A bone-targeting biomimetic biomineral nanocomposite material, its preparation method and application

Abstract

This application provides a bone-targeting biomimetic biomineral nanocomposite material, its preparation method, and its application, relating to the field of medical materials. The method includes the following steps: S1: Providing water-soluble phosphate and water-soluble calcium salt; S2: Mixing the aqueous solutions of the water-soluble phosphate and water-soluble calcium salt, followed by magnetic stirring and centrifugation to obtain calcium phosphate; S3: Washing the calcium phosphate and freeze-drying for storage; S4: Providing GLG1+ NIH-3T3 cells and expanding them; S5: Extracting the cell membrane from the GLG1+ cells and resuspending the cell membrane in pre-cooled PBS buffer to obtain a cell membrane solution, which is stored at -80°C for later use; S6: Mixing the calcium phosphate from S1 with the GLG1+ cell membrane solution and sonicating to obtain cell membrane-coated calcium phosphate nanoparticles; S7: Centrifuging the calcium phosphate nanoparticles to remove excess cell membrane, resuspending them in ultrapure water and washing at least twice to finally obtain the bone-targeting biomimetic biomineral nanocomposite material, which is then stored at 4°C.

Description

Technical Field

[0001] This application relates to the field of medical materials, and in particular to a bone-targeting biomimetic biomineral nanocomposite material, its preparation method, and its application. Background Technology

[0002] Studies indicate that delayed or impaired bone mineralization is a significant cause of osteoporosis and poor fracture healing. For bone repair biomaterials, exploring the processes and mechanisms of hard tissue biomineralization and mimicking the structure and function of natural hard tissues holds promise for improving their repair performance. Bone mineralization is the process by which amorphous calcium phosphate crystals develop into hydroxyapatite crystals embedded in the interstitial spaces of bone's organic matter. During biomineralization, mitochondrial minerals within osteoblasts are encapsulated in vesicles and secreted into the extracellular matrix, interacting with type I collagen and promoting its mineralization. These extracellular vesicles may play a crucial role in their entry into the type I collagen molecular chain. Research has confirmed that extracellular vesicles secreted by osteoblasts, primarily composed of amorphous calcium phosphate encapsulated in vesicles, can promote osteogenic differentiation and collagen mineralization of bone marrow mesenchymal stem cells. Therefore, preparing phospholipid-membrane-encapsulated calcium phosphate biomineral materials by mimicking the structure and composition of extracellular vesicles secreted by osteoblasts may have a positive effect on the treatment of osteoporosis.

[0003] Calcium phosphate is a major component of extracellular vesicles secreted by osteoblasts and also a major inorganic component of human bone tissue. It possesses high biocompatibility, biodegradability, and osteoinductive properties, and is widely used in bone regeneration and repair. Calcium phosphate biomaterials can be classified into various crystalline and amorphous phases based on their chemical phases, such as hydroxyapatite, calcium hydrogen phosphate, and octacalcium phosphate. Amorphous calcium phosphate materials exhibit better bioactivity and biodegradability than crystalline calcium phosphate materials. However, as a metastable phase, amorphous calcium phosphate is unstable in aqueous solutions and readily transforms into its corresponding crystalline form through processes such as dissolution, nucleation, and crystallization.

[0004] Creatine phosphate (CP), guanine dinucleotide phosphate (GDP), guanine trinucleotide phosphate (GTP), adenine trinucleotide phosphate (ATP), adenine dinucleotide phosphate (ADP), and adenine mononucleotide phosphate (AMP) are phosphate biomolecules present in the human body. They can regulate the size and morphology of calcium phosphate materials, maintaining their original amorphous phase structure, which is essential as a precursor for biomineralization. Currently, inspired by the biomineralization mechanism, many domestic and international scholars have biomimeticly synthesized calcium phosphate / hydroxyapatite biomineral materials. However, these biomaterials lack bone targeting and have poor therapeutic effects in osteoporotic bone repair.

[0005] Therefore, there is an urgent need to develop an improved composite material to achieve rapid repair of osteoporotic sites. Summary of the Invention

[0006] This application provides a method for preparing bone-targeting biomimetic biomineral nanocomposite materials. By adopting a biomimetic strategy, highly bioactive nano-calcium phosphate is prepared and combined with a phospholipid membrane with bone-targeting properties to form a bone-targeting biomimetic biomineral nanocomposite material.

[0007] On the one hand, this application provides a method for preparing bone-targeting biomimetic biomineral nanocomposite materials, including the following steps:

[0008] S1: Provides water-soluble phosphates and water-soluble calcium salts;

[0009] S2: Mix an aqueous solution of water-soluble phosphate and water-soluble calcium salt, and then perform magnetic stirring and centrifugation to obtain calcium phosphate;

[0010] S3: Washing: Wash the obtained calcium phosphate with ultrapure water and anhydrous ethanol at least once each, and freeze-dry for storage;

[0011] S4: Provides GLG1 + CXCR4 + Phospholipid membrane solutions of NIH-3T3 cell membranes or other complex bone-targeting proteins or peptides;

[0012] S5: The calcium phosphate in S1 and the phospholipid membrane solution in S4 are mixed in a mass ratio of 50:1 to 1:50 and subjected to ultrasonic treatment to obtain phospholipid membrane-coated calcium phosphate nanoparticles.

[0013] S6: Centrifuge the calcium phosphate nanoparticles to remove excess phospholipid membrane, resuspend them in ultrapure water and wash the bottom at least twice to finally obtain the bone-targeting biomimetic biomineral nanocomposite material, and store the composite material in an environment below 25°C.

[0014] Preferably, the water-soluble calcium salt in S1 includes calcium chloride and / or its hydrate, calcium nitrate and / or its hydrate, and / or calcium acetate and / or its hydrate.

[0015] Preferably, the water-soluble phosphate in S1 includes sodium phosphate and / or its hydrate, sodium hydrogen phosphate and / or its hydrate.

[0016] Preferably, the water-soluble calcium salt in S1 is a 40 mL aqueous solution containing 100–500 mg CaCl2.

[0017] Preferably, the water-soluble phosphate in S1 is prepared by mixing an aqueous solution of 10-100 mg Na3PO4 and 10-100 mg Na2HPO4 with a mixed solution containing 50-200 mg of high-energy phosphate molecules in 20 mL.

[0018] Preferably, the phospholipid membrane in step S4 is the cell membrane of GLG1+ cells. The method for preparing the phospholipid membrane solution is as follows: After providing a bone-targeting phospholipid membrane, the phospholipid membrane is extracted and resuspended in pre-cooled PBS buffer to obtain a buffer solution, which is then stored at -80℃ for later use. The specific steps are as follows:

[0019] S41: Discard the cell culture medium and add PBS buffer to wash the cells;

[0020] S42: Collect cultured cells using a cell scraper;

[0021] S43: After centrifugation and discarding the supernatant, resuspend the cell pellet in pre-cooled membrane protein extraction reagent A containing protease inhibitors, and lyse it on ice for 15-30 min.

[0022] S44: Add two steel balls and use a tissue homogenizer to break the cells for 1-5 minutes at 70-100 Hz and -20°C.

[0023] S45: Remove the steel ball and centrifuge at 500-1000g for 5-30 minutes at 4°C. Transfer the supernatant to a new centrifuge tube to remove cell nuclei and unbroken cells. Centrifuge at 10000-15000g for 15-60 minutes at 4°C. The precipitate is the cell membrane.

[0024] Preferably, the high-energy phosphate molecules include creatine phosphate, guanine dinucleotide phosphate, guanine trinucleotide phosphate, adenine trinucleotide phosphate, adenine dinucleotide phosphate, and adenine mononucleotide phosphate, with a molar concentration of 0.01–100 mmol / L in the final reaction solution.

[0025] Preferably, the specific steps for removing excess phospholipid film in S6 are as follows:

[0026] S61: Mix the phospholipid membrane solution from step S5 and the calcium phosphate prepared in S2 at a mass ratio of 50:1 to 1:50;

[0027] S62: Calcium phosphate nanoparticles coated with phospholipid membranes were prepared by ultrasonic treatment in an ice-water bath for 5–30 min.

[0028] S63: Centrifuge at 5000-10000 rpm for 5-30 minutes.

[0029] On the other hand, this application also provides a bone-targeting biomimetic biomineral nanocomposite material, characterized in that it is prepared by the above-mentioned method for preparing a bone-targeting biomimetic biomineral nanocomposite material.

[0030] On the other hand, this application also provides the application of the above-mentioned preparation method or the above-mentioned bone-targeting biomimetic biomineral nanocomposite material in the preparation of bone repair and bone regeneration materials.

[0031] Preferably, the application involves using the bone-targeting biomimetic biomineral nanocomposite material as an injectable agent, which is then intravenously injected and targeted to the vicinity of the site to be repaired.

[0032] Beneficial effects:

[0033] This invention employs a biomimetic strategy to prepare highly bioactive nano-calcium phosphate, which is then combined with a bone-targeting phospholipid membrane to form a bone-targeting biomimetic biomineral nanocomposite material. Its main characteristics are bone targeting and high biocompatibility. The encapsulated active calcium phosphate material can rapidly and accurately target bone tissue. On the one hand, the active calcium phosphate material can be directly deposited in the extracellular matrix to promote collagen mineralization. On the other hand, the free active calcium phosphate material can be phagocytosed by bone marrow mesenchymal stem cells and promote their osteogenic differentiation, thereby achieving rapid repair of osteoporotic sites. Attached Figure Description

[0034] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 Transmission electron microscopy image of amorphous calcium phosphate (ACP / CP) prepared by phosphocreatine regulation in this invention;

[0036] Figure 2 This is a transmission electron microscope image of the bone-targeting phospholipid membrane-coated ACP / CP nanocomposite (ACP / CP@CM) in this invention;

[0037] Figure 3 This is a transmission electron microscope image of amorphous calcium phosphate (ACP), the control sample in this invention.

[0038] Figure 4 This is a transmission electron microscope image of hydroxyapatite (HAP), the control sample in this invention.

[0039] Figure 5 This is a Zeta potential diagram of ACP / CP, ACP / CP@CM, ACP, HAP, and bone-targeting phospholipid membrane vesicles (CM) in this invention;

[0040] Figure 6 The FTIR spectra of ACP / CP, ACP / CP@CM, ACP, HAP, and CM in this invention are shown below.

[0041] Figure 7 The XRD patterns of ACP / CP, ACP / CP@CM, ACP, and HAP in this invention are shown below.

[0042] Figure 8 This is a Western blot image of the functionalized membrane protein GLG1 on the surface of the ACP / CP@CM sample in this invention and the CM sample in Example 5;

[0043] Figure 9 This is a CCK-8 result diagram showing the effects of ACP / CP, ACP / CP@CM, ACP, HAP, and CM on cell proliferation in this invention.

[0044] Figure 10 Alkaline phosphatase staining images showing the effects of ACP / CP, ACP / CP@CM, ACP, HAP, and CM on promoting osteogenic differentiation of bone marrow mesenchymal stem cells in this invention.

[0045] Figure 11 Alizarin Red staining diagram showing the regulation of osteogenic differentiation of bone marrow mesenchymal stem cells by ACP / CP, ACP / CP@CM, ACP, HAP, and CM in this invention;

[0046] Figure 12 This is a diagram showing the RT-qPCR results of key genes in osteogenic differentiation of bone marrow mesenchymal stem cells using ACP / CP, ACP / CP@CM, ACP, HAP, and CM in this invention.

[0047] Figure 13 These are CLSM images showing the effects of ACP / CP@CM in this invention and HAP in Example 4 on collagen mineralization;

[0048] Figure 14 Fluorescence imaging images of the femur and tibia after tail vein injection of ACP / CP@Con-CM and ACP / CP@CM in mice according to the present invention;

[0049] Figure 15 These are Micro-CT three-dimensional reconstructed images of the femur in the sagittal and horizontal planes after tissue samples were taken from each group of animals in this invention.

[0050] Figure 16 This is a flowchart of the preparation method of the present invention. Detailed Implementation

[0051] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0052] The following detailed description of some embodiments of this application is provided in conjunction with the accompanying drawings. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0053] Example 1

[0054] refer to Figure 1 This embodiment provides a method for preparing bone-targeting biomimetic biomineral nanocomposite materials, including the following steps:

[0055] S1: Provides water-soluble phosphate and water-soluble calcium salt. In this example, the water-soluble calcium salt is 40 mL of an aqueous solution containing 100–500 mg CaCl2. The water-soluble phosphate is prepared by mixing 20 mL of an aqueous solution containing 10–100 mg Na3PO4 and 10–100 mg Na2HPO4 with 20 mL of a mixed solution containing 50–200 mg of high-energy phosphate molecules.

[0056] The high-energy phosphate molecules in this embodiment include creatine phosphate, guanine dinucleotide phosphate, guanine trinucleotide phosphate, adenine trinucleotide phosphate, adenine dinucleotide phosphate, and adenine mononucleotide phosphate, with a molar concentration of 0.01–100 mmol / L in the final reaction solution.

[0057] S2: Mix the aqueous solutions of water-soluble phosphate and water-soluble calcium salt, stir magnetically for 5 min-6 h, and then centrifuge at 8000 rpm for 5-30 min to obtain calcium phosphate;

[0058] It should be noted that the calcium phosphate obtained in this application can be either amorphous calcium phosphate (ACP) or crystalline calcium phosphate.

[0059] S3: Washing: Wash the obtained calcium phosphate with ultrapure water and anhydrous ethanol at least once each, and freeze-dry for storage;

[0060] S4: GLG1 + Taking NIH-3T3 cell membrane coating as an example: providing GLG1 + NIH-3T3 cells were expanded to a density of 90%.

[0061] S5: For GLG1 +Cell membranes were extracted from the cells and resuspended in pre-cooled PBS buffer to obtain a cell membrane solution, which was stored at -80°C for later use.

[0062] In this embodiment, in step S4, GLG1 + The specific steps for cell membrane extraction are as follows:

[0063] S41: Discard the cell culture medium and add PBS buffer to wash the cells;

[0064] S42: Collect cultured cells using a cell scraper;

[0065] S43: After centrifugation and discarding the supernatant, resuspend the cell pellet in pre-cooled membrane protein extraction reagent A containing protease inhibitors, and lyse it on ice for 15-30 min.

[0066] S44: Add two steel balls and use a tissue homogenizer to break the cells for 1-5 minutes at 70-100 Hz and -20°C.

[0067] S45: Remove the steel ball and centrifuge at 500-1000g for 5-30 minutes at 4°C. Transfer the supernatant to a new centrifuge tube to remove cell nuclei and unbroken cells. Centrifuge at 10000-15000g for 15-60 minutes at 4°C. The precipitate is the cell membrane.

[0068] S5: Combine the calcium phosphate in S1 with GLG1 + Cell membrane solutions were mixed in a mass ratio of 5:1 to 1:5 and subjected to ultrasonic treatment to obtain cell membrane-coated calcium phosphate nanoparticles.

[0069] The cell membrane-coated calcium phosphate nanoparticles finally obtained in this embodiment are bone-targeting biomimetic biomineral nanocomposite materials.

[0070] Specifically, refer to Figure 1 The image shows a transmission electron microscope (TEM) image of the prepared calcium phosphate nanoparticles. It indicates that the material consists of spherical nanoparticles with uneven size and significant nanoparticle aggregation. Individual nanoparticles have a diameter of approximately 10 nm, while the aggregated size is approximately 100-180 nm, providing possibilities for subsequent cell membrane modification and systemic injection.

[0071] S6: Centrifuge the calcium phosphate nanoparticles to remove excess cell membranes, resuspend them in ultrapure water and wash the bottom at least twice to finally obtain the bone-targeting biomimetic biomineral nanocomposite material (ACP / CP@CM), and store the composite material in an environment of 4°C.

[0072] In this embodiment, the specific steps for removing excess cell membranes in S6 are as follows:

[0073] S61: Extract the GLG1 obtained in step S5 + The cell membrane solution and the calcium phosphate prepared in S2 were mixed in a mass ratio of 5:1 to 1:5.

[0074] S62: Cell membrane-coated calcium phosphate nanoparticles were prepared by sonication in an ice-water bath for 5–30 min.

[0075] S63: Centrifuge at 5000-10000 rpm for 5-30 minutes.

[0076] Specifically, refer to Figure 2 Transmission electron microscopy (TEM) images of the cell membrane-encapsulated calcium phosphate composite material are presented. The results show that the surface of the calcium phosphate composite material is coated with a cell membrane, and the nanoparticle size is slightly increased, exhibiting a core-shell structure. These results preliminarily indicate the successful preparation of the calcium phosphate composite material.

[0077] Figure 5 The zeta potential of the material is given, showing that the ACP / CP surface carries a negative charge of -7.7 mV.

[0078] Figure 6 The FTIR spectrum of the material is presented, indicating that ACP / CP contains its corresponding components.

[0079] Figure 7 The XRD pattern of the material is presented. ACP / CP shows a broad weak diffraction peak at 30°, indicating that the formed calcium phosphate nanoparticles are an amorphous phase.

[0080] Figure 9 The image shows the CCK-8 results illustrating the effect of ACP / CP on cell proliferation. Similar to the control group, there was no significant difference in BMSC proliferation in the ACP / CP group, indicating that the nanoparticles exhibit good biocompatibility over a longer period.

[0081] Figure 10 Alkaline phosphatase staining image showing the effect of ACP / CP on promoting osteogenic differentiation of bone marrow mesenchymal stem cells. Compared with the control group, the ACP / CP group had a higher content of alkaline phosphatase, indicating that the material has a promoting effect on osteogenic differentiation.

[0082] Figure 11 Alizarin Red staining image showing the effect of ACP / CP on promoting osteogenic differentiation of bone marrow mesenchymal stem cells. Compared with the control group, the ACP / CP group had a higher content of calcium nodules, indicating that it can effectively promote cell maturation and calcification.

[0083] Figure 12This image shows the RT-qPCR results of ACP / CP on key genes in osteogenic differentiation of bone marrow mesenchymal stem cells. Compared to the control group, ACP / CP upregulated the expression of osteogenic factors RUNX2 and BMP2.

[0084] Example 2

[0085] This embodiment provides a method for preparing bone-targeted biomimetic biomineral nanocomposite materials. The difference between this embodiment and Example 1 is that:

[0086] 1. In step S4, for GLG1 + The specific steps for cell membrane extraction are as follows:

[0087] S41: Discard the cell culture medium and add PBS buffer to wash the cells;

[0088] S42: Collect cultured cells using a cell scraper;

[0089] S43: After centrifugation and discarding the supernatant, resuspend the cell pellet in pre-cooled membrane protein extraction reagent A containing protease inhibitors, and lyse it on ice for 15 min.

[0090] S44: Add two steel balls and use a tissue homogenizer to break the cells for 1 min at 70 Hz and -20 ℃;

[0091] S45: Remove the steel ball, centrifuge at 700g for 10 minutes at 4°C, and transfer the supernatant to a new centrifuge tube to remove cell nuclei and unbroken cells; centrifuge at 14000g for 30 minutes at 4°C, and the precipitate is the cell membrane.

[0092] 2. The specific steps for removing excess cell membrane in S6 are as follows:

[0093] S61: Mix the GLG1+ cell membrane solution extracted in step S5 and the calcium phosphate prepared in S2 at a mass ratio of 2:1.

[0094] S62: Cell membrane-coated calcium phosphate nanoparticles were prepared by sonication in an ice-water bath for 10 min.

[0095] S63: Centrifuge at 8000 rpm for 5 min.

[0096] Figure 2 Transmission electron microscopy (TEM) images of the cell membrane-encapsulated calcium phosphate composite material are presented. The results show that the surface of ACP / CP@CM is coated with a cell membrane, and the nanoparticle size is slightly increased, exhibiting a core-shell structure. These results preliminarily indicate that ACP / CP@CM has been successfully prepared.

[0097] Figure 5The zeta potential of the material is given, showing that the ACP / CP@CM surface carries a negative charge of -10.2 mV. Comparison of ACP / CP, CM, and ACP / CP@CM reveals that the negative charge of ACP / CP@CM increases, exhibiting a charge similar to that of CM. This also indirectly proves that the cell membrane is successfully coated on the surface of the bioactive mineral ACP / CP.

[0098] Figure 6 The infrared spectrum of the material is presented, further confirming that ACP / CP@CM contains its corresponding components.

[0099] Figure 7 The X-ray diffraction pattern of the material is shown. ACP / CP@CM shows a relatively broad weak diffraction peak at 30°, indicating that its physical form is an amorphous phase.

[0100] Figure 8 The results of Western blot analysis of GLG1 protein on ACP / CP@CM show that GLG1 protein is expressed on ACP / CP@CM, and this material can well preserve the functional protein GLG1 on cells with high GLG1 expression.

[0101] Figure 9 The image shows the CCK-8 results illustrating the effect of ACP / CP@CM on cell proliferation. Similar to the control group, there was no significant difference in BMSC proliferation in the ACP / CP@CM group, further demonstrating that the nanoparticles exhibit good biocompatibility over a longer period.

[0102] Figure 10 Alkaline phosphatase staining image showing the effect of ACP / CP@CM on promoting osteogenic differentiation of bone marrow mesenchymal stem cells. Compared with the ACP / CP group in Example 1, the ACP / CP@CM group showed a larger positive area for alkaline phosphatase, mainly due to the encapsulation of bone-targeting cell membranes, which promoted the targeted delivery of the material and enhanced its osteogenic differentiation capacity.

[0103] Figure 11 Alizarin Red staining image showing the effect of ACP / CP@CM on promoting osteogenic differentiation of bone marrow mesenchymal stem cells. Compared with the ACP / CP group in Example 1, the ACP / CP@CM group had a higher content of calcium nodules and a stronger bone-forming ability.

[0104] Figure 12 The image shows the RT-qPCR results of key genes in osteogenic differentiation of bone marrow mesenchymal stem cells induced by ACP / CP@CM. Compared with the ACP / CP group in Example 1, ACP / CP@CM further upregulated the expression of osteogenic factors RUNX2 and BMP2, showing a significant difference compared with the control group.

[0105] Figure 13This is a CLSM image showing the effect of ACP / CP@CM on collagen mineralization. First, collagen molecules were stained red with Cy3 dye. Then, they were placed in a solution containing ACP / CP@CM and mineralized at 37°C. Calcein was used to label the mineralized calcium phosphate on the collagen. The results showed that collagen molecules co-incubated with ACP / CP@CM exhibited significant green fluorescence, indicating that ACP / CP@CM deposited within the collagen molecules and promoted collagen mineralization, resulting in calcium phosphate minerals.

[0106] Example 3

[0107] This embodiment provides a method for preparing bone-targeted biomimetic biomineral nanocomposite materials. The difference between this embodiment and Example 1 is that:

[0108] S1: Water-soluble phosphate and water-soluble calcium salt are provided. In this example, the water-soluble calcium salt is a 40 mL aqueous solution containing 246.7 mg CaCl2. The water-soluble phosphate is prepared by mixing 40 mL of an aqueous solution containing 109.3 mg Na3PO4 and 94.7 mg Na2HPO4.

[0109] S2: In an ice-water bath, mix the aqueous solutions of water-soluble phosphate and water-soluble calcium salt, stir magnetically for 5 min, and then centrifuge at 8000 rpm for 5 min to obtain amorphous calcium phosphate (ACP).

[0110] S3: Wash twice with ultrapure water and anhydrous ethanol respectively, disperse in anhydrous ethanol or freeze-dry and store at -20℃.

[0111] Reference Figure 3 The transmission electron microscopy (TEM) images of the amorphous calcium phosphate (ACP) nanoparticles are presented, showing that the material consists of spherical nanoparticles of uneven size with obvious nanoparticle aggregation, and the diameters range from 10 to 80 nm. Compared with Example 1, the diameter of individual nanoparticles is larger, and the aggregation state is more obvious.

[0112] Example 4

[0113] This embodiment provides a method for preparing bone-targeted biomimetic biomineral nanocomposite materials. The difference between this embodiment and Example 1 is that:

[0114] S1: Water-soluble phosphate and water-soluble calcium salt are provided. In this example, the water-soluble calcium salt is a 40 mL aqueous solution containing 246.7 mg CaCl2. The water-soluble phosphate is prepared by mixing 40 mL of an aqueous solution containing 109.3 mg Na3PO4 and 94.7 mg Na2HPO4.

[0115] S2: At room temperature, an aqueous solution of water-soluble phosphate and water-soluble calcium salt was mixed and magnetically stirred for 60 min, then centrifuged at 8000 rpm for 5 min to obtain hydroxyapatite (HAP).

[0116] S3: Wash twice with ultrapure water and anhydrous ethanol respectively, disperse in anhydrous ethanol or freeze-dry and store at -20℃.

[0117] Figure 4 Transmission electron microscopy (TEM) images of the material are presented, showing that HAP exhibits a nanosheet-like structure with a length of approximately 120 nm.

[0118] Figure 5 The zeta potential of the material is given, showing that the HAP surface carries a negative charge of -8.2 mV.

[0119] Figure 6 The infrared spectrum of the material is given, indicating that HAP contains its corresponding components.

[0120] Figure 7 The X-ray diffraction pattern of the material is shown. Sharp crystalline HAP diffraction peaks were observed at 25.2° and 30.4° for 2θ.

[0121] Figure 9 The image shows the CCK-8 results illustrating the effect of HAP on cell proliferation. Similar to the control group, there was no significant difference in BMSC proliferation in the HAP group, further demonstrating the good biocompatibility of this nanoparticle over a longer period.

[0122] Figure 10 Alkaline phosphatase staining image showing the effect of HAP on promoting osteogenic differentiation of bone marrow mesenchymal stem cells. Compared with the ACP group in Example 3, the content of alkaline phosphatase in the HAP group was reduced. This is mainly because HAP is in crystalline form, which reduces its biological activity and thus reduces its osteogenic differentiation capacity.

[0123] Figure 11 Alizarin Red staining image showing the effect of HAP on promoting osteogenic differentiation of bone marrow mesenchymal stem cells. Compared with the ACP group in Example 3, the calcium nodules in the HAP group stained darker. This is mainly because the HAP material is not easily degraded, and the large amount of calcium in the material chelates with the alizarin Red dye.

[0124] Figure 12 This is a graph showing the RT-qPCR results of key genes in osteogenic differentiation of bone marrow mesenchymal stem cells by HAP. Compared with the ACP group in Example 3, the expression levels of osteogenic factors RUNX2 and BMP2 were reduced in the HAP group.

[0125] Figure 13This is a CLSM image showing the effect of HAP on collagen mineralization. Collagen molecules were initially labeled with red fluorescence. After co-incubation with HAP, no green fluorescent calcium phosphate was observed, indicating that HAP treatment failed to induce intra-collagen mineralization.

[0126] Example 5

[0127] GLG1 was prepared + The NIH-3T3 cell membrane was designated as the cell membrane (CM) group.

[0128] Specifically, this embodiment can be considered as a blank embodiment of embodiments 1-4.

[0129] Figure 5 The zeta potential of the cell membrane is given, showing that the cell membrane surface carries a negative charge of -13.8 mV.

[0130] Figure 6 The infrared spectrum of the cell membrane is given, indicating that the cell membrane contains its corresponding components.

[0131] Figure 8 The results of Western blot analysis of GLG1 protein on the cell membrane show that the extracted cell membrane effectively preserves the functional protein GLG1 on cells with high GLG1 expression.

[0132] Figure 9 This is a CCK-8 assay showing the effect of the cell membrane on cell proliferation. Similar to the control group, there was no significant difference in BMSC proliferation in the cell membrane (CM) group.

[0133] Figure 10 Alkaline phosphatase staining image showing the effect of cell membrane on promoting osteogenic differentiation of bone marrow mesenchymal stem cells. Compared with the control group, the alkaline phosphatase content in the CM group was increased, mainly due to the protein contained in the cell membrane promoting osteogenic differentiation.

[0134] Figure 11 Alizarin Red staining image showing the effect of cell membrane on promoting osteogenic differentiation of bone marrow mesenchymal stem cells. No significant difference was observed compared to the control group.

[0135] Figure 12 This image shows the RT-qPCR results of key genes in bone marrow mesenchymal stem cell osteogenic differentiation via cell membrane. Compared to the control group, there was no significant difference in the expression levels of osteogenic factors RUNX2 and BMP2 in the CM group.

[0136] Example 6

[0137] This embodiment describes the application of the non-targeting biomimetic biomineral nanocomposite material (ACP / CP@Con-CM) and the bone-targeting biomimetic biomineral nanocomposite material (ACP / CP@CM) prepared by the preparation methods described in Examples 1-4 in laboratory mice.

[0138] (1) Osteoporosis Modeling. Healthy female C57BL / 6 mice were selected and ovariectomized to establish a mouse osteoporosis model. First, mice were anesthetized by intraperitoneal injection of 3% pentobarbital at a dose of 40 mg / kg. After the mice became sluggish, they were placed prone with their limbs fixed on the operating table. The hair on their backs was shaved off with an electric shaver and disinfected with 75% alcohol. Next, a longitudinal incision was made on both sides of the kidney area on the back of the mouse. The muscles and peritoneum were separated, and the pink ovarian tissue attached to the adipose tissue was located. Both ovarian tissues were completely removed. After surgery, the wounds were sutured in layers and disinfected with povidone-iodine. The mice were kept warm on a heating pad and separated into different cages after waking up. The wound healing of the mice was carefully observed after surgery. The bedding was changed regularly to keep the cages clean and prevent postoperative infection.

[0139] (2) Administration and sampling. Four weeks after modeling, mice were injected weekly with 200 μL of ACP / CP@CM at a dose of 20 mg / kg via the tail vein. Other administration groups were set up in parallel, including ACP / CP in Example 1, ACP in Example 3, HAP in Example 4, and CM in Example 5, with 6 mice in each group. After eight weeks of treatment, samples were collected from the mice in each group for analysis.

[0140] Figure 14 Fluorescence imaging of the femur and tibia after tail vein injection of ACP / CP@Con-CM and ACP / CP@CM in mice. Using untransfected normal NIH-3T3 cell membranes as a control, ACP / CP@Con-CM was prepared by modifying the surface of ACP / CP. The ACP / CP@CM group showed strong fluorescence in both the femur and tibia, exhibiting significant bone-targeting activity compared to ACP / CP@Con-CM.

[0141] Figure 15 Micro-CT three-dimensional reconstruction images of the femur in the sagittal and horizontal planes after injection of different materials into the tail vein of mice. The bone loss in the HAP, ACP, and CM groups was similar to that in the OVX group in Comparative Example 2, indicating no therapeutic effect. The ACP / CP group showed a slight increase in bone mass, suggesting that ACP / CP has a certain positive effect on osteoporosis repair, but this effect is not significant. The ACP / CP@CM group, with its modified bone-targeting cell membrane, recovered bone mass to a level similar to that of the Sham group in Comparative Example 1, demonstrating excellent osteoporosis repair efficacy.

[0142] Example 7 (Comparative Example 1 to Example 6):

[0143] (1) Sham-operated control group. Mice in the sham-operated group were anesthetized by intraperitoneal injection of 3% pentobarbital at a dose of 40 mg / kg. After the mice became sluggish, they were placed prone with their limbs fixed on the operating table. The hair on their backs was shaved off with an electric shaver, and the area was disinfected with 75% alcohol. Next, a longitudinal incision was made on both sides of the kidney area on the back of the mouse. The muscles and peritoneum were separated, and the pink ovarian tissue attached to the adipose tissue was located. Only the adipose tissue of the same size near the ovary was removed. After the operation, the wound was sutured in layers and disinfected with povidone-iodine. The mice were kept warm on a heating pad and separated into different cages after waking up. The wound healing of the mice was carefully observed after the operation. The bedding was changed regularly to keep the cage clean and prevent postoperative infection.

[0144] (2) Administration and sample collection. Four weeks after modeling, 200 μL of physiological saline was injected into the tail vein of mice weekly. After eight weeks of treatment, the mice were sampled for analysis.

[0145] Example 8 (Comparative Example 2 to Example 6):

[0146] (1) Same as Example 6(1).

[0147] (2) Drug administration and tissue collection. Four weeks after modeling, 200 μL of physiological saline was injected into the tail vein of mice weekly. After eight weeks of treatment, tissue samples were collected from the mice for analysis. This group was designated as the osteoporosis modeling (OVX) group.

[0148] Figure 15 It can be seen that compared with the Sham group, the OVX group has significantly less bone mass and a more porous cancellous bone reticular structure in the distal femur, which indicates the success of osteoporosis modeling.

[0149] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing bone-targeting biomimetic biomineral nanocomposite materials, characterized in that, Includes the following steps: S1: Provides water-soluble phosphates and water-soluble calcium salts; S2: Mix an aqueous solution of water-soluble phosphate and water-soluble calcium salt, and then perform magnetic stirring and centrifugation to obtain calcium phosphate; S3: Washing: Wash the obtained calcium phosphate at least once with ultrapure water and at least once with anhydrous ethanol, and freeze-dry for storage; S4: Provides GLG1 + Phospholipid membrane solution of NIH-3T3 cell membrane; In step S4, the phospholipid membrane is GLG1. + Preparation of cell phospholipid membrane solution: After providing a bone-targeting phospholipid membrane, extract the phospholipid membrane and resuspend it in pre-chilled PBS buffer to obtain a buffer solution, which is stored at -80℃. The specific steps are as follows: Discard the cell culture medium and add PBS buffer to wash the cells; collect the cultured cells with a cell scraper; after centrifugation and discarding the supernatant, resuspend the cell pellet with pre-chilled membrane protein extraction reagent A containing protease inhibitors, and lyse on ice for 15-30 min; add two steel balls and homogenize the cells using a tissue homogenizer at 70-100 Hz at -20℃ for 1-5 min; remove the steel balls and centrifuge at 500-1000g at 4℃ for 5-30 min, transfer the supernatant to a new centrifuge tube to remove cell nuclei and unlysaturated cells; centrifuge at 10000-15000g at 4℃ for 15-60 min, and the pellet is the cell membrane. S5: The calcium phosphate in S2 and the phospholipid membrane solution in S4 are mixed in a mass ratio of 50:1 to 1:50 and subjected to ultrasonic treatment to obtain phospholipid membrane-coated calcium phosphate nanoparticles. S6: Centrifuge the calcium phosphate nanoparticles to remove excess phospholipid membranes, resuspend and wash them with ultrapure water at least twice to finally obtain the bone-targeting biomimetic biomineral nanocomposite material, and store the composite material in an environment below 25°C.

2. The method for preparing a bone-targeting biomimetic biomineral nanocomposite material according to claim 1, characterized in that, The water-soluble calcium salt in S1 includes at least one of calcium chloride, calcium chloride hydrate, calcium nitrate, calcium nitrate hydrate, calcium acetate, and calcium acetate hydrate.

3. The method for preparing a bone-targeting biomimetic biomineral nanocomposite material according to claim 1, characterized in that, The water-soluble phosphate in S1 includes at least one of sodium phosphate, sodium phosphate hydrate, sodium hydrogen phosphate, and sodium hydrogen phosphate hydrate.

4. A method for preparing a bone-targeting biomimetic biomineral nanocomposite material according to claim 1 or 2, characterized in that, The water-soluble calcium salt in S1 is a 40 mL aqueous solution containing 100–500 mg of CaCl2.

5. A method for preparing a bone-targeting biomimetic biomineral nanocomposite material according to claim 1 or 3, characterized in that, The specific preparation method of water-soluble phosphate in S1 is as follows: 20 mL of an aqueous solution containing 10–100 mg Na3PO4 and 10–100 mg Na2HPO4 is mixed with 20 mL of a solution containing 50–200 mg of high-energy phosphate molecules.

6. The method for preparing a bone-targeting biomimetic biomineral nanocomposite material according to claim 5, characterized in that, The high-energy phosphate molecule includes any one of creatine phosphate, guanine dinucleotide phosphate, guanine trinucleotide phosphate, adenine trinucleotide phosphate, adenine dinucleotide phosphate, and adenine mononucleotide phosphate, with a molar concentration of 0.01 to 100 mmol / L in the final reaction solution.

7. The method for preparing a bone-targeting biomimetic biomineral nanocomposite material according to claim 1, characterized in that, In step S5, the ultrasonic treatment is performed in an ice-water bath for 5–30 min, and in step S6, the centrifugation is performed at 5000–10000 rpm for 5–30 min.

8. A bone-targeting biomimetic biomineral nanocomposite material, characterized in that, The material is prepared by any one of the bone-targeting biomimetic biomineral nanocomposite materials as described in claims 1 to 5.

9. The bone-targeting biomimetic biomineral nanocomposite material prepared by the preparation method according to any one of claims 1-7 or the bone-targeting biomimetic biomineral nanocomposite material according to claim 8, in the preparation of bone repair and bone regeneration materials.

10. The application of the bone-targeting biomimetic biomineral nanocomposite material prepared by the preparation method according to any one of claims 1-7 or the bone-targeting biomimetic biomineral nanocomposite material according to claim 8 in the preparation of injectable formulations, characterized in that, The injection is administered intravenously and targeted to the vicinity of the site to be repaired.

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