CN112675130B - Application of hollow nano particles in preparation of osteoporosis treatment medicine - Google Patents
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Abstract
The invention discloses application of hollow nano particles in preparation of osteoporosis treatment medicines, and belongs to the technical field of preparation of medical medicines. Firstly, the application of hollow nano particles taking in-vitro shock wave response zoledronic acid as a framework component in the preparation of osteoporosis treatment medicines is provided; secondly, the application of the hollow nano-particles with zoledronic acid and/or the composition containing zoledronic acid as skeleton components in the preparation of the osteoporosis therapeutic drug is provided, in the application, the hollow nano-particles are interfered by external shock waves, and the hollow nano-particles are locally and in situ crushed by the external shock waves, so that the osteoporosis therapeutic drug has an obvious osteoporosis therapeutic effect. The invention can enhance the responsiveness of the external shock wave of the particles by controlling the shell thickness of the hollow nano hydroxyapatite particles, realize local controlled release, crush the particles in specific bone regions, improve local bone density and play a role in treating osteoporosis.
Description
Technical Field
The invention belongs to the technical field of preparation of medical medicaments, and particularly relates to application of an in-vitro shock wave as an intervention method in preparation of a medicament for treating osteoporosis by locally and in-situ crushing hollow nano particles.
Background
Osteoporotic fracture is a brittle fracture caused by low energy or non-violent factors, is a serious consequence of Osteoporosis (OP), is a serious hazard, and is one of the main causes of disability and death for middle-aged and elderly patients. How to prevent OP fractures has therefore not only been a medical problem to be solved urgently, but also one of the serious social problems.
Fortunately, pharmacological intervention of OP has made rapid and significant progress over the past half century. Current clinical therapeutic agents for OP mainly include estrogens, bisphosphonates and selective estrogen receptor modulators. Studies have shown that these interventions can be effective in reducing the incidence of brittle fractures. Among these drugs, the most clinically used is the Bisphosphate (BPs). BPs are the most effective anti-OP drugs commonly used in clinic, which can inhibit the recruitment and differentiation of osteoclasts, reduce their absorptive activity and increase their rate of apoptosis, thereby achieving the effect of inhibiting bone resorption; chemically, BPs are characterized by two phosphonate groups (PCPs) sharing carbon atoms, which have strong affinity for inorganic components of bone tissue such as Hydroxyapatite (HA). Related studies have demonstrated that zoledronic acid (ZOL) is the biphosphate with the highest affinity for bone tissue, while because of its nitrogen-containing heterocycles, the anti-bone resorption effect of ZOL is also trending in the BP family. ZOL is a 3 rd generation bisphosphonate drug that shows better efficacy compared to other first-line bisphosphonate drugs such as alendronate sodium. However, ZOL, like other drugs, is not efficiently focused on the site of the fracture. Moreover, it is reported in literature that adverse reactions such as fever, mandibular necrosis, hypercalcemia, renal injury and the like often occur in patients under the conditions of the existing dosages and frequencies of use. In order to increase the local tissue drug concentration and reduce complications, the drug is delivered to the fracture site and released in large quantity by using a delivery tool, and the best choice for rapidly and greatly improving the local bone density and bone strength is possible.
There have been studies designed for systemic administration and local enrichment strategies for BPs, and the preparation of HA nanoparticles seems to be a good choice. HA HAs been one of the most popular materials in many biomedical applications, especially in bone tissue engineering. Thanks to the fact that the inorganic components of the HAP are similar to natural bone matrix, the HA HAs excellent biocompatibility, osteoinductive capacity, osteoconductive capacity, osseointegration capacity and excellent biodegradability, HAP can form good chemical bonding with collagen in bone, and the HAP is widely applied to implant surface modification and modification in the orthopaedics field and is a medical nanomaterial approved by FDA. Meanwhile, the HA HAs bone promoting capability complementary to the bone absorption resisting capability of ZOL, ZOL is adopted to directly synthesize ZOL-HA nano particles, and the material is proved to be degradable in vivo, so that the purposes of inhibiting bone fracture and promoting bone formation are expected to be achieved. The ZOL-HA coating is directly deposited on the nano-liposome by adopting a template method, so that the hollow ZOL-HA nano-particle is synthesized.
More importantly, the ZOL-HA nanoparticles with hollow structures can be controlled released by in vitro shock wave (ESW) mediation. ESW is a sound wave with mechanical properties that acts on the body through its mechanical, cavitation and thermal effects to produce corresponding biological effects, and has been approved by the FDA for the treatment of a variety of diseases. The unique cavitation effect can efficiently mediate the breakage of the hollow nano particles, and provides possibility for realizing the local drug release of ZOL-HA nano particles. The release of the nano-particles which are used for coating the antitumor drugs by using ESW mediation at the treatment target point of tumor tissues is studied abroad, but the research on the prevention of OP fracture is not yet seen at home and abroad. Therefore, we consider that this property can be utilized to locally release the anti-OP nanoparticles at the therapeutic target of bone tissue, increasing the local drug concentration. In addition, in the treatment of OP, the importance of local mechanical stimulation is not neglected, and it can maintain bone remodeling, repair bone microdamage, avoid microdamage accumulation and fracture. The mechanical stimulation change can penetrate through lamellar bones or microtubule systems, and up-regulate endogenous nitric oxide synthase mRNA expression to induce migration and differentiation of osteoclast precursors by down-regulating mRNA expression of bone absorption markers such as RANK, RANKL and the like, thereby playing a role in inhibiting bone absorption. As a local mechanical stimulation mode, ESWT can reduce bone mass loss, induce new bone formation and improve the microstructure of bone tissue, and strengthen local bone mass, thus being an effective method for preventing OP.
Disclosure of Invention
The main technical problems to be solved by the invention are as follows:
at present, the first-line medicine for clinically treating osteoporosis is biphosphate represented by zoledronic acid, but zoledronic acid cannot be concentrated at the position easy to fracture, so that adverse reactions such as fever, mandibular necrosis, hypocalcemia, kidney injury and the like can be frequently generated by patients under the conditions of the existing use dosage and frequency. Therefore, it is important to develop a drug delivery system that can efficiently concentrate zoledronic acid at a site prone to fracture to achieve better osteoporosis treatment while reducing adverse effects in patients.
In order to solve the technical problems, the invention aims to provide an application of hollow nano particles in preparation of osteoporosis treatment drugs, wherein the hollow nano particles are subjected to in-situ crushing through in-vitro shock wave, and the hollow nano hydroxyapatite particles taking the in-vitro shock wave response zoledronic acid as a framework component are prepared, so that the zoledronic acid can be enriched at the fracture site easily, the local bone density is improved, and a good osteoporosis treatment effect is achieved.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the application of hollow nano particles with in-vitro shock wave response zoledronic acid as a framework component in the preparation of medicaments for treating osteoporosis.
As a preferable scheme of the invention, the hollow nano-particles with the external shock wave response zoledronic acid as a framework component are applied to the preparation of the osteoporosis therapeutic drug, the hollow nano-particles are hollow nano-hydroxyapatite particles, and the shell thickness of the hollow nano-hydroxyapatite particles is 1-50 nm.
It is another object of the present invention to provide the use of hollow nanoparticles of zoledronic acid and/or zoledronic acid-containing compositions as a backbone component in the preparation of a medicament for the treatment of osteoporosis, wherein the hollow nanoparticles are subjected to an in vitro shock wave intervention.
Preferably, the hollow nano particles with zoledronic acid and/or the composition containing zoledronic acid as skeleton components are used in preparing osteoporosis treating medicine, and the intervention refers to local in-situ crushing of the hollow nano particles through the external shock wave, which is in-vitro divergent shock wave.
Preferably, the application of the hollow nano-particles with zoledronic acid and/or the composition containing zoledronic acid as skeleton components in the preparation of osteoporosis treatment medicines is that the hollow nano-particles are hollow nano-hydroxyapatite particles, and the shell thickness of the hollow nano-hydroxyapatite particles is 1-50 nm.
Preferably, the application of the hollow nano-particles with zoledronic acid and/or the composition containing zoledronic acid as skeleton components in the preparation of the osteoporosis therapeutic drug is that the preparation method of the hollow nano-hydroxyapatite particles is as follows: the DOPA template is used, so that zoledronic acid and calcium chloride are deposited on the DOPA template to form a hollow shell structure.
Preferably, the hollow nano particles with zoledronic acid and/or the composition containing zoledronic acid as skeleton components are used for preparing osteoporosis treatment medicines, and the zoledronic acid and the composition containing zoledronic acid also comprise solvents and/or pharmaceutically acceptable carriers.
The preparation method of the hollow nano hydroxyapatite particles comprises the following steps: and a DOPA template with uniform particle size is used, so that zoledronic acid and calcium chloride are deposited on the DOPA template to form a hollow shell structure.
The application of the hollow nano particles with the external shock wave response zoledronic acid as a framework component in the preparation of the osteoporosis therapeutic drug is that after a hollow shell structure is formed, hydroxy-polyethylene glycol-phosphoric acid is added for a period of time to terminate a deposition reaction, a mixed system is obtained, the shell thickness of the hollow nano hydroxyapatite particles is controlled by controlling the pH of the mixed system and/or the time of adding OH-PEG-PA, and the preferred shell thickness is 1-50 nm.
The application of the hollow nano-particles with the external shock wave response zoledronic acid as a framework component in the preparation of the osteoporosis therapeutic drug adopts intravenous injection or local administration.
The mechanism of the application of the hollow nano-particles in the preparation of the osteoporosis treatment medicine is as follows:
zoledronic acid is a first-line medicine for clinically treating osteoporosis at present, hydroxyapatite is a bone affinity material which is the first index in bone tissue engineering, and in-vitro shock waves can crush hollow nano particles in situ through cavitation effect, so that the zoledronic acid has a bone promoting effect. Therefore, the hollow nano hydroxyapatite particles with the in-vitro shock wave responsive zoledronic acid as a framework component are used for treating osteoporosis, and the local bone density can be effectively improved.
Compared with the prior art, the invention has the following beneficial technical effects:
(1) The hollow nano hydroxyapatite particles taking the zoledronic acid as the skeleton component can effectively improve the local concentration of the zoledronic acid at the position where the fracture is easy, the zoledronic acid has the high-efficiency osteoclast inhibition effect, and simultaneously, the hydroxyapatite and the external shock wave have the effect of promoting osteoblasts; compared with the current clinical treatment method, the invention reduces the clinical side effect of zoledronic acid and simultaneously realizes the compound treatment effects of inhibiting the bone fracture and promoting the bone formation.
(2) The existing clinical method for treating osteoporosis has great side effects and can not effectively improve local bone density. Based on the above, the hollow nano hydroxyapatite particles with the external shock wave responsive zoledronic acid as a framework component are constructed, and the particles with specific shell thickness are found to be locally crushed in situ by external shock waves, so that the systemic side effect can be effectively avoided, the local bone density is improved, and the purpose of treating osteoporosis is achieved.
Drawings
The invention is further described below with reference to the accompanying drawings:
FIG. 1 is a schematic representation of the construction and characterization of a hollow nano-hydroxyapatite particle system with an in vitro shock wave responsive zoledronic acid as a scaffold component in accordance with an embodiment of the present invention, wherein:
a is the hydration particle diameter of the nano particles with different shell thicknesses after dynamic light scattering detection and shock wave treatment;
b is SEM and TEM images of the nano particles with different shell thicknesses after the nano particles with different shell thicknesses are treated by shock waves;
c is the in vitro drug release curve of the nano-particles with different shell thicknesses and the nano-particles with different shell thicknesses after the shock wave treatment;
d is the infrared spectrum of the constituent components and the nano particles with different shell thicknesses; BMSC;
FIG. 2 is a graph showing the effect of different shell thickness nanoparticles on BMSC cell and BMMC cell survival after treatment with shock waves;
FIG. 3 is a graph showing the safety evaluation of nanoparticles with different shell thicknesses in rats after treatment with shock waves, wherein A is the HE staining of important organs, and B is the hemolysis test;
FIG. 4 is a graph showing targeting and metabolism of different shell thickness nanoparticles in rats after treatment with shock waves;
fig. 5-10 show the effect of different shell thickness nanoparticles on osteoporosis treatment with different shell thickness nanoparticles after shock wave treatment.
Wherein FIG. 5 shows mainly Micro-CT imaging;
fig. 6 and 7 mainly show the results of the three-point bending experiment;
fig. 8 and 9 mainly show the nanoindentation experimental results;
FIG. 10 shows mainly the results of HE staining, TRAP-specific staining, BMP-2 immunohistochemical staining.
Detailed Description
The invention provides application of hollow nano particles in preparation of osteoporosis treatment medicines, and in order to make the advantages and the technical scheme of the invention clearer and more definite, the invention is described in detail below with reference to specific embodiments.
The raw materials required by the invention can be purchased through commercial sources.
Example 1:
preparing hollow nano hydroxyapatite particles with in-vitro shock wave responsive zoledronic acid as a framework component:
the components are as follows: zoledronic acid, calcium chloride, DOPA, OH-PEG-PA.
The preparation method comprises the following steps:
the synthesis was performed in a 300 ml glass flask, which was washed with a small amount of glacial acetic acid, rinsed 5 times with hot tap water, rinsed 2 times with deionized water, rinsed 5 times with acetone, rinsed 5 times with hot tap water, rinsed 5 times with deionized water, and dried in an oven at 60 ℃ for 2 hours. Liposome templates were prepared by extruding a total of 6mg DOPA lipid in 6mL deionized water solution at room temperature through a 100-nm PC filter until the average size distribution as measured by Dynamic Light Scattering (DLS) was uniform. After extrusion, the liposomes were allowed to stand for at least 1 hour and used within 48 hours after extrusion. 100mL of deionized water was mixed with 3mL (24 mg) of ZOL aqueous solution and 300. Mu.L of 1M NaOH. 3mg CaCl was applied to 3mL aliquots using a volumetric pipette 2 And 3mL of the liposome prepared as described above were added to each other within 5s of each other while magnetically stirring the suspension at 400rpm at room temperature. After 40 minutes, 1mL of an aqueous solution containing 10mg of OH-PEG-PA was pipetted into the stirred solution to stop the reaction and stabilize the shell suspension. After adding OH-PEG-PA, stirring the suspension for 20 min, collecting the mixed solution, and adding the mixed system into dialysis bag with molecular weight cut-off of 3500 for dialysis to obtain NP1.
Example 2:
preparing hollow nano hydroxyapatite particles with in-vitro shock wave responsive zoledronic acid as a framework component:
the components are as follows: zoledronic acid, calcium chloride, DOPA, OH-PEG-PA.
The preparation method comprises the following steps:
the synthesis was performed in a 300 ml glass flask, which was washed with a small amount of glacial acetic acid, rinsed 5 times with hot tap water, rinsed 2 times with deionized water, rinsed 5 times with acetone, rinsed 5 times with hot tap water, rinsed 5 times with deionized water, and dried in an oven at 60 ℃ for 2 hours. Liposome templates were prepared by extruding a total of 6mg DOPA lipid in 6mL deionized water solution at room temperature through a 100-nm PC filter until the average size distribution as measured by Dynamic Light Scattering (DLS) was uniform. After extrusion, the liposomes were allowed to stand for at least 1 hour and used within 48 hours after extrusion. 100mL of deionized water was added3mL (24 mg) of ZOL aqueous solution and 300. Mu.L of 1M NaOH were mixed. 3mg CaCl was applied to 3mL aliquots using a volumetric pipette 2 And 3mL of the liposome prepared as described above were added to each other within 5s of each other while magnetically stirring the suspension at 400rpm at room temperature. After 120 minutes, 1mL of an aqueous solution containing 10mg of OH-PEG-PA was pipetted into the stirred solution to stop the reaction and stabilize the shell suspension. After adding OH-PEG-PA, stirring the suspension for 20 min, collecting the mixed solution, and adding the mixed system into dialysis bag with molecular weight cut-off of 3500 for dialysis to obtain NP2.
Example 3:
preparing hollow nano hydroxyapatite particles with in-vitro shock wave responsive zoledronic acid as a framework component:
the components are as follows: zoledronic acid, calcium chloride, DOPA, OH-PEG-PA.
The preparation method comprises the following steps:
the synthesis was performed in a 300 ml glass flask, which was washed with a small amount of glacial acetic acid, rinsed 5 times with hot tap water, rinsed 2 times with deionized water, rinsed 5 times with acetone, rinsed 5 times with hot tap water, rinsed 5 times with deionized water, and dried in an oven at 60 ℃ for 2 hours. Liposome templates were prepared by extruding a total of 6mg DOPA lipid in 6mL deionized water solution at room temperature through a 100-nm PC filter until the average size distribution as measured by Dynamic Light Scattering (DLS) was uniform. After extrusion, the liposomes were allowed to stand for at least 1 hour and used within 48 hours after extrusion. 100mL of deionized water was mixed with 3mL (24 mg) of ZOL aqueous solution and 300. Mu.L of 1M NaOH. 3mg CaCl was applied to 3mL aliquots using a volumetric pipette 2 And 3mL of the liposome prepared as described above were added to each other within 5s of each other while magnetically stirring the suspension at 400rpm at room temperature. After 240 minutes, 1mL of an aqueous solution containing 10mg of OH-PEG-PA was pipetted into the stirred solution to stop the reaction and stabilize the shell suspension. After adding OH-PEG-PA, stirring the suspension for 20 min, collecting the mixed solution, and adding the mixed system into dialysis bag with molecular weight cut-off of 3500 for dialysis to obtain NP3.
The following describes the specific experimental steps of the present invention in detail with reference to the accompanying drawings:
1.1 construction and characterization of hollow nanohydroxyapatite particle System with in vitro shock wave responsive zoledronic acid as skeleton component
NP1, NP2, NP3 were obtained as in example 1-example 3 above, and three kinds of particles were treated with an in vitro shock wave, respectively, and the hydrated particle size was analyzed by a particle size analyzer; scanning Electron Microscopy (SEM) to detect morphology; identifying the shell thickness by a Transmission Electron Microscope (TEM); identifying the particle components by infrared spectroscopy; ICP-OES measures the in vitro release rate of the particles. As shown in FIG. 1, wherein A is the variation of the hydration particle size of three different particles and the particle after the shock wave treatment, it can be seen that the hydration particle size of the three particles is changed after the shock wave treatment, the original hydration particle size of the particles has a single particle size distribution around 100nm, and the particle size distribution around 10nm and 1000nm respectively appears after the shock treatment, which suggests that large particle fusion occurs in addition to small particle size fragments; b is particle morphology and shell thickness displayed by SEM and TEM, and confirms the experimental result of hydration particle size, wherein three particles with particle size of about 100nm are fused with large particles after shock wave treatment, and meanwhile TEM results show that the shell thickness of three particles NP1, NP2 and NP3 is gradually thickened; c is the in vitro drug release result of the particles, and shows that after the particles are treated by shock waves, the in vitro release speed of NP1 and NP2 is obviously improved. D is the infrared spectrum of the nano particles and the raw materials, and proves that the active ingredients such as ZOL are combined on the particles.
1.2 Effect of particles on BMSC and BMMC cell survival
Cell viability and death staining: rat bone marrow mesenchymal stem cells (BMSCs) and bone marrow mononuclear macrophages (BMMC) are inoculated into a 12-hole plate, different medicines are added for treatment, the mixture is incubated for 24 hours, and a cell living dying staining kit is used for detecting the living state of cells; as shown in fig. 2, various degrees of death occurred in the BMMC of each group after particle treatment, with more death in the NP1 and NP2 groups after shock wave treatment, while no significant tendency of cell death occurred in the BMSCs after particle treatment of each group compared to the control group.
1.3 evaluation of the safety of particles in rats
Three different particles are respectively injected into a rat body from tail vein, shock wave intervention is carried out on the particles or not according to the group, the intervention part is the far end of femur of the rat, the heart, liver, spleen, lung and kidney of the rat are respectively obtained when materials are obtained after 2 months, 4% paraformaldehyde is fixed, paraffin embedding slice and HE staining are carried out, and main viscera HE staining slice is obtained, and the result is shown in figure 3A. To study the risk of hemolysis of different particles by intravenous injection, rat hearts were taken, blood cells were obtained by centrifugation with anticoagulation of 20Mm EDTA.2Na, three different particles and three particles after shock wave treatment were added respectively, after resting for 5min, centrifugation was performed, and the supernatant was taken out and absorbance at 541nm was measured in a microplate reader, and the results are shown in FIG. 3B.
1.4 targeting and metabolism of particles in rats
Three different particles of the cy5.5 dye-linked are injected into the rat body from the tail vein respectively, fluorescence imaging is carried out in a small animal living body imager according to time points, shock wave intervention is carried out or not according to the group, the intervention part is the far end of the femur of the rat, and the result is shown in figure 4.
1.5, the effect of the particles on treating osteoporosis. Wherein fig. 5 mainly shows Micro-CT imaging; fig. 6 and 7 mainly show the results of the three-point bending experiment; fig. 8 and 9 mainly show the nanoindentation experimental results; FIG. 10 shows mainly the results of HE staining, TRAP-specific staining, BMP-2 immunohistochemical staining.
Molding for osteoporosis: female SD rats of 3 months old are selected, sodium pentobarbital (30 mg/kg) is injected into the abdominal cavity, after the animals are anesthetized, the animals are taken to be in a prone position and fixed on an operating table, and iodine tincture is sterilized by alcohol after skin preparation, and conventional towel spreading is performed. Selecting an incision position at the position of 0.5cm at the left side of the spinal column and about 0.8cm below the rib, cutting the skin and the subcutaneous tissue, cutting the incision size to be 0.5cm, performing blunt free fascia to the muscle layer, performing blunt separation on the left side of the lumbar vertebra by 0.5cm, lifting abdominal fat by a hemostatic forceps, searching ovarian tissue, ligating the No. 1 thread at the oviduct, cutting off the ovarian tissue, checking that the residual oviduct and fat are brought back into the abdominal cavity after no active bleeding, and suturing the muscles and fascia layer by layer. The right ovary was excised by the same method, a small amount of penicillin powder was spread subcutaneously, the skin was sutured, and the wound was disinfected again with iodophor. Until the animals are completely resuscitated and returned to the animal house. Penicillin 2 ten thousand units were given for 3 days of intramuscular injection after surgery. The rats were fed (200 g/d) with free water at 23℃and relative humidity of 40% -70%. The rat is sacrificed by 8w neck removal after the molding operation, femur Micro-CT scanning and three-dimensional reconstruction, three-point bending experiments and nanoindentation experiments are taken, paraffin embedding and slicing are simultaneously carried out on the far end of femur, HE staining, TRAP specific staining and BMP-2 immunohistochemical staining are simultaneously taken, and the result is shown in figure 10.
The parts not described in the invention can be realized by referring to the prior art.
It should be noted that: any equivalent or obvious modifications made by those skilled in the art under the teachings of this specification shall fall within the scope of this invention.
Claims (4)
1. The application of hollow nano particles taking in-vitro shock wave response zoledronic acid as a framework component in preparing osteoporosis treatment medicines is characterized in that: the hollow nano-particles are hollow nano-hydroxyapatite particles, and the shell thickness of the hollow nano-hydroxyapatite particles is 1-50 nm;
the preparation method of the hollow nano hydroxyapatite particles comprises the following steps:
the required components are zoledronic acid, calcium chloride and OH-PEG-PA, and DOPA templates with uniform particle sizes are used, so that the zoledronic acid and the calcium chloride are deposited on the DOPA templates to form a hollow shell structure;
wherein the synthesis was performed in a 300 ml glass flask, which was washed with a small amount of glacial acetic acid, rinsed 5 times with hot tap water, rinsed 2 times with deionized water, rinsed 5 times with acetone, rinsed 5 times with hot tap water, rinsed 5 times with deionized water, and dried in an oven at 60 ℃ for 2 hours;
liposome templates were prepared by extruding a total of 6mg DOPA lipid in 6mL deionized water solution at room temperature through a 100-nm PC filter until the average size distribution as measured by dynamic light scattering was uniform;
after extrusion, the liposomes were allowed to stand for at least 1 hour and used within 48 hours after extrusion;
100mL of deionized water was mixed with 3mL, i.e., 24mg, of ZOL aqueous solution and 300. Mu.L of 1M NaOH;
3mL portions of 3mg CaCl2 and 3mL of the liposome prepared as described above were added to each other within 5s of each other using a volume pipette while magnetically stirring the suspension at 400rpm at room temperature;
after a hollow shell structure is formed, 1mL of the mixture containing 10mg of OH-PEG-PA is added for a period of time to terminate the deposition reaction, a mixed system is obtained, and the shell thickness of the hollow nano hydroxyapatite particles is controlled by controlling the pH of the mixed system and/or the time of adding the OH-PEG-PA;
wherein, after adding OH-PEG-PA, the suspension is stirred for 20 minutes, the mixed solution is collected, and the mixed system is added into a dialysis bag with 3500 molecular weight cut-off for dialysis, thus obtaining NP.
2. The use of hollow nanoparticles of an in vitro shock wave responsive zoledronic acid as a scaffold component according to claim 1 for the preparation of a medicament for the treatment of osteoporosis, characterized in that: the administration mode is intravenous injection or local administration.
3. The application of zoledronic acid and/or a composition containing zoledronic acid as a skeleton component in the preparation of a medicament for treating osteoporosis is characterized in that: the hollow nano particles are interfered by external shock waves;
the intervention refers to local in-situ crushing of the hollow nano particles through the external shock wave, wherein the external shock wave is external divergent shock wave;
the hollow nano-particles are hollow nano-hydroxyapatite particles, and the shell thickness of the hollow nano-hydroxyapatite particles is 1-50 nm;
the preparation method of the hollow nano hydroxyapatite particles comprises the following steps:
the required components are zoledronic acid, calcium chloride and OH-PEG-PA, and DOPA templates with uniform particle sizes are used, so that the zoledronic acid and the calcium chloride are deposited on the DOPA templates to form a hollow shell structure;
wherein the synthesis was performed in a 300 ml glass flask, which was washed with a small amount of glacial acetic acid, rinsed 5 times with hot tap water, rinsed 2 times with deionized water, rinsed 5 times with acetone, rinsed 5 times with hot tap water, rinsed 5 times with deionized water, and dried in an oven at 60 ℃ for 2 hours;
liposome templates were prepared by extruding a total of 6mg DOPA lipid in 6mL deionized water solution at room temperature through a 100-nm PC filter until the average size distribution as measured by dynamic light scattering was uniform;
after extrusion, the liposomes were allowed to stand for at least 1 hour and used within 48 hours after extrusion;
100mL of deionized water was mixed with 3mL, i.e., 24mg, of ZOL aqueous solution and 300. Mu.L of 1M NaOH;
3mL portions of 3mg CaCl2 and 3mL of the liposome prepared as described above were added to each other within 5s of each other using a volume pipette while magnetically stirring the suspension at 400rpm at room temperature;
after a hollow shell structure is formed, 1mL of the mixture containing 10mg of OH-PEG-PA is added for a period of time to terminate the deposition reaction, a mixed system is obtained, and the shell thickness of the hollow nano hydroxyapatite particles is controlled by controlling the pH of the mixed system and/or the time of adding the OH-PEG-PA;
wherein, after adding OH-PEG-PA, the suspension is stirred for 20 minutes, the mixed solution is collected, and the mixed system is added into a dialysis bag with 3500 molecular weight cut-off for dialysis, thus obtaining NP.
4. Use of hollow nanoparticles of zoledronic acid and/or zoledronic acid-containing compositions as framework components according to claim 3 for the preparation of medicaments for the treatment of osteoporosis, characterized in that: the zoledronic acid and compositions containing zoledronic acid also include a solvent and/or a pharmaceutically acceptable carrier.
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