[0019] The beneficial effects of the present invention are as follows: The invention provides a primary osteoporosis diagnosis and treatment nanoprobe with in-situ catalysis and on-demand gas production. The nanoprobe uses rare earth core-shell nanoparticles with bidirectional conversion as a carrier, and on the basis of the nanoprobe, amorphous calcium carbonate and romumab are coated to synthesize a RE@Ca-Ig nanoprobe with uniform dispersion and uniform particle size and integrated fluorescence imaging and treatment. The nanoprobe can be locally treated with enhanced penetration and retention effects and active targeted accumulation of romumab in primary osteoporosis, and the released romumab, a commonly used osteoporosis drug, can play a therapeutic effect locally. At the same time, due to the characteristic of microenvironmental acidic environment degrading CaCO3 nanoparticles, the nanoprobe is responsively degraded, and the released fluorescent molecule RENP has the imaging characteristics of high tissue resolution and spatial resolution in the down-conversion near-infrared region, and up-converts emission in the ultraviolet region to stimulate GSNO of blood sinusoid endothelial cells adjacent to the bone microenvironment to release NO, and the released NO can promote bone formation, thereby realizing a new treatment strategy of in-situ catalysis and on-demand gas production to effectively improve the osteoporosis microenvironment. That is, the nanoprobe of the present invention can be used to perform on-demand "gas-drug-drug-antibody" combined treatment on osteoporosis sites under the guidance of activatable near-infrared imaging. The preparation method is simple and easy to implement, and can achieve integrated diagnosis and treatment of primary osteoporosis with obvious effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a transmission electron microscope image of the nanoprobe synthesized in Example 1 of the present invention.

[0021] Figure 2 This is the up-conversion fluorescence emission spectrum of the nanoprobe synthesized in Example 1 of the present invention - GSNO ultraviolet absorption spectrum.

[0022] Figure 3 This is the in vitro NO generation ability of the nanoprobe synthesized in Example 1 of the present invention.

[0023] Figure 4 The neutralization performance of the nanoprobe synthesized in Example 1 of the present invention and the Ca 2+ Unleash performance.

[0024] Figure 5 This is the fluorescence uptake spectrum of the nanoprobe synthesized in Example 1 of the present invention in osteoblast precursor cells 3T3-E1.

[0025] Figure 6 This is an in vivo fluorescence imaging image of the nanoprobe synthesized in Example 1 of the present invention.

[0026] Figure 7 This is a MicroCT effect diagram of the in vivo anti-osteoporosis treatment of the distal femur by the nanoprobe synthesized in Example 1 of the present invention. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Example 1 The preparation of a nanoprobe for diagnosis and treatment of primary osteoporosis with in-situ catalysis and on-demand gas production comprises the following steps: S1. Synthesis of NaYF4:20%Yb / 0.5%Tm nanoparticles: C6H9O6Y·6H2O (0.795 mmol), Yb(C2H3O2)3·6H2O (0.2 mmol) and Tm(OOCCH3)3·6H2O (0.005 mmol) were placed in a mixture containing 6 mL of oleic acid (90%) and 15 mL of 1-octadecene (90%). The mixture was heated to 140°C and maintained under nitrogen protection for 1 h. Then, the solution was cooled to room temperature, and then 8 mL of methanol solution containing 2.5 mmol NaOH and 4 mmol NH4F was slowly added. After stirring for 1 hour, the mixture was heated to 80°C and maintained for 1 hour to remove excess methanol. The temperature was then increased to 290°C, rising at about 15°C / min and maintained under nitrogen protection for 1 hour. The final solution was cooled to room temperature, and then the precipitate was collected by centrifugation at 10,000 rpm for 5 min, washed twice with excess ethanol and centrifuged. Finally, the resulting precipitate was redispersed in 5 mL of cyclohexane and centrifuged at low speed (3,000 rpm) to remove NaF. The supernatant was stored for later use; S2. The synthesis of core-shell nanoparticles was performed by epitaxial growth. A certain amount of C6H9O6Y·6H2O (0.8 mmol), C6H9O6Nd·6H2O (0.2 mmol), 6mL oleic acid (90%) and 15mL 1-octadecene (90%) were added to a 100 mL three-necked flask. The solution was then heated to 140°C under nitrogen protection and maintained for about 1 hour until an optically transparent solution was formed. When the solution temperature dropped to room temperature, 8mL of a methanol solution containing 2.5mmol NaOH and 4mmol NH4F and 5mL of a cyclohexane solution containing the synthesized NaYF4: 20%Yb / 0.5%Tm core were added. After stirring for 1 hour, the mixture was heated to 80°C to remove excess volatile solvents, and then the solution was heated to 300°C and maintained for 1 hour under nitrogen protection. After cooling to room temperature, excess ethanol was poured into the solution to precipitate NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd. The resulting product was centrifuged and washed twice with ethanol. The resulting nanocrystals could be easily redispersed in cyclohexane; S3, 0.5mmol Y(CH3CO2)3 was used to replace RE(CH3CO2)3, and the synthesis process was the same as the NaYF4:Nd shell growth process in S2; S4, hydrophilic NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd@NaYF4 was prepared by washing with hydrochloric acid to remove the oleic acid ligands on the surface. Then, the nanocrystals and 10% PAA (2 mL) were added to H2O (3 mL) and stirred for 12 h. Finally, the PAA-RENPs were collected by centrifugation; S5. In the RENP@CaCO3 particles, 5 mg of CaCl2 and 10 mg of PAA-RENPs were pre-dissolved in 100 ml of ethanol and placed in a glass bottle. The bottle mouth was covered with aluminum foil and pierced with fine holes. Then, the glass bottle was placed in a vacuum oven containing 5 g of dry ammonium bicarbonate (NH4HCO3). After keeping the entire system in a vacuum environment for 24 hours, the temperature was set to 30-70 ° C to obtain RENPs nanoparticles containing CaCO3, which could be separated by centrifugation at 8000 rpm. These nanoparticles were redispersed in anhydrous ethanol for further modification.

[0029] Furthermore, the modification of the above nanoparticles was carried out in two steps. First, 20 mg of RENP@CaCO3 solution (ethanol solution) and 1 mL of DOPA solution (2 mg / mL, chloroform) were mixed under ultrasound for 20 min. The resulting turbid solution was centrifuged to remove unbound free DOPA and then redispersed in chloroform. Then, DSPE-PEG-COOH was mixed with the chloroform solution of RENP@CaCO3-DOPA under vigorous stirring overnight to PEGylate the nanoparticles. Then, the chloroform was evaporated and the resulting RENP@CaCO3-PEG nanoparticles were dissolved in an aqueous solution for use; S6. Wash the prepared RENP@CaCO3-PEG with water several times and centrifuge (8000 rpm, 5 min). After purification, take 5 mg of the purified RENP@CaCO3-PEG solution and add it to 50 μL of EDC / NHS mixture to activate the carboxyl group for 40 min. Add 10 μL of 5μM Romosozumab and incubate at 37 °C overnight. Centrifuge (5000 rpm, 5 min) and disperse in 80 μL of water to obtain RE@Ca-Ig.

[0030] Depend on Figure 1 It can be seen that RE@Ca-Ig nanoparticles are spherical and have an average particle size of 40 nm.

[0031] Depend on Figure 2 It can be seen that nitric oxide is stored in the body in the form of S-sulfinylglutathione (GSNO). The ultraviolet absorption spectrum of GSNO overlaps with the upconversion emission spectrum of RE@Ca-Ig, which theoretically indicates that GSNO effectively absorbs the ultraviolet energy emitted by RE@Ca-Ig, thereby leading to the release of NO.

[0032] Depend on Figure 3 It can be seen that in order to test the controlled release ability of NO, five laser switching experiments were carried out on the RE@Ca-Ig material within 10 minutes. The results show that when the laser is turned on, NO accumulates rapidly, while when the laser is turned off, the NO content remains stable. When RE@Ca-Ig is excited by lasers with different power densities (0.5, 1.0 and 2.0 W / cm²), the release rate of NO is positively correlated with the laser intensity.

[0033] Depend on Figure 4It can be seen that the pH value changes after introducing different volumes of RE@Ca-Ig solution into PBS buffer (pH=4.5). With the addition of RE@Ca-Ig, the pH value of the solution gradually returned to normal levels, indicating that it has effective acid neutralization ability. Controlling degradation in an acidic environment is essential to maintain the efficacy of RE@Ca-Ig in vivo. After 36 hours, the release was almost saturated, reaching 90% at pH 4.5 and only 29% at pH 7.4. This significant difference in release indicates that RE@Ca-Ig can be specifically activated at the lesion site, thereby improving the local microenvironment. The degradation of CaCO3 affected the RE@Ca-Ig particle size, and the average hydrodynamic particle size decreased after 72 hours of acid treatment at pH 4.5. Overall, RE@CR showed significant sensitivity and specificity to pH stimulation, effectively neutralized acid, created an optimal environment for reconstructing the bone microenvironment, and provided sufficient material for bone mineralization.

[0034] Depend on Figure 5 It can be seen that: using 3T3 cells as a model, the NIR-II fluorescence of RE@Ca-Ig was used to evaluate the cellular internalization at different incubation times. NIR-II fluorescence microscopy images showed that the fluorescence intensity of RE@CR increased with incubation time, indicating enhanced cellular uptake.

[0035] Depend on Figure 6 It can be seen that RE@Ca-Ig can achieve NIR-II fluorescence imaging in the primary osteoporosis mouse model.

[0036] Depend on Figure 7 It can be seen that trabeculae are microscopic structures within bone tissue and are essential for maintaining bone strength and stability. MicroCT three-dimensional and two-dimensional reconstructions showed that compared with the healthy group, the bone density of mice in the osteoporosis modeling group was significantly reduced, the trabeculae were thinner, and the trabecular gaps were enlarged.

[0037] Example 2 A preparation method of a primary osteoporosis diagnosis and treatment nanoprobe with in-situ catalysis and on-demand gas production comprises the following steps: S1. Synthesis of NaYF4: 20% Yb / 0.5% Tm nanoparticles. C6H9O6Y·6H2O (0.8 mmol), Yb(C2H3O2)3·6H2O (0.2 mmol) and Tm(OOCCH3)3·6H2O (0.005 mmol) were placed in a mixture containing 6 mL of oleic acid (90%) and 15 mL of 1-octadecene (90%). The mixture was heated to 140°C and maintained under nitrogen protection for 1 h. Then, the solution was cooled to room temperature, and then 8 mL of methanol solution containing 2.5 mmol NaOH and 4 mmol NH4F was slowly added. After stirring for 1 h, the mixture was heated to 80°C and maintained for 1 h to remove excess methanol. The temperature was then increased to 290°C, rising at about 15°C / min and maintained under nitrogen protection for 1 h. The final solution was cooled to room temperature, and then the precipitate was collected by centrifugation at 10000 rpm for 5 min, washed twice with excess ethanol and centrifuged. Finally, the resulting precipitate was redispersed in 5 mL of cyclohexane and centrifuged at low speed (3000 rpm) to remove NaF. The supernatant was stored for later use; S2. The synthesis of core-shell nanoparticles was performed by epitaxial growth. A certain amount of C6H9O6Y·6H2O (0.8 mmol), C6H9O6Nd·6H2O (0.2 mmol), 6mL oleic acid (90%) and 15mL 1-octadecene (90%) were added to a 100 mL three-necked flask. The solution was then heated to 140°C under nitrogen protection and maintained for about 1 hour until an optically transparent solution was formed. When the solution temperature dropped to room temperature, 8mL of a methanol solution containing 2.5mmol NaOH and 4mmol NH4F and 5mL of a cyclohexane solution containing the synthesized NaYF4:20%Yb / 0.5%Tm core were added. After stirring for 1 hour, the mixture was heated to 80°C to remove excess volatile solvents, and then the solution was heated to 300°C and maintained for 1 hour under nitrogen protection. After cooling to room temperature, excess ethanol was poured into the solution to precipitate NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd. The resulting product was centrifuged and washed twice with ethanol. The resulting nanocrystals could be easily redispersed in cyclohexane; S3, using 0.5mmol Y(CH3CO2)3 to replace RE(CH3CO2)3, the synthesis process is the same as the NaYF4:Nd shell growth process in S2 above; S4, hydrophilic NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd@NaYF4 was prepared by washing with hydrochloric acid to remove the oleic acid ligands on the surface. Then, the nanocrystals and 10% PAA (2 mL) were added to H2O (3 mL) and stirred for 12 h. Finally, the PAA-RENPs were collected by centrifugation; S5. The RENP@CaCO3 particles were synthesized by gas diffusion reaction. 5 mg of CaCl2 and 10 mg of PAA-RENPs were pre-dissolved in 100 ml of ethanol and placed in a glass bottle. The bottle mouth was covered with aluminum foil and pierced with fine holes. Then, the glass bottle was placed in a vacuum oven containing 5 g of dry ammonium bicarbonate (NH4HCO3). After keeping the entire system in a vacuum environment for 24 hours, the temperature was set to 30-70°C to obtain RENPs nanoparticles containing CaCO3, which could be separated by centrifugation at 8000 rpm. These nanoparticles were redispersed in anhydrous ethanol for further modification.

[0038] Furthermore, the modification of the above nanoparticles was carried out in two steps. First, 20 mg of RENP@CaCO3 solution (ethanol solution) and 1 mL of DOPA solution (2 mg / mL, chloroform) were mixed under ultrasound for 20 min. The resulting turbid solution was centrifuged to remove unbound free DOPA and then redispersed in chloroform. Then, DSPE-PEG-COOH was mixed with the chloroform solution of RENP@CaCO3-DOPA under vigorous stirring overnight to PEGylate the nanoparticles. Then, the chloroform was evaporated and the resulting RENP@CaCO3-PEG nanoparticles were dissolved in aqueous solution for use; S6. Wash the prepared RENP@CaCO3-PEG with water several times and centrifuge (8000 rpm, 5 min). After purification, take 5 mg of the purified RENP@CaCO3-PEG solution and add it to 50 μL of EDC / NHS mixed solution to activate the carboxyl group for 40 min. Add 10 μL of 5μM Romosozumab, incubate at 37 °C overnight, centrifuge (5000 rpm, 5 min), and disperse in 80 μL of water to obtain RE@Ca-Ig.

[0039] Example 3 The preparation of a rheumatoid arthritis microenvironment-responsive integrated diagnosis and treatment nanoprobe comprises the following steps: S1. Synthesis of NaYF4:20%Yb / 0.5%Tm nanoparticles. YCl3·6H2O (0.795 mmol), YbCl3·6H2O (0.2 mmol) and TmCl3·6H2O (0.005 mmol) were placed in a mixture containing 6 mL of oleic acid (90%) and 15 mL of 1-octadecene (90%). The mixture was heated to 140°C and maintained under nitrogen protection for 1 h. Then, the solution was cooled to room temperature, and 8 mL of methanol solution containing 2.5 mmol NaOH and 4 mmol NH4F was slowly added. After stirring for 1 h, the mixture was heated to 80°C and maintained for 1 h to remove excess methanol. The temperature was then raised to 290°C, rising at about 15°C / min and maintained under nitrogen protection for 1 h. The final solution was cooled to room temperature, and the precipitate was collected by centrifugation at 10,000 rpm for 5 min, washed twice with excess ethanol and centrifuged. Finally, the resulting precipitate was redispersed in 5 mL of cyclohexane and centrifuged at low speed (3000 rpm) to remove NaF. The supernatant was stored for later use; S2. The synthesis of core-shell nanoparticles adopts epitaxial growth method. A certain amount of YCl3·6H2O (0.8 mmol), C6H9O6Nd·6H2O (0.2 mmol), 6mL oleic acid (90%) and 15mL 1-octadecene (90%) were added to a 100 mL three-necked flask. The solution was then heated to 140°C under nitrogen protection and maintained for about 1 hour until an optically transparent solution was formed. When the solution temperature dropped to room temperature, 8mL of methanol solution containing 2.5mmol NaOH and 4mmol NH4F and 5mL of cyclohexane solution containing the synthesized NaYF4:20%Yb / 0.5%Tm core were added respectively. After stirring for 1 hour, the mixture was heated to 80°C to remove excess volatile solvents, and then the solution was heated to 300°C and maintained for 1 hour under nitrogen protection. After cooling to room temperature, excess ethanol was poured into the solution to precipitate NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd. The resulting product was centrifuged and washed twice with ethanol. The resulting nanocrystals could be easily redispersed in cyclohexane; S3, except that 0.5mmol YCl3 was used to replace RECl3, the synthesis process was the same as the NaYF4:Nd shell growth process in S2 above; S4, hydrophilic NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd@NaYF4 was prepared by washing with hydrochloric acid to remove the oleic acid ligands on the surface. Then, the nanocrystals and 10% PAA (2 mL) were added to H2O (3 mL) and stirred for 12 h. Finally, the PAA-RENPs were collected by centrifugation; S5. The RENP@CaCO3 particles were synthesized by gas diffusion reaction. 5 mg of CaCl2 and 10 mg of PAA-RENPs were pre-dissolved in 100 ml of ethanol and placed in a glass bottle. The bottle mouth was covered with aluminum foil and pierced with fine holes. Then, the glass bottle was placed in a vacuum oven containing 5 g of dry ammonium bicarbonate (NH4HCO3). After keeping the entire system in a vacuum environment for 24 hours, the temperature was set to 30-70°C to obtain RENPs nanoparticles containing CaCO3, which could be separated by centrifugation at 8000 rpm. These nanoparticles were redispersed in anhydrous ethanol for further modification.

[0040] Furthermore, the modification of the above nanoparticles was carried out in two steps. First, 20 mg of RENP@CaCO3 solution (ethanol solution) and 1 mL of DOPA solution (2 mg / mL, chloroform) were mixed under ultrasound for 20 min. The resulting turbid solution was centrifuged to remove unbound free DOPA and then redispersed in chloroform. Then, DSPE-PEG-COOH was mixed with the chloroform solution of RENP@CaCO3-DOPA under vigorous stirring overnight to PEGylate the nanoparticles. Then, the chloroform was evaporated and the resulting RENP@CaCO3-PEG nanoparticles were dissolved in aqueous solution for use; S6. Wash the prepared RENP@CaCO3-PEG with water several times and centrifuge (8000 rpm, 5 min). After purification, take 5 mg of the purified RENP@CaCO3-PEG solution and add it to 50 μL of EDC / NHS mixed solution to activate the carboxyl group for 40 min. Add 10 μL of 5μM Romosozumab, incubate at 37 °C overnight, centrifuge (5000 rpm, 5 min), and disperse in 80 μL of water to obtain RE@Ca-Ig.

[0041] Example 4 A preparation method of a primary osteoporosis diagnosis and treatment nanoprobe with in-situ catalysis and on-demand gas production comprises the following steps: S1. Synthesis of NaYF4:20%Yb / 0.5%Tm nanoparticles. C6H9O6Y·6H2O (0.795 mmol), Yb(C2H3O2)3·6H2O (0.2 mmol) and Tm(OOCCH3)3·6H2O (0.005 mmol) were placed in a mixture containing 6 mL of oleic acid (90%) and 15 mL of 1-octadecene (90%). The mixture was heated to 140°C and maintained under nitrogen protection for 1 h. Then, the solution was cooled to room temperature, and then 8 mL of methanol solution containing 2.5 mmol NaOH and 4 mmol NH4F was slowly added. After stirring for 1 hour, the mixture was heated to 80°C and maintained for 1 hour to remove excess methanol. The temperature was then increased to 290°C, rising at about 15°C / min and maintained under nitrogen protection for 1 hour. The final solution was cooled to room temperature, and then the precipitate was collected by centrifugation at 10,000 rpm for 5 min, washed twice with excess ethanol and centrifuged. Finally, the resulting precipitate was redispersed in 5 mL of cyclohexane and centrifuged at low speed (3,000 rpm) to remove NaF. The supernatant was stored for later use; S2. The synthesis of core-shell nanoparticles was performed by epitaxial growth. A certain amount of C6H9O6Y·6H2O (0.8 mmol), C6H9O6Nd·6H2O (0.2 mmol), 6mL oleic acid (90%) and 15mL 1-octadecene (90%) were added to a 100 mL three-necked flask. The solution was then heated to 140°C under nitrogen protection and maintained for about 1 hour until an optically transparent solution was formed. When the solution temperature dropped to room temperature, 8mL of a methanol solution containing 2.5mmol NaOH and 4mmol NH4F and 5mL of a cyclohexane solution containing the synthesized NaYF4: 20%Yb / 0.5%Tm core were added. After stirring for 1 hour, the mixture was heated to 80°C to remove excess volatile solvents, and then the solution was heated to 300°C and maintained for 1 hour under nitrogen protection. After cooling to room temperature, excess ethanol was poured into the solution to precipitate NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd. The resulting product was centrifuged and washed twice with ethanol. The resulting nanocrystals could be easily redispersed in cyclohexane; S3, using 0.5mmol Y(CH3CO2)3 to replace RE(CH3CO2)3, the synthesis process is the same as the NaYF4:Nd shell growth process in S2 above; S4, hydrophilic NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd@NaYF4 was prepared by washing with 0.1M hydrochloric acid to remove the oleic acid ligands on the surface, and the dispersion washing was performed using an ultrasonic disperser at a frequency of 50 kHz. Then, the nanocrystals and 10% PAA (2 mL) were added to H2O (3 mL) and stirred for 12 hours. Finally, the PAA-RENPs were collected by centrifugation; S5. In the RENP@CaCO3 particles, 5 mg of CaCl2 and 10 mg of PAA-RENPs were pre-dissolved in 100 ml of ethanol and placed in a glass bottle. The bottle mouth was covered with aluminum foil and pierced with fine holes. Then, the glass bottle was placed in a vacuum oven containing 5 g of dry ammonium bicarbonate (NH4HCO3). After keeping the entire system in a vacuum environment for 24 hours, the temperature was set to 30-70 ° C to obtain RENPs nanoparticles containing CaCO3, which could be separated by centrifugation at 8000 rpm. These nanoparticles were redispersed in anhydrous ethanol for further modification.

[0042] Furthermore, the modification of the above nanoparticles was carried out in two steps. First, 20 mg of RENP@CaCO3 solution (ethanol solution) and 1 mL of DOPA solution (2 mg / mL, chloroform) were mixed under ultrasound for 20 min. The resulting turbid solution was centrifuged to remove unbound free DOPA and then redispersed in chloroform. Then, DSPE-PEG-COOH was mixed with the chloroform solution of RENP@CaCO3-DOPA under vigorous stirring overnight to PEGylate the nanoparticles. Then, the chloroform was evaporated and the resulting RENP@CaCO3-PEG nanoparticles were dissolved in an aqueous solution for use.

[0043] S6. Wash the prepared RENP@CaCO3-PEG with water several times and centrifuge (8000 rpm, 5 min). After purification, take 5 mg of the purified RENP@CaCO3-PEG solution and add it to 50 μL of EDC / NHS mixture to activate the carboxyl group for 40 min. Add 10 μL of 5μM Romosozumab and incubate at 37 °C overnight. Centrifuge (5000 rpm, 5 min) and disperse in 80 μL of water to obtain RE@Ca-Ig.

[0044] Example 5 A preparation method of a primary osteoporosis diagnosis and treatment nanoprobe with in-situ catalysis and on-demand gas production comprises the following steps: S1. Synthesis of NaYF4:20%Yb / 0.5%Tm nanoparticles. C6H9O6Y·6H2O (0.795 mmol), Yb(C2H3O2)3·6H2O (0.2 mmol) and Tm(OOCCH3)3·6H2O (0.005 mmol) were placed in a mixture containing 6 mL of oleic acid (90%) and 15 mL of 1-octadecene (90%). The mixture was heated to 140°C and maintained under nitrogen protection for 1 h. Then, the solution was cooled to room temperature, and then 8 mL of methanol solution containing 2.5 mmol NaOH and 4 mmol NH4F was slowly added. After stirring for 1 hour, the mixture was heated to 80°C and maintained for 1 hour to remove excess methanol. The temperature was then increased to 290°C, rising at about 15°C / min and maintained under nitrogen protection for 1 hour. The final solution was cooled to room temperature, and then the precipitate was collected by centrifugation at 10000 rpm for 5 min, washed twice with excess ethanol and centrifuged. Finally, the resulting precipitate was redispersed in 5 mL of cyclohexane and centrifuged at low speed (3000 rpm) to remove NaF. The supernatant was stored for later use. ; S2. The synthesis of core-shell nanoparticles was performed by epitaxial growth. A certain amount of C6H9O6Y·6H2O (0.8 mmol), C6H9O6Nd·6H2O (0.2 mmol), 6mL oleic acid (90%) and 15mL 1-octadecene (90%) were added to a 100 mL three-necked flask. The solution was then heated to 140°C under nitrogen protection and maintained for about 1 hour until an optically transparent solution was formed. When the solution temperature dropped to room temperature, 8mL of a methanol solution containing 2.5mmol NaOH and 4mmol NH4F and 5mL of a cyclohexane solution containing the synthesized NaYF4: 20%Yb / 0.5%Tm core were added. After stirring for 1 hour, the mixture was heated to 80°C to remove excess volatile solvents, and then the solution was heated to 300°C and maintained for 1 hour under nitrogen protection. After cooling to room temperature, excess ethanol was poured into the solution to precipitate NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd. The resulting product was centrifuged and washed twice with ethanol. The resulting nanocrystals could be easily redispersed in cyclohexane; S3, using 0.5mmol Y(CH3CO2)3 to replace RE(CH3CO2)3, the synthesis process is the same as the NaYF4:Nd shell growth process in S2 above; S4, hydrophilic NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd@NaYF4 was prepared by washing with hydrochloric acid to remove the oleic acid ligands on the surface. Then, the nanocrystals and 10% PAA (2 mL) were added to H2O (3 mL) and stirred for 12 h. Finally, the PAA-RENPs were collected by centrifugation; S5. In the RENP@CaCO3 particles, which were synthesized by gas diffusion reaction, 5 mg of CaCl2 and 5 mg of PAA-RENPs were pre-dissolved in 100 ml of ethanol and placed in a glass bottle. The bottle mouth was covered with aluminum foil and pierced with fine holes. Then, the glass bottle was placed in a vacuum oven containing 5 g of dry ammonium bicarbonate (NH4HCO3). After keeping the entire system in a vacuum environment for 24 hours, the temperature was set to 30-70 ° C to obtain RENPs nanoparticles containing CaCO3, which could be separated by centrifugation at 8000 rpm. These nanoparticles were redispersed in anhydrous ethanol for further modification.

[0045] Furthermore, the modification of the above nanoparticles was carried out in two steps. First, 20 mg of RENP@CaCO3 solution (ethanol solution) and 1 mL of DOPA solution (2 mg / mL, chloroform) were mixed under ultrasound for 20 min. The resulting turbid solution was centrifuged to remove unbound free DOPA and then redispersed in chloroform. Then, DSPE-PEG-COOH was mixed with the chloroform solution of RENP@CaCO3-DOPA under vigorous stirring overnight to PEGylate the nanoparticles. Then, the chloroform was evaporated and the resulting RENP@CaCO3-PEG nanoparticles were dissolved in an aqueous solution for use.

[0046] S6. Wash the prepared RENP@CaCO3-PEG with water several times and centrifuge (8000 rpm, 5 min). After purification, take 5 mg of the purified RENP@CaCO3-PEG solution and add it to 50 μL of EDC / NHS mixture to activate the carboxyl group for 40 min. Add 10 μL of 5μM Romosozumab and incubate at 37 °C overnight. Centrifuge (5000 rpm, 5 min) and disperse in 80 μL of water to obtain RE@Ca-Ig.

[0047] Example 6 The preparation of a rheumatoid arthritis microenvironment-responsive integrated diagnosis and treatment nanoprobe comprises the following steps: S1. Synthesis of NaYF4:20%Yb / 0.5%Tm nanoparticles. C6H9O6Y·6H2O (0.795 mmol), Yb(C2H3O2)3·6H2O (0.2 mmol) and Tm(OOCCH3)3·6H2O (0.005 mmol) were placed in a mixture containing 6 mL of oleic acid (90%) and 15 mL of 1-octadecene (90%). The mixture was heated to 140°C and maintained under nitrogen protection for 1 h. Then, the solution was cooled to room temperature, and then 8 mL of methanol solution containing 2.5 mmol NaOH and 4 mmol NH4F was slowly added. After stirring for 1 hour, the mixture was heated to 80°C and maintained for 1 hour to remove excess methanol. The temperature was then increased to 290°C, rising at about 15°C / min and maintained under nitrogen protection for 1 hour. The final solution was cooled to room temperature, and then the precipitate was collected by centrifugation at 10,000 rpm for 5 min, washed twice with excess ethanol and centrifuged. Finally, the resulting precipitate was redispersed in 5 mL of cyclohexane and centrifuged at low speed (3,000 rpm) to remove NaF. The supernatant was stored for later use; S2. The synthesis of core-shell nanoparticles was performed by epitaxial growth. A certain amount of C6H9O6Y·6H2O (0.8 mmol), C6H9O6Nd·6H2O (0.2 mmol), 6mL oleic acid (90%) and 15mL 1-octadecene (90%) were added to a 100 mL three-necked flask. The solution was then heated to 140°C under nitrogen protection and maintained for about 1 hour until an optically transparent solution was formed. When the solution temperature dropped to room temperature, 8mL of a methanol solution containing 2.5mmol NaOH and 4mmol NH4F and 5mL of a cyclohexane solution containing the synthesized NaYF4: 20%Yb / 0.5%Tm core were added. After stirring for 1 hour, the mixture was heated to 80°C to remove excess volatile solvents, and then the solution was heated to 300°C and maintained for 1 hour under nitrogen protection. After cooling to room temperature, excess ethanol was poured into the solution to precipitate NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd. The resulting product was centrifuged and washed twice with ethanol. The resulting nanocrystals could be easily redispersed in cyclohexane; S3, using 0.5mmol Y(CH3CO2)3 to replace RE(CH3CO2)3, the synthesis process is the same as the NaYF4:Nd shell growth process in S2 above; S4, hydrophilic NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd@NaYF4 was prepared by washing with hydrochloric acid to remove the oleic acid ligands on the surface. Then, the nanocrystals and 10% PAA (2 mL) were added to H2O (3 mL) and stirred for 12 h. Finally, the PAA-RENPs were collected by centrifugation; S5. The RENP@CaCO3 particles were synthesized by gas diffusion reaction. 5 mg of CaCl2 and 10 mg of PAA-RENPs were pre-dissolved in 100 ml of ethanol and placed in a glass bottle. The bottle mouth was covered with aluminum foil and pierced with fine holes. Then, the glass bottle was placed in a vacuum oven containing 5 g of dry ammonium bicarbonate (NH4HCO3). After keeping the entire system in a vacuum environment for 24 hours, the temperature was set to 30-70°C to obtain RENPs nanoparticles containing CaCO3, which could be separated by centrifugation at 8000 rpm. These nanoparticles were redispersed in anhydrous ethanol for further modification.

[0048] Further, the modification of the above nanoparticles was carried out in two steps. First, 20 mg of RENP@CaCO3 solution (ethanol solution) and 1 ml of DOPA solution (0.1 mg / ml, chloroform) were mixed under ultrasound for 20 min. The resulting turbid solution was centrifuged to remove unbound free DOPA and then redispersed in chloroform. Then, 10~100 mg DSPE-PEG-COOH and chloroform solution of RENP@CaCO3-DOPA were dissolved and dispersed using an ultrasonic disperser for 10 min at a frequency of 50 kHz and stirred overnight under vigorous stirring to PEGylate the nanoparticles. Then, the chloroform was evaporated and the obtained RENP@CaCO3-PEG nanoparticles were dissolved in an aqueous solution for use.

[0049] S6. Wash the prepared RENP@CaCO3-PEG with water several times and centrifuge (8000 rpm, 5 min). After purification, take 5 mg of the purified RENP@CaCO3-PEG solution and add it to 50 μL of EDC / NHS mixture to activate the carboxyl group for 40 min. Add 10 μL of 5μM Romosozumab and incubate at 37 °C overnight. Centrifuge (5000 rpm, 5 min) and disperse in 80 μL of water to obtain RE@Ca-Ig.

[0050] Example 7 A preparation method of a primary osteoporosis diagnosis and treatment nanoprobe with in-situ catalysis and on-demand gas production comprises the following steps: S1. Synthesis of NaYF4:20%Yb / 0.5%Tm nanoparticles. C6H9O6Y·6H2O (0.795 mmol), Yb(C2H3O2)3·6H2O (0.2 mmol) and Tm(OOCCH3)3·6H2O (0.005 mmol) were placed in a mixture containing 6 mL of oleic acid (90%) and 15 mL of 1-octadecene (90%). The mixture was heated to 140°C and maintained under nitrogen protection for 1 h. Then, the solution was cooled to room temperature, and then 8 mL of methanol solution containing 2.5 mmol NaOH and 4 mmol NH4F was slowly added. After stirring for 1 hour, the mixture was heated to 80°C and maintained for 1 hour to remove excess methanol. The temperature was then increased to 290°C, rising at about 15°C / min and maintained under nitrogen protection for 1 hour. The final solution was cooled to room temperature, and then the precipitate was collected by centrifugation at 10,000 rpm for 5 min, washed twice with excess ethanol and centrifuged. Finally, the resulting precipitate was redispersed in 5 mL of cyclohexane and centrifuged at low speed (3,000 rpm) to remove NaF. The supernatant was stored for later use; S2. The synthesis of core-shell nanoparticles was performed by epitaxial growth. A certain amount of C6H9O6Y·6H2O (0.8 mmol), C6H9O6Nd·6H2O (0.2 mmol), 6mL oleic acid (90%) and 15mL 1-octadecene (90%) were added to a 100 mL three-necked flask. The solution was then heated to 140°C under nitrogen protection and maintained for about 1 hour until an optically transparent solution was formed. When the solution temperature dropped to room temperature, 8mL of a methanol solution containing 2.5mmol NaOH and 4mmol NH4F and 5mL of a cyclohexane solution containing the synthesized NaYF4: 20%Yb / 0.5%Tm core were added. After stirring for 1 hour, the mixture was heated to 80°C to remove excess volatile solvents, and then the solution was heated to 300°C and maintained for 1 hour under nitrogen protection. After cooling to room temperature, excess ethanol was poured into the solution to precipitate NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd. The resulting product was centrifuged and washed twice with ethanol. The resulting nanocrystals could be easily redispersed in cyclohexane; S3, using 0.5mmol Y(CH3CO2)3 to replace RE(CH3CO2)3, the synthesis process is the same as the NaYF4:Nd shell growth process in S2 above; S4, hydrophilic NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd@NaYF4 was prepared by washing with hydrochloric acid to remove the oleic acid ligands on the surface. Then, the nanocrystals and 10% PAA (2 mL) were added to H2O (3 mL) and stirred for 12 h. Finally, the PAA-RENPs were collected by centrifugation; S5. The RENP@CaCO3 particles were synthesized by gas diffusion reaction. 5 mg of CaCl2 and 10 mg of PAA-RENPs were pre-dissolved in 100 ml of ethanol and placed in a glass bottle. The bottle mouth was covered with aluminum foil and pierced with fine holes. Then, the glass bottle was placed in a vacuum oven containing 5 g of dry ammonium bicarbonate (NH4HCO3). After keeping the entire system in a vacuum environment for 24 hours, the temperature was set to 30-70°C to obtain RENPs nanoparticles containing CaCO3, which could be separated by centrifugation at 8000 rpm. These nanoparticles were redispersed in anhydrous ethanol for further modification.

[0051] Furthermore, the modification of the above nanoparticles was carried out in two steps. First, 20 mg of RENP@CaCO3 solution (ethanol solution) and 1 mL of DOPA solution (2 mg / mL, chloroform) were mixed under ultrasound for 20 min. The resulting turbid solution was centrifuged to remove unbound free DOPA and then redispersed in chloroform. Then, DSPE-PEG-COOH was mixed with the chloroform solution of RENP@CaCO3-DOPA under vigorous stirring overnight to PEGylate the nanoparticles. Then, the chloroform was evaporated and the resulting RENP@CaCO3-PEG nanoparticles were dissolved in an aqueous solution for use.

[0052] S6. Prepare EDC / NHS mixed solution, the mass volume ratio of EDC / NHS mixed solution is 1mg:1-6mL; the mass ratio of EDC to NHS is 1mg:1-10mg, and the volume dissolved in deionized water is 1mL. Wash the prepared RENP@CaCO3-PEG with water several times and centrifuge (8000 rpm, 5 min). After purification, take 5mg of purified RENP@CaCO3-PEG solution and add it to 50 μL EDC / NHS mixed solution to activate the carboxyl group for 40 min, add 10 μL 5μM Romosozumab, incubate at 37 °C overnight, centrifuge (5000 rpm, 5min), and disperse in 80 μL water to obtain RE@Ca-Ig.

[0053] Only the preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in this field without departing from the purpose of the present invention, and various changes should be included in the protection scope of the present invention.

Claims

1. A primary osteoporosis diagnosis and treatment nanoprobe with in-situ catalysis and on-demand gas production, characterized in that: The nanoprobe is a spherical structure, denoted as RENP@CaCO3-Ig, including a rare earth core-shell structure at the core, CaCO3 constituting the shell layer, and romumab connected to the outermost layer; the diameter of the nanoprobe is 50-75 nm.

2. A method for preparing a nanoprobe for diagnosis and treatment of primary osteoporosis with in-situ catalysis and on-demand gas production as claimed in claim 1, characterized in that: The steps include: S1. Synthesis of NaYF4:20%Yb / 0.5%Tm nanoparticles: put acetate hydrate or chloride hydrate of Y, acetate hydrate or chloride hydrate of Yb and acetate hydrate or chloride hydrate of Tm into a container containing oleic acid and 1-octadecene, heat the mixture to 140°C and keep it for 1 hour under nitrogen protection, then cool it to room temperature, and then slowly add methanol solution containing NaOH and NH4F, after stirring for 1 hour, heat the mixture to 80°C and keep it for 1 hour To remove excess methanol, the temperature was then raised to 290°C at a rate of 15°C / min and maintained for 1 h under nitrogen protection. The final solution was cooled to room temperature and then centrifuged at 10,000 rpm for 5 min to collect the precipitate, which was washed twice with excess ethanol and centrifuged. Finally, the precipitate was redispersed in cyclohexane and centrifuged at 3,000 rpm to remove NaF. The supernatant, i.e., the cyclohexane solution containing the NaYF4:20%Yb / 0.5%Tm core, was stored for later use. S2. Synthesis of core-shell nanoparticles: using the epitaxial growth method, add acetate hydrate or chloride hydrate of Y, acetate hydrate or chloride hydrate of Nd, oleic acid and 1-octadecene in a three-necked flask, then heat the solution to 140°C under nitrogen protection and keep it for 1 hour until an optically transparent solution is formed. When the solution temperature drops to room temperature, add a methanol solution containing NaOH and NH4F and a cyclohexane solution containing the synthesized NaYF4:20%Yb / 0.5%Tm core. After stirring for 1 hour, the mixture is heated to 80°C to remove excess methanol, and then the solution is heated to 300°C and kept for 1 hour under nitrogen protection. After cooling to room temperature, pour excess ethanol into the solution to precipitate NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd. The resulting product is centrifuged and washed twice with ethanol. The obtained nanocrystals are redispersed in cyclohexane. S3. Add acetate hydrate or chloride hydrate of Y, oleic acid and 1-octadecene to a three-necked flask, and then heat the solution to 140°C under nitrogen protection and keep it for 1 hour until an optically transparent solution is formed. When the solution temperature drops to room temperature, add a methanol solution containing NaOH and NH4F, and a cyclohexane solution containing the synthesized NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd core. After stirring for 1 hour, heat the mixture to 80°C to remove excess methanol, and then heat the solution to 300°C and keep it for 1 hour under nitrogen protection. After cooling to room temperature, pour excess ethanol into the solution to precipitate NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd@NaYF4. Centrifuge the resulting product and wash it twice with ethanol. The obtained nanocrystals are redispersed in cyclohexane. S4, preparation of hydrophilic NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd@NaYF4: washing NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd@NaYF4 nanocrystals with hydrochloric acid to remove the oleic acid ligands on the surface, then adding the nanocrystals and PAA into H2O and stirring for 12 h, finally, centrifuging to collect PAA-RENPs; S5. Synthesis of RENP@CaCO3 particles: CaCl2 and PAA-RENPs were pre-dissolved in ethanol through gas diffusion reaction and placed in a glass bottle. The bottle mouth was covered with aluminum foil and pierced with fine holes. Then, the glass bottle was placed in a vacuum drying oven filled with dry ammonium bicarbonate. After the whole system was kept in a vacuum environment for 24 h, the temperature was set to 30-70 °C to obtain RENPs nanoparticles containing CaCO3. The particles were separated by centrifugation at 8000 rpm, redispersed in anhydrous ethanol, and further modified. The modification of the above nanoparticles was carried out in two steps: first, the ethanol solution of RENP@CaCO3 and the chloroform solution of DOPA were mixed under ultrasound for 20 min, the resulting turbid solution was centrifuged to remove unbound free DOPA, and then redispersed in chloroform to obtain RENP@CaCO3-DOPA; then, DSPE-PEG-COOH and the chloroform solution of RENP@CaCO3-DOPA were stirred vigorously overnight to PEGylate the nanoparticles, and then, the chloroform was evaporated and the obtained RENP@CaCO3-PEG nanoparticles were dissolved in aqueous solution for use; S6. The prepared RENP@CaCO3-PEG was washed with water for multiple times and purified by centrifugation. The purified RENP@CaCO3-PEG solution was added to an EDC / NHS mixture to activate the carboxyl group for 40 min, and romumab was added. The mixture was incubated at 37 °C overnight, and dispersed in water by centrifugation at 5000 rpm for 5 min to obtain RE@Ca-Ig.

3. The method for preparing a nanoprobe for diagnosis and treatment of primary osteoporosis with in-situ catalysis and on-demand gas production according to claim 2, characterized in that: In S1, the acetate hydrate or chloride hydrate of Y, the acetate hydrate or chloride hydrate of Yb and the acetate hydrate or chloride hydrate of Tm are C6H9O6Y·6H2O or YCl3·6H2O, Yb(C2H3O2)3·6H2O or YbCl3·6H2O and Tm(OOCCH3)3·6H2O or TmCl3·6H2O respectively; The molar ratio of the acetate hydrate or chloride hydrate of Y, the acetate hydrate or chloride hydrate of Yb and the acetate hydrate or chloride hydrate of Tm in S1 is 0.795-0.8:0.2:0.005; The mass concentration of oleic acid is 90%, the mass concentration of 1-octadecene is 90%, and the volume ratio of oleic acid to 1-octadecene is 6:15; The dosage ratio of NaOH, NH4F and methanol is 2.5mmol:4mmol:8mL; The amount of cyclohexane used is 5 mL.

4. The method for preparing a nanoprobe for diagnosis and treatment of primary osteoporosis with in-situ catalysis and on-demand gas production according to claim 2, characterized in that: The acetate hydrate or chloride hydrate of Y and the acetate hydrate or chloride hydrate of Nd in S2 are C6H9O6Y·6H2O or YCl3·6H2O, C6H9O6Nd·6H2O or NdCl3·6H2O respectively; Further, the usage ratio of the acetate hydrate or chloride hydrate of Y, the acetate hydrate or chloride hydrate of Nd, oleic acid and 1-octadecene in S2 is 0.795-0.8 mmol: 0.2 mmol: 6 mL: 15 mL; The dosage ratio of NaOH, NH4F and methanol is 2.5mmol:4mmol:8mL; The amount of cyclohexane solution containing NaYF4:20%Yb / 0.5%Tm core was 5mL; The amount of cyclohexane used is 5 mL.

5. The method for preparing a nanoprobe for diagnosis and treatment of primary osteoporosis with in-situ catalysis and on-demand gas production according to claim 2, characterized in that: The acetate hydrate or chloride hydrate of Y in S3 is Y(CH3CO2)3 or YCl3; The usage ratio of the acetate hydrate or chloride hydrate of Y, oleic acid and 1-octadecene is 0.5 mmol: 6 mL: 15 mL; The dosage ratio of NaOH, NH4F and methanol is 2.5mmol:4mmol:8mL; The amount of cyclohexane solution containing NaYF4:20%Yb / 0.5%Tm@NaYF4:20%Nd core was 5mL; The amount of cyclohexane used is 5 mL.

6. The method for preparing a nanoprobe for diagnosis and treatment of primary osteoporosis with in-situ catalysis and on-demand gas production according to claim 2, characterized in that: The concentration of the hydrochloric acid in S4 is 0.1 M, and an ultrasonic disperser is used for washing at a frequency of 50 kHz; the mass concentration of PAA is 10%, the amount used is 2 mL, and the amount of H2O used is 3 mL.

7. The method for preparing a nanoprobe for diagnosis and treatment of primary osteoporosis with in-situ catalysis and on-demand gas production according to claim 2, characterized in that: The dosage ratio of CaCl2, PAA-RENPs and ethanol described in S5 is 5 mg: 10 mg: 100 mL; the dosage of ammonium bicarbonate is 5 g; The dosage ratio of the RENP@CaCO3 ethanol solution and the DOPA chloroform solution is 20 mg:1 mL; wherein the DOPA chloroform solution contains 2 mg of DOPA per mL of chloroform; The dosage of the DSPE-PEG-COOH is 10-100 mg.

8. The method for preparing a nanoprobe for diagnosis and treatment of primary osteoporosis with in-situ catalysis and on-demand gas production according to claim 2, characterized in that: The dosage ratio of the purified RENP@CaCO3-PEG solution, EDC / NHS mixed solution, romumab, and water in S6 is 5 mg: 50 μL: 10 μL: 80 μL; among them, the concentration of romumab is 5 μM; the mass ratio of EDS to NHS is 1 mg: 1-10 mg.

9. A primary osteoporosis diagnosis and treatment nanoprobe with in-situ catalysis and on-demand gas production as claimed in claim 1 is used for the combined treatment of primary osteoporosis with "gas, medicine and dual resistance" under the guidance of near-infrared second-zone fluorescence imaging.

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