Bone peptide composition for osteoporosis repair and preparation method thereof

Abstract

The invention discloses an osteoporosis repair type bone peptide composition and a preparation method thereof, and relates to the technical field of biological agents. The bone peptide composition for osteoporosis repair is prepared from targeted metal composite nanoparticles loaded with bone collagen peptide, an injectable gel delivery matrix and calcium chloride, tuna bone collagen peptide is encapsulated in ZIF-8 nanoparticles, and copper peroxide black phosphorus composite particles are adsorbed on the surfaces of the ZIF-8 nanoparticles, so that the encapsulation effect is enhanced, the inactivation and degradation effects of the external environment on the tuna bone collagen peptide are reduced, and bone targeting and good biocompatibility are also endowed; then, the targeting metal composite nanoparticles loaded with the bone collagen peptide are attached to an injectable gel delivery matrix to form a microsphere structure, so that the stability of the tuna bone collagen peptide is effectively protected, and the adverse reaction is reduced and the treatment effect is improved in the action modes of slow release and targeting release.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological agents, and specifically refers to an osteoporotic repair type bone peptide composition and a preparation method thereof. Background Art

[0002] Osteoporosis is a metabolic bone disease that mostly occurs in the elderly and menopausal women, mainly manifested as reduced bone mass, decreased bone density and quality, resulting in damage to the bone microstructure, increased bone brittleness, increased fracture risk, and bone necrosis in severe cases. Generally, osteoporosis is caused by an imbalance in bone metabolism due to reduced bone formation and increased bone resorption; osteoblasts participate in the dynamic process of bone formation and are the most critical cells in the bone remodeling process. They can secrete various extracellular matrix proteins, such as alkaline phosphatase and osteocalcin, and can also produce collagen and new bone tissue through matrix calcification; therefore, promoting the proliferation and differentiation of osteoblasts, improving the function of osteoblasts, and regulating bone remodeling are the keys to preventing osteoporosis and promoting bone health.

[0003] Conventional drug treatments maintain bone homeostasis by inhibiting osteoclastogenesis or promoting osteogenesis through antiresorptive agents, anabolic agents, and selective estrogen receptor modulators. However, long-term use of antiresorptive drugs will inhibit bone turnover, damage natural bone repair, produce obvious adverse reactions and affect compliance; therefore, finding safe and effective bioactive peptides that can regulate bone metabolism has become a research hotspot.

[0004] The current existing technologies mainly have the following problems:

[0005] Due to the poor stability and bioavailability of bioactive peptides, the therapeutic effect on osteoporosis is reduced, and multiple administrations are likely to cause adverse reactions. Summary of the Invention

[0006] In view of the above situation, to overcome the defects of the prior art, the present invention provides an osteoporotic repair type bone peptide composition, which comprises the following components in parts by weight: 50 - 60 parts of targeted metal composite nanoparticles loaded with bone collagen peptide, 20 - 30 parts of injectable gel delivery matrix, and 10 - 20 parts of calcium chloride.

[0007] The targeted metal composite nanoparticles loaded with bone collagen peptide comprise the following components in parts by weight: 20 - 30 parts of tuna bone collagen peptide, 10 - 20 parts of ZIF-8 nanoparticles, and 8 - 10 parts of cupric peroxide black phosphorus composite particles.

[0008] The injectable gel delivery matrix comprises the following components in parts by weight: 6 - 10 parts of a biological crosslinking agent, 1 - 5 parts of gold nanorods, and 10 - 20 parts of sodium alginate.

[0009] The preparation method of the target metal composite nanoparticles loaded with bone collagen peptide specifically includes the following steps:

[0010] (1) Add 8.0 - 9.0 g of tuna bone meal into 100 mL of water, add animal protease, and carry out enzymatic hydrolysis at pH 7.0 and a temperature of 50 - 55 °C for 6 - 8 h. After the enzymatic hydrolysis is completed, inactivate the enzyme at 90 °C for 30 min, then centrifuge at a speed of 6000 - 8000 rpm for 10 - 15 min, take the supernatant, and use the membrane separation method to intercept the enzymatic hydrolysate, collect the bone collagen peptide with a molecular weight of 250 - 1000 Da, and freeze-dry it. The low-molecular-weight tuna bone collagen peptide can significantly improve the activities of alkaline phosphatase and osteocalcin in osteoblasts, thus facilitating the differentiation of osteoblasts, promoting the secretion of type I collagen, the main component of the organic matrix of bone, and contributing to the formation of bone matrix. Therefore, the prepared tuna bone collagen peptide has good ability to promote the proliferation, differentiation, and mineralization of osteoblasts, and obtain tuna bone collagen peptide;

[0011] (2) Add 2.0 g of zinc nitrate hexahydrate into 20 mL of deionized water to obtain a zinc nitrate hexahydrate solution for later use. Then add 8.0 g of 2-methylimidazole into 40 mL of deionized water, and then add the tuna bone collagen peptide described in step (1). Stir at a speed of 60 - 80 rpm for 5 - 10 min. Rapidly drop the zinc nitrate hexahydrate solution into the 2-methylimidazole solution containing tuna bone collagen peptide, and stir at a speed of 600 - 800 rpm for 20 - 30 min. Collect and centrifuge the suspension, and wash the precipitate with deionized water 3 - 5 times. The porous structure and pH sensitivity of ZIF-8 make it an ideal drug delivery carrier. It can encapsulate active substances through its pore structure, protect the stability of tuna bone collagen peptide, reduce the risk of its inactivation or degradation in the external environment, and achieve the slow release of drugs in a weakly acidic environment, improving the bioavailability of tuna bone collagen peptide. And ZIF-8 is stable under physiological conditions, has low toxicity to cells and tissues, and can also inhibit the destruction of bone tissue by inflammatory factors by releasing zinc ions, and obtain ZIF-8 nanoparticles loaded with bone collagen peptide;

[0012] (3) Add 0.5 g of polyvinylpyrrolidone to 5 mL of a copper(II) chloride dihydrate solution with a mass fraction of 0.1 - 0.2%, ultrasonically dissolve and stir evenly, then add 5 mL of a sodium hydroxide solution with a mass fraction of 0.08%, magnetically stir evenly, and then slowly drop in 10 μL of hydrogen peroxide with a mass fraction of 30%, stir for 20 - 30 min, subject the reaction mixture to ultra-high-speed centrifugation at a centrifugation speed of 5000 - 6000 rpm for 10 - 20 min, collect the precipitate, wash it with ultrapure water 3 - 5 times, and freeze-dry. The metal peroxide can react with the excessive hydrogen ions in the osteoporotic bone tissue, and the simultaneously released copper ions help induce and stimulate osteoblasts, which is beneficial to bone mineralization and bone regeneration, obtaining cupric peroxide nanoparticles;

[0013] (4) Disperse 1.0 g of sodium hydroxide in 40 mL of N-methylpyrrolidone, ultrasonically treat for 30 min, then add black phosphorus powder, and ultrasonically treat in an ice bath environment for 8 - 10 h. Centrifuge the suspension 2 - 3 times, collect the supernatant, continue to centrifuge 3 - 4 times, collect the precipitate, and freeze-dry to obtain black phosphorus nanosheets. Disperse the cupric peroxide nanoparticles described in step (3) in 10 mL of ultrapure water, then add black phosphorus nanosheets, and magnetically stir for 2 - 3 h. Using electrostatic interaction, the positively charged cupric peroxide nanoparticles are adsorbed onto the surface of the negatively charged black phosphorus nanosheets, forming a composite material with anti-inflammatory and osteogenic promoting effects. In the inflammatory and slightly acidic environment of osteoporosis, the cupric peroxide nanoparticles are decomposed into Cu 2+ and hydroxyl radicals, where Cu 2+ can have a certain degree of osteogenic promoting effect on osteoblasts, and the hydroxyl radicals can accelerate the degradation of black phosphorus nanosheets in the light-free environment in vivo, thereby undergoing self-mineralization to produce calcium phosphate nanoparticles. Collect the solid substance to obtain cupric peroxide black phosphorus composite particles;

[0014] (5) Disperse the ZIF-8 nanoparticles loaded with bone collagen peptides described in step (2) in 20 mL of ultrapure water, add the cupric peroxide black phosphorus composite particles described in step (4), magnetically stir for 3 - 5 h, collect the precipitate, and attach the cupric peroxide black phosphorus composite particles to the surface of the ZIF-8 nanoparticles loaded with bone collagen peptides to form a protective layer, increasing the stability of the ZIF-8 nanoparticles loaded with bone collagen peptides, improving the biocompatibility and bone targeting performance of the nanoparticles, further enhancing the effective release of bone collagen peptides, and being beneficial to improving the treatment effect of osteoporosis, obtaining targeted metal composite nanoparticles loaded with bone collagen peptides;

[0015] Preferably, in step (1), the addition amount of animal protease is 1.5 - 2.5% of the addition amount of tuna bone meal, and the enzyme activity is 300,000 U / g. The animal protease can decompose macromolecular proteins such as collagen in bone tissue into small molecule peptides and amino acids, improving the extraction rate and bioavailability of bone peptides;

[0016] Preferably, in step (4), the addition amount of black phosphorus powder is 10.0 - 20.0 mg. The biodegradation product of black phosphorus can be transformed into calcium phosphate nanoparticles through self-mineralization, which has natural affinity with hydroxyapatite in bone tissue, thereby promoting the enrichment of black phosphorus in bone tissue and showing a certain bone-targeting effect.

[0017] The present invention also provides a preparation method for an osteoporosis-repairing bone peptide composition, which specifically includes the following steps:

[0018] S1. Dissolve 100.0 mg of chitosan in 5 mL of a 30% dimethyl sulfoxide aqueous solution, adjust the pH to 5.0, and stir until completely dissolved for later use. Chitosan can improve the activity of alkaline phosphatase, accelerate bone formation, inhibit the activity of osteoclasts, reduce bone resorption, and also enhance bone density by promoting calcium absorption. Then dissolve chlorogenic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide in 5 mL of dimethyl sulfoxide, stir and activate for 1 - 2 h, then mix the activated chitosan solution and chlorogenic acid solution, stir overnight, transfer to a dialysis bag, first dialyze with an acidic buffer solution of pH 5.0, change the buffer solution every 12 h, and change to ultrapure water of pH 7.0 for dialysis for 3 - 4 h on the third day. Collect the reaction product and perform freeze-drying. The carboxyl group of chlorogenic acid reacts with the amino group of chitosan to synthesize a non-toxic biocrosslinker with a catechol structure, effectively improving the disadvantages of poor stability and water solubility of chlorogenic acid. Among them, the catechol structure can mimic the effect of estrogen, be beneficial to maintaining bone density, and also show high reactivity in the crosslinking reaction to obtain a biocrosslinker;

[0019] S2. Dissolve 1.0 - 2.0 g of sodium alginate in 100 mL of ultrapure water, stir evenly, add gold nanorods, stir magnetically for 1 - 2 h, and then add the biocrosslinker described in step S1, and mix evenly by vibration. Under the action of the biocrosslinker and gold nanorods, the loading amount and stability of the delivery matrix are improved, and it also has an auxiliary effect of promoting bone formation and inhibiting bone resorption, further improving the stability, release effectiveness, and therapeutic effect of a single active substance to obtain an injectable gel delivery matrix;

[0020] S3. Add the targeted metal composite nanoparticles loaded with bone collagen peptides into the injectable gel delivery matrix described in step S2, and ultrasonically treat for 2 - 3 h. Then, use a portable electrospray device to spray it into 100 mL of calcium chloride solution with a mass fraction of 1 - 2%. Let it stand for 6 - 8 h, and then rinse the gel product with ultrapure water 3 - 5 times. Through this process, a tightly complex porous microsphere structure is prepared. The pores of the microspheres adsorb the targeted metal composite nanoparticles loaded with bone collagen peptides, effectively protecting the stability of bone collagen peptides, reducing the inactivation risk caused by the external environment, and also significantly improving the bioavailability of bone collagen peptides in a slow-release and targeted-release mode of action. At the same time, by synergistically acting with other components, it plays a therapeutic role in osteoporosis in multiple ways and achieves excellent therapeutic effects, obtaining an osteopeptide composition for osteoporosis repair;

[0021] Preferably, in step S1, the addition amount of chlorogenic acid is 170.0 - 178.0 mg, the addition amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 0.1 g, and the addition amount of N-hydroxysuccinimide is 80.0 mg. Chlorogenic acid is not only used as a cross-linking agent but also helps to prevent and treat osteoporosis by regulating bone metabolism-related hormones (such as estrogen and parathyroid hormone) and maintaining bone metabolism balance;

[0022] Preferably, in step S2, the addition amount of gold nanorods is 0.1 - 0.5 g. The addition of gold nanorods can form a large coherent network structure with a high cross-linking degree, which can accurately deliver active substances to bone tissue, improve the curative effect and reduce side effects.

[0023] The beneficial effects achieved by the present invention are as follows:

[0024] In the present invention, tuna bone collagen peptide is encapsulated in ZIF-8 nanoparticles, and copper peroxide black phosphorus composite particles are adsorbed on the surface to form a protective layer, which further enhances the encapsulation effect, reduces the inactivation and degradation of tuna bone collagen peptide by the external environment, and at the same time endows bone targeting and good biocompatibility. Then, the targeted metal composite nanoparticles loaded with bone collagen peptide are attached to the complex network of the injectable gel delivery matrix to form a microsphere structure, effectively protecting the stability of tuna bone collagen peptide. In the form of slow release and targeted release, the bioavailability of tuna bone collagen peptide is significantly improved. Multiple substances act together to enrich the diseased part of osteoporosis, reducing the adverse reactions caused by multiple administrations and obtaining an efficient therapeutic effect. In the targeted metal composite nanoparticles loaded with bone collagen peptide, ZIF-8 nanoparticles are used as carriers to encapsulate the active substance tuna bone collagen peptide, protecting the stability of tuna bone collagen peptide and realizing the slow release of the drug in a weakly acidic environment. Then, the copper peroxide black phosphorus composite particles are attached to the surface of the ZIF-8 nanoparticles loaded with bone collagen peptide to protect the stability of tuna bone collagen peptide in an encapsulated manner, reducing the inactivation effect of the external environment and also reducing the adverse stimulation to the human body. It also has a certain bone targeting effect, improving the enrichment of tuna bone collagen peptide at the osteoporosis site, thereby enhancing the bioavailability and improving the therapeutic effectiveness for osteoporosis. In the injectable gel delivery matrix, under the action of a biocrosslinking agent and gold nanorods, a highly crosslinked large and tightly coherent network structure is formed, which can slowly release and accurately deliver the active substance to the bone tissue, further improving the encapsulation amount and encapsulation stability of the targeted metal composite nanoparticles loaded with bone collagen peptide, reducing the adverse stimulation of tuna bone collagen peptide, and at the same time improving the bioavailability and therapeutic effect of tuna bone collagen peptide. Among them, the biocrosslinking agent and gold nanorods also have an auxiliary effect of promoting bone formation and inhibiting bone resorption, which helps to improve the therapeutic effect. The present invention uses the targeted metal composite nanoparticles loaded with bone collagen peptide, the injectable gel delivery matrix and calcium chloride to prepare an osteopeptide composition for osteoporosis repair, which can slowly release tuna bone collagen peptide in a targeted manner, improve the stability and bioavailability of the active substance, reduce the occurrence of adverse reactions, and also have an efficient therapeutic effect. Description of the Drawings

[0025] Figure 1 It is a scanning electron micrograph of the osteopeptide composition for osteoporosis repair prepared in Example 1 of the present invention;

[0026] Figure 2 It is a graph of the encapsulation efficiency results of Examples 1-4 and Comparative Examples 1-3 of the present invention;

[0027] Figure 3 It is a graph of the allergy rate results of Examples 1-4 and Comparative Examples 1-3 of the present invention;

[0028] Figure 4 This is the graph of the treatment effective rates for Examples 1-4 and Comparative Examples 1-3 of the present invention. Detailed implementation manners

[0029] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0030] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are only for demonstration purposes, but cannot limit the content of this application.

[0031] The experimental methods in the following embodiments are all conventional methods unless otherwise specified; the test materials used in the following embodiments are all obtained from commercial channels unless otherwise specified.

[0032] Example 1

[0033] This example provides an osteoporotic repair type bone peptide composition, which comprises the following components in parts by weight: 60 parts of targeted metal composite nanoparticles loaded with bone collagen peptide, 30 parts of injectable gel delivery matrix, and 20 parts of calcium chloride.

[0034] The targeted metal composite nanoparticles loaded with bone collagen peptide comprise the following components in parts by weight: 30 parts of tuna bone collagen peptide, 20 parts of ZIF-8 nanoparticles, and 10 parts of cupric peroxide black phosphorus composite particles.

[0035] The injectable gel delivery matrix comprises the following components in parts by weight: 10 parts of biocrosslinking agent, 5 parts of gold nanorods, and 20 parts of sodium alginate.

[0036] The preparation method of the targeted metal composite nanoparticles loaded with bone collagen peptide specifically comprises the following steps:

[0037] (1) Add 9.0 g of tuna bone powder to 100 mL of water, and add animal protease. The addition amount of animal protease is 2.5% of the added amount of tuna bone powder, and the enzyme activity is 300,000 U / g. Animal protease can decompose macromolecular proteins such as collagen in bone tissue into small molecule peptides and amino acids, improve the extraction rate and bioavailability of bone peptides. Enzymolysis is carried out at pH 7.0 and a temperature of 55 °C for 8 h. After the enzymolysis is completed, inactivate the enzyme at 90 °C for 30 min, then centrifuge at 8000 rpm for 15 min, take the supernatant, and use membrane separation method to intercept the enzymolysis solution, collect bone collagen peptides with a molecular weight of 250 - 1000 Da, and freeze-dry. The low molecular weight tuna bone collagen peptides can significantly improve the activities of alkaline phosphatase and osteocalcin in osteoblasts, thus facilitating the differentiation of osteoblasts, promoting the secretion of type I collagen, the main component of bone organic matrix, and thus contributing to the formation of bone matrix. Therefore, the prepared tuna bone collagen peptides have good ability to promote the proliferation, differentiation and mineralization of osteoblasts, and obtain tuna bone collagen peptides;

[0038] (2) Add 2.0 g of zinc nitrate hexahydrate to 20 mL of deionized water to obtain a zinc nitrate hexahydrate solution for later use. Then add 8.0 g of 2-methylimidazole to 40 mL of deionized water, and then add the tuna bone collagen peptides described in step (1). Stir at 80 rpm for 10 min. Quickly drop the zinc nitrate hexahydrate solution into the 2-methylimidazole solution containing tuna bone collagen peptides, and stir at 800 rpm for 30 min. Collect the suspension by centrifugation, wash the precipitate 5 times with deionized water. The porous structure and pH sensitivity of ZIF-8 make it an ideal drug delivery carrier. It can encapsulate active substances through its pore structure, protect the stability of tuna bone collagen peptides, reduce the risk of inactivation or degradation in the external environment, and achieve slow release of drugs in a weakly acidic environment, improving the bioavailability of tuna bone collagen peptides. And ZIF-8 is stable under physiological conditions, has low toxicity to cells and tissues, and can also inhibit the damage of inflammatory factors to bone tissue by releasing zinc ions, and obtain ZIF-8 nanoparticles loaded with bone collagen peptides;

[0039] (3) Add 0.5 g of polyvinylpyrrolidone to 5 mL of a 0.2% copper(II) chloride dihydrate solution, dissolve it by ultrasonic wave and stir evenly. Then add 5 mL of a 0.08% sodium hydroxide solution, stir evenly with a magnetic stirrer, and then slowly drop 10 μL of 30% hydrogen peroxide solution, stir for 30 min. Carry out ultra-high speed centrifugation on the reaction mixture, with a centrifugation speed of 6000 rpm and a centrifugation time of 20 min. Collect the precipitate, wash it 5 times with ultrapure water, and freeze-dry. Metal peroxides can react with excessive hydrogen ions in osteoporotic bone tissue, and at the same time, the released copper ions help to induce and stimulate osteoblasts, which is beneficial to bone mineralization and bone regeneration, and obtain cuprous oxide nanoparticles;

[0040] (4) Disperse 1.0 g of sodium hydroxide in 40 mL of N-methylpyrrolidone, ultrasonically treat for 30 min, then add black phosphorus powder with an addition amount of 20.0 mg. The biodegradation product of black phosphorus can be converted into calcium phosphate nanoparticles through self-mineralization, which has natural affinity with hydroxyapatite in bone tissue, thereby promoting the enrichment of black phosphorus in bone tissue, showing a certain bone-targeting effect. Then ultrasonically treat for 10 h in an ice bath environment. Centrifuge the suspension 3 times, collect the supernatant, continue to centrifuge 4 times, collect the precipitate, and freeze-dry to obtain black phosphorus nanosheets. Disperse the cuprous oxide nanoparticles described in step (3) in 10 mL of ultrapure water, then add the black phosphorus nanosheets, and magnetically stir for 3 h. Utilize electrostatic interaction to adsorb the positively charged cuprous oxide nanoparticles onto the surface of the negatively charged black phosphorus nanosheets to form a composite material with anti-inflammatory and osteogenic promoting effects. In the inflammatory and slightly acidic environment of osteoporosis, the cuprous oxide nanoparticles are decomposed into Cu 2+ and hydroxyl radicals, where Cu 2+ can have a certain degree of osteogenic promoting effect on osteoblasts, and the hydroxyl radicals can accelerate the degradation of black phosphorus nanosheets in the light-free environment in vivo, thereby undergoing self-mineralization to produce calcium phosphate nanoparticles. Collect the solid matter to obtain cuprous oxide-black phosphorus composite particles;

[0041] (5) Disperse the ZIF-8 nanoparticles loaded with bone collagen peptide described in step (2) in 20 mL of ultrapure water, add the cuprous oxide-black phosphorus composite particles described in step (4), and magnetically stir for 5 h. Collect the precipitate. Attach the cuprous oxide-black phosphorus composite particles to the surface of the ZIF-8 nanoparticles loaded with bone collagen peptide to form a protective layer, which increases the stability of the ZIF-8 nanoparticles loaded with bone collagen peptide, improves the biocompatibility and bone-targeting performance of the nanoparticles, further enhances the effective release of bone collagen peptide, and is beneficial to improving the treatment effect of osteoporosis, to obtain targeted metal composite nanoparticles loaded with bone collagen peptide.

[0042] This example provides a preparation method for an osteoporosis repair-type bone peptide composition, specifically including the following steps:

[0043] S1. Dissolve 100.0 mg of chitosan in 5 mL of a 30% (mass fraction) aqueous dimethyl sulfoxide solution, adjust the pH to 5.0, and stir until completely dissolved for later use. Chitosan can increase the activity of alkaline phosphatase, accelerate bone formation, inhibit the activity of osteoclasts, reduce bone resorption, and also enhance bone density by promoting calcium absorption. Then dissolve chlorogenic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide in 5 mL of dimethyl sulfoxide and stir for activation for 2 h. The addition amount of chlorogenic acid is 178.0 mg, the addition amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 0.1 g, and the addition amount of N-hydroxysuccinimide is 80.0 mg. Chlorogenic acid is not only used as a cross-linking agent but also can maintain bone metabolism balance by regulating bone metabolism-related hormones (such as estrogen and parathyroid hormone), which helps prevent and treat osteoporosis. Then mix the activated chitosan solution and chlorogenic acid solution, stir overnight, transfer to a dialysis bag, first dialyze with an acidic buffer solution of pH 5.0, change the buffer solution every 12 h, and on the third day, change to ultrapure water of pH 7.0 for dialysis for 4 h. Collect the reaction product and perform freeze-drying. The carboxyl group of chlorogenic acid reacts with the amino group of chitosan to synthesize a non-toxic biocrosslinker with a catechol structure, effectively improving the disadvantages of chlorogenic acid being unstable and having poor water solubility. Among them, the catechol structure can mimic the role of estrogen, is beneficial to maintaining bone density, and can also show high reactivity in the cross-linking reaction to obtain a biocrosslinker;

[0044] S2. Dissolve 2.0 g of sodium alginate in 100 mL of ultrapure water, stir evenly, add 0.5 g of gold nanorods. The addition of gold nanorods can form a large coherent network structure with a high degree of cross-linking, which can accurately deliver active substances to bone tissue, improve the curative effect and reduce side effects. Stir magnetically for 2 h, then add the biocrosslinker described in step S1 and mix evenly by vibration. Under the action of the biocrosslinker and gold nanorods, the loading capacity and stability of the delivery matrix are improved, and it also has an auxiliary effect of promoting bone formation and inhibiting bone resorption, further improving the stability, release effectiveness, and therapeutic effect of a single active substance to obtain an injectable gel delivery matrix;

[0045] S3. Add the targeted metal composite nanoparticles loaded with bone collagen peptide into the injectable gel delivery matrix described in step S2, and ultrasonically treat for 3 h. Then, spray it into 100 mL of a calcium chloride solution with a mass fraction of 2% using a portable electrospray device, let it stand for 8 h, and then rinse the gel product 5 times with ultrapure water. Through this process, a tightly complex porous microsphere structure is prepared. The pores of the microspheres adsorb the targeted metal composite nanoparticles loaded with bone collagen peptide, effectively protecting the stability of bone collagen peptide and reducing the inactivation risk caused by the external environment. Also, in the form of slow release and targeted release, it significantly improves the bioavailability of bone collagen peptide. At the same time, by synergistically acting with other components, it exerts a therapeutic effect on osteoporosis in multiple ways and obtains excellent therapeutic effects, resulting in an osteopeptide composition for osteoporosis repair.

[0046] In this example, the prepared osteopeptide composition for osteoporosis repair was subjected to scanning electron microscopy to observe its microscopic morphology. Figure 1 Figure 5 shows the SEM image of the osteopeptide composition for osteoporosis repair prepared in Example 1 magnified 1000 times. As Figure 1 , the osteopeptide composition for osteoporosis repair prepared in this example presents a microsphere structure with high surface crosslinking.

[0047] Example 2

[0048] This example proposes an osteopeptide composition for osteoporosis repair, which includes the following components in parts by weight: 50 parts of targeted metal composite nanoparticles loaded with bone collagen peptide, 20 parts of injectable gel delivery matrix, and 10 parts of calcium chloride.

[0049] The targeted metal composite nanoparticles loaded with bone collagen peptide include the following components in parts by weight: 20 parts of tuna bone collagen peptide, 10 parts of ZIF-8 nanoparticles, and 8 parts of copper peroxide black phosphorus composite particles.

[0050] The injectable gel delivery matrix includes the following components in parts by weight: 6 parts of biocrosslinking agent, 1 part of gold nanorods, and 10 parts of sodium alginate.

[0051] The preparation method of the targeted metal composite nanoparticles loaded with bone collagen peptide specifically includes the following steps:

[0052] (1) Add 8.0 g of tuna bone powder to 100 mL of water, and add animal protease. The addition amount of animal protease is 1.5% of the added amount of tuna bone powder, and the enzyme activity is 300,000 U / g. Animal protease can decompose macromolecular proteins such as collagen in bone tissue into small peptides and amino acids, improve the extraction rate and bioavailability of bone peptides. Enzymolysis is carried out at pH 7.0 and a temperature of 50 °C for 6 h. After the enzymolysis is completed, inactivate the enzyme at 90 °C for 30 min, then centrifuge at a speed of 6000 rpm for 10 min, take the supernatant, and use the membrane separation method to intercept the enzymolysis solution, collect the bone collagen peptide with a molecular weight of 250 - 1000 Da, and freeze-dry it. The low-molecular-weight tuna bone collagen peptide can significantly improve the activities of alkaline phosphatase and osteocalcin of osteoblasts, thus facilitating the differentiation of osteoblasts, promoting the secretion of type I collagen, the main component of the organic matrix of bone, and thus contributing to the formation of bone matrix. Therefore, the prepared tuna bone collagen peptide has good ability to promote the proliferation, differentiation and mineralization of osteoblasts, and obtain tuna bone collagen peptide;

[0053] (2) Add 2.0 g of zinc nitrate hexahydrate to 20 mL of deionized water to obtain a zinc nitrate hexahydrate solution for later use. Then add 8.0 g of 2-methylimidazole to 40 mL of deionized water, and then add the tuna bone collagen peptide described in step (1). Stir at a speed of 60 rpm for 5 min. Quickly drop the zinc nitrate hexahydrate solution into the 2-methylimidazole solution containing tuna bone collagen peptide, and stir at a speed of 600 rpm for 20 min. Collect the suspension by centrifugation, and wash the precipitate 3 times with deionized water. The porous structure and pH sensitivity of ZIF-8 make it an ideal drug delivery carrier. It can encapsulate active substances through its pore structure, protect the stability of tuna bone collagen peptide, reduce the risk of inactivation or degradation in the external environment, and achieve the slow release of drugs in a weakly acidic environment, improve the bioavailability of tuna bone collagen peptide. And ZIF-8 is stable under physiological conditions, has low toxicity to cells and tissues, and can also inhibit the damage of inflammatory factors to bone tissue by releasing zinc ions, and obtain ZIF-8 nanoparticles loaded with bone collagen peptide;

[0054] (3) Add 0.5 g of polyvinylpyrrolidone to 5 mL of a 0.1% copper chloride dihydrate solution, dissolve it by ultrasonic wave and stir evenly, then add 5 mL of a 0.08% sodium hydroxide solution, stir evenly by magnetic force, and then slowly drop 10 μL of 30% hydrogen peroxide, stir for 20 min, perform ultra-high-speed centrifugation on the reaction mixture, the centrifugation speed is 5000 rpm, and the centrifugation time is 10 min. Collect the precipitate, wash it 3 times with ultrapure water, and freeze-dry it. Metal peroxides can react with excessive hydrogen ions in osteoporotic bone tissue, and the released copper ions help to induce and stimulate osteoblasts, which is beneficial to bone mineralization and bone regeneration, and obtain cupric peroxide nanoparticles;

[0055] (4) Disperse 1.0 g of sodium hydroxide in 40 mL of N-methylpyrrolidone, ultrasonically treat for 30 min, then add black phosphorus powder with an addition amount of 10.0 mg. The biodegradation product of black phosphorus can be transformed into calcium phosphate nanoparticles through self-mineralization, which has a natural affinity with hydroxyapatite in bone tissue, thereby promoting the enrichment of black phosphorus in bone tissue, showing a certain bone-targeting effect. Then ultrasonically treat for 8 h in an ice bath environment, centrifuge the suspension twice, collect the supernatant, continue to centrifuge three times, collect the precipitate, and freeze-dry to obtain black phosphorus nanosheets. Disperse the cuprous oxide nanoparticles described in step (3) in 10 mL of ultrapure water, then add the black phosphorus nanosheets, and magnetically stir for 2 h. Utilize the electrostatic interaction to adsorb the positively charged cuprous oxide nanoparticles onto the surface of the negatively charged black phosphorus nanosheets to form a composite material with anti-inflammatory and osteogenic promoting effects. In the inflammatory and slightly acidic environment of osteoporosis, the cuprous oxide nanoparticles are decomposed into Cu 2+ and hydroxyl radicals, where Cu 2+ can have a certain degree of osteogenic promoting effect on osteoblasts, and the hydroxyl radicals can accelerate the degradation of black phosphorus nanosheets in the light-free environment in vivo, thereby undergoing self-mineralization to produce calcium phosphate nanoparticles. Collect the solid substance to obtain cuprous oxide-black phosphorus composite particles;

[0056] (5) Disperse the ZIF-8 nanoparticles loaded with bone collagen peptides described in step (2) in 20 mL of ultrapure water, add the cuprous oxide-black phosphorus composite particles described in step (4), magnetically stir for 3 h, collect the precipitate, and attach the cuprous oxide-black phosphorus composite particles to the surface of the ZIF-8 nanoparticles loaded with bone collagen peptides to form a protective layer, which increases the stability of the ZIF-8 nanoparticles loaded with bone collagen peptides, improves the biocompatibility and bone-targeting performance of the nanoparticles, further enhances the effective release of bone collagen peptides, and is beneficial to improving the treatment effect of osteoporosis, to obtain targeted metal composite nanoparticles loaded with bone collagen peptides.

[0057] This example provides a preparation method for an osteoporosis repair-type bone peptide composition, specifically including the following steps:

[0058] S1. Dissolve 100.0 mg of chitosan in 5 mL of a 30% (mass fraction) aqueous dimethyl sulfoxide solution, adjust the pH to 5.0, and stir until completely dissolved for later use. Chitosan can enhance the activity of alkaline phosphatase, accelerate bone formation, inhibit the activity of osteoclasts, reduce bone resorption, and also enhance bone density by promoting calcium absorption. Then dissolve chlorogenic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide in 5 mL of dimethyl sulfoxide and stir for activation for 1 h. The addition amount of chlorogenic acid is 170.0 mg, the addition amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 0.1 g, and the addition amount of N-hydroxysuccinimide is 80.0 mg. Chlorogenic acid is not only used as a cross-linking agent but also can maintain bone metabolism balance by regulating bone metabolism-related hormones (such as estrogen and parathyroid hormone), which helps prevent and treat osteoporosis. Then mix the activated chitosan solution and chlorogenic acid solution, stir overnight, transfer to a dialysis bag, first dialyze with an acidic buffer solution of pH 5.0, change the buffer solution every 12 h, and on the third day, change to ultrapure water of pH 7.0 for dialysis for 3 h. Collect the reaction product and perform freeze-drying. The carboxyl group of chlorogenic acid reacts with the amino group of chitosan to synthesize a non-toxic biocrosslinking agent with a catechol structure, effectively improving the disadvantages of chlorogenic acid being unstable and having poor water solubility. Among them, the catechol structure can mimic the action of estrogen, which is beneficial to maintaining bone density and can also show high reactivity in the cross-linking reaction to obtain a biocrosslinking agent;

[0059] S2. Dissolve 1.0 g of sodium alginate in 100 mL of ultrapure water, stir evenly, add 0.1 g of gold nanorods. The addition of gold nanorods can form a large, coherent network structure with a high degree of cross-linking, which can accurately deliver active substances to bone tissue, improve the curative effect and reduce side effects. Stir magnetically for 1 h, then add the biocrosslinking agent described in step S1 and mix evenly by vibration. Under the action of the biocrosslinking agent and gold nanorods, the loading capacity and stability of the delivery matrix are improved, and it also has an auxiliary effect of promoting bone formation and inhibiting bone resorption, further improving the stability, release effectiveness, and therapeutic effect of a single active substance to obtain an injectable gel delivery matrix;

[0060] S3. Add the target metal composite nanoparticles loaded with bone collagen peptide into the injectable gel delivery matrix described in step S2, and ultrasonically treat for 2 h. Then, use a portable electrospray device to spray it into 100 mL of a calcium chloride solution with a mass fraction of 1%, and let it stand for 6 h. Then, rinse the gel product 3 times with ultrapure water. Through this process, a tightly complex porous microsphere structure is prepared. The pores of the microspheres adsorb the target metal composite nanoparticles loaded with bone collagen peptide, effectively protecting the stability of bone collagen peptide, reducing the inactivation risk caused by the external environment, and also significantly improving the bioavailability of bone collagen peptide in a slow-release and targeted-release manner. At the same time, combined with the synergistic effect of other components, multiple measures are taken to play a therapeutic role in osteoporosis, and excellent therapeutic effects are obtained, resulting in an osteopeptide composition for osteoporosis repair.

[0061] Example 3

[0062] This example presents an osteopeptide composition for osteoporosis repair, which includes the following components in parts by weight: 55 parts of target metal composite nanoparticles loaded with bone collagen peptide, 25 parts of injectable gel delivery matrix, and 15 parts of calcium chloride.

[0063] The target metal composite nanoparticles loaded with bone collagen peptide include the following components in parts by weight: 25 parts of tuna bone collagen peptide, 15 parts of ZIF-8 nanoparticles, and 9 parts of copper peroxide black phosphorus composite particles.

[0064] The injectable gel delivery matrix includes the following components in parts by weight: 8 parts of biocrosslinker, 3 parts of gold nanorods, and 15 parts of sodium alginate.

[0065] The preparation method of the target metal composite nanoparticles loaded with bone collagen peptide specifically includes the following steps:

[0066] (1) Add 8.5 g of tuna bone powder to 100 mL of water, and add animal protease. The addition amount of animal protease is 2.0% of the added amount of tuna bone powder, and the enzyme activity is 300,000 U / g. Animal protease can decompose macromolecular proteins such as collagen in bone tissue into small molecule peptides and amino acids, improving the extraction rate and bioavailability of bone peptides. Enzymolysis is carried out at pH 7.0 and a temperature of 52.5 °C for 7 h. After the enzymolysis is completed, inactivate the enzyme at 90 °C for 30 min, then centrifuge at a speed of 7000 rpm for 12.5 min, take the supernatant, and use membrane separation method to intercept the enzymolysis solution, collect bone collagen peptides with a molecular weight of 250 - 1000 Da, and freeze-dry. The low molecular weight tuna bone collagen peptides can significantly improve the activities of alkaline phosphatase and osteocalcin in osteoblasts, thus facilitating the differentiation of osteoblasts, promoting the secretion of type I collagen, the main component of bone organic matrix, and thus contributing to the formation of bone matrix. Therefore, the prepared tuna bone collagen peptides have good ability to promote the proliferation, differentiation and mineralization of osteoblasts, and obtain tuna bone collagen peptides;

[0067] (2) Add 2.0 g of zinc nitrate hexahydrate to 20 mL of deionized water to obtain a zinc nitrate hexahydrate solution for later use. Then add 8.0 g of 2-methylimidazole to 40 mL of deionized water, and then add the tuna bone collagen peptides described in step (1). Stir at a speed of 70 rpm for 7.5 min. Quickly drop the zinc nitrate hexahydrate solution into the 2-methylimidazole solution containing tuna bone collagen peptides, and stir at a speed of 700 rpm for 25 min. Collect and centrifuge the suspension, wash the precipitate 4 times with deionized water. The porous structure and pH sensitivity of ZIF-8 make it an ideal drug delivery carrier. It can encapsulate active substances through its pore structure, protect the stability of tuna bone collagen peptides, reduce the risk of inactivation or degradation in the external environment, and achieve slow release of drugs in a weakly acidic environment, improving the bioavailability of tuna bone collagen peptides. And ZIF-8 is stable under physiological conditions, has low toxicity to cells and tissues, and can also inhibit the destruction of bone tissue by inflammatory factors by releasing zinc ions, obtaining ZIF-8 nanoparticles loaded with bone collagen peptides;

[0068] (3) Add 0.5 g of polyvinylpyrrolidone to 5 mL of a 0.15% copper(II) chloride dihydrate solution, dissolve it by ultrasound and stir evenly, then add 5 mL of a 0.08% sodium hydroxide solution, stir evenly with a magnetic stirrer, and then slowly drop 10 μL of a 30% hydrogen peroxide solution, stir for 25 min. Carry out ultra-high speed centrifugation on the reaction mixture, with a centrifugation speed of 5500 rpm and a centrifugation time of 15 min. Collect the precipitate, wash it 4 times with ultrapure water, and freeze-dry. Metal peroxides can react with excessive hydrogen ions in osteoporotic bone tissue, and at the same time the released copper ions help to induce and stimulate osteoblasts, which is beneficial to bone mineralization and bone regeneration, obtaining cuprous oxide nanoparticles;

[0069] (4) Disperse 1.0 g of sodium hydroxide in 40 mL of N-methylpyrrolidone, ultrasonically treat for 30 min, then add black phosphorus powder, and the addition amount of black phosphorus powder is 15.0 mg. The biodegradation product of black phosphorus can be transformed into calcium phosphate nanoparticles through self-mineralization, which has natural affinity with hydroxyapatite in bone tissue, thereby promoting the enrichment of black phosphorus in bone tissue, showing a certain bone-targeting effect. Then ultrasonically treat for 9 h in an ice bath environment, centrifuge the suspension twice, collect the supernatant, continue to centrifuge three times, collect the precipitate, and freeze-dry to obtain black phosphorus nanosheets. Disperse the cuprous oxide nanoparticles described in step (3) in 10 mL of ultrapure water, then add black phosphorus nanosheets, and magnetically stir for 2.5 h. Using electrostatic interaction, adsorb the positively charged cuprous oxide nanoparticles onto the surface of the negatively charged black phosphorus nanosheets to form a composite material with anti-inflammatory and osteogenic promotion effects. In the inflammatory and slightly acidic environment of osteoporosis, the cuprous oxide nanoparticles are decomposed into Cu 2+ and hydroxyl radicals, where Cu 2+ can produce a certain degree of osteogenic promotion effect on osteoblasts, and hydroxyl radicals can accelerate the degradation of black phosphorus nanosheets in the light-free environment in vivo, thereby undergoing self-mineralization to produce calcium phosphate nanoparticles. Collect the solid matter to obtain cuprous oxide-black phosphorus composite particles;

[0070] (5) Disperse the ZIF-8 nanoparticles loaded with bone collagen peptide described in step (2) in 20 mL of ultrapure water, add the cuprous oxide-black phosphorus composite particles described in step (4), magnetically stir for 4 h, collect the precipitate, and attach the cuprous oxide-black phosphorus composite particles to the surface of the ZIF-8 nanoparticles loaded with bone collagen peptide to form a protective layer, increasing the stability of the ZIF-8 nanoparticles loaded with bone collagen peptide, improving the biocompatibility and bone-targeting performance of the nanoparticles, further enhancing the effective release of bone collagen peptide, and being beneficial to improving the treatment effect of osteoporosis to obtain targeted metal composite nanoparticles loaded with bone collagen peptide.

[0071] This example provides a preparation method for an osteoporosis-repairing bone peptide composition, specifically including the following steps:

[0072] S1. Dissolve 100.0 mg of chitosan in 5 mL of a 30% (mass fraction) aqueous dimethyl sulfoxide solution, adjust the pH to 5.0, and stir until completely dissolved for later use. Chitosan can enhance the activity of alkaline phosphatase, accelerate bone formation, inhibit the activity of osteoclasts, reduce bone resorption, and also enhance bone density by promoting calcium absorption. Then dissolve chlorogenic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide in 5 mL of dimethyl sulfoxide and stir for activation for 1.5 h. The addition amount of chlorogenic acid is 174.0 mg, the addition amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 0.1 g, and the addition amount of N-hydroxysuccinimide is 80.0 mg. Chlorogenic acid is not only used as a cross-linking agent but also can maintain bone metabolism balance by regulating bone metabolism-related hormones (such as estrogen and parathyroid hormone), which helps prevent and treat osteoporosis. Then mix the activated chitosan solution and chlorogenic acid solution and stir overnight. Transfer it to a dialysis bag, first dialyze with an acidic buffer solution of pH 5.0, change the buffer solution every 12 h, and on the third day, change to ultrapure water of pH 7.0 for dialysis for 3.5 h. Collect the reaction product and perform freeze-drying. The carboxyl group of chlorogenic acid reacts with the amino group of chitosan to synthesize a non-toxic biocompatible cross-linking agent with a catechol structure, effectively improving the disadvantages of chlorogenic acid being unstable and having poor water solubility. Among them, the catechol structure can mimic the role of estrogen, which is beneficial to maintaining bone density and can also show high reactivity in the cross-linking reaction to obtain a biocompatible cross-linking agent;

[0073] S2. Dissolve 1.5 g of sodium alginate in 100 mL of ultrapure water, stir evenly, add 0.3 g of gold nanorods. The addition of gold nanorods can form a large, coherent network structure with a high degree of cross-linking, which can accurately deliver active substances to bone tissue, improve the curative effect and reduce side effects. Stir magnetically for 1.5 h, then add the biocompatible cross-linking agent described in step S1 and mix evenly by vibration. Under the action of the biocompatible cross-linking agent and gold nanorods, the loading capacity and stability of the delivery matrix are improved, and it also has an auxiliary effect of promoting bone formation and inhibiting bone absorption, further improving the stability, release effectiveness, and therapeutic effect of a single active substance to obtain an injectable gel delivery matrix;

[0074] S3. Add the targeted metal composite nanoparticles loaded with bone collagen peptide into the injectable gel delivery matrix described in step S2, and ultrasonically treat for 2.5 h. Then, use a portable electrospray device to spray it into 100 mL of calcium chloride solution with a mass fraction of 1.5%, and let it stand for 7 h. Then, rinse the gel product 4 times with ultrapure water. Through this process, a tightly complex porous microsphere structure is prepared. The pores of the microspheres adsorb the targeted metal composite nanoparticles loaded with bone collagen peptide, effectively protecting the stability of bone collagen peptide and reducing the inactivation risk caused by the external environment. Also, in the form of slow release and targeted release, it significantly improves the bioavailability of bone collagen peptide. At the same time, by synergistically acting with other components, it exerts a therapeutic effect on osteoporosis in multiple ways and obtains excellent therapeutic effects, resulting in an osteopeptide composition for osteoporosis repair.

[0075] Example 4

[0076] This example presents an osteopeptide composition for osteoporosis repair, including the following components in parts by weight: 60 parts of targeted metal composite nanoparticles loaded with bone collagen peptide, 20 parts of injectable gel delivery matrix, and 20 parts of calcium chloride.

[0077] The targeted metal composite nanoparticles loaded with bone collagen peptide include the following components in parts by weight: 30 parts of tuna bone collagen peptide, 20 parts of ZIF-8 nanoparticles, and 8 parts of copper peroxide black phosphorus composite particles.

[0078] The injectable gel delivery matrix includes the following components in parts by weight: 10 parts of biocrosslinker, 1 part of gold nanorods, and 20 parts of sodium alginate.

[0079] The preparation method of the targeted metal composite nanoparticles loaded with bone collagen peptide specifically includes the following steps:

[0080] (1) Add 9.0 g of tuna bone meal to 100 mL of water, and add animal protease. The addition amount of animal protease is 2.5% of the added amount of tuna bone meal, and the enzyme activity is 300,000 U / g. Animal protease can decompose macromolecular proteins such as collagen in bone tissue into small peptides and amino acids, improve the extraction rate and bioavailability of bone peptides. Enzymolysis is carried out at pH 7.0 and a temperature of 55 °C for 6 h. After the enzymolysis is completed, inactivate the enzyme at 90 °C for 30 min, then centrifuge at 8000 rpm for 10 min, take the supernatant, and use membrane separation method to intercept the enzymolysis solution, collect bone collagen peptides with a molecular weight of 250 - 1000 Da, and freeze-dry. The low-molecular-weight tuna bone collagen peptides can significantly improve the activities of alkaline phosphatase and osteocalcin of osteoblasts, thus facilitating the differentiation of osteoblasts, promoting the secretion of type I collagen, the main component of bone organic matrix, and thus contributing to the formation of bone matrix. Therefore, the prepared tuna bone collagen peptides have good ability to promote the proliferation, differentiation and mineralization of osteoblasts, and obtain tuna bone collagen peptides;

[0081] (2) Add 2.0 g of zinc nitrate hexahydrate to 20 mL of deionized water to obtain a zinc nitrate hexahydrate solution for later use. Then add 8.0 g of 2-methylimidazole to 40 mL of deionized water, and then add the tuna bone collagen peptides described in step (1). Stir at 80 rpm for 5 min. Quickly drip the zinc nitrate hexahydrate solution into the 2-methylimidazole solution containing tuna bone collagen peptides, and stir at 800 rpm for 20 min. Collect the suspension by centrifugation, wash the precipitate 5 times with deionized water. The porous structure and pH sensitivity of ZIF-8 make it an ideal drug delivery carrier. It can encapsulate active substances through its pore structure, protect the stability of tuna bone collagen peptides, reduce the risk of inactivation or degradation in the external environment, and achieve slow release of drugs in a weakly acidic environment, improve the bioavailability of tuna bone collagen peptides. And ZIF-8 is stable under physiological conditions, has low toxicity to cells and tissues, and can also inhibit the destruction of bone tissue by inflammatory factors by releasing zinc ions, and obtain ZIF-8 nanoparticles loaded with bone collagen peptides;

[0082] (3) Add 0.5 g of polyvinylpyrrolidone to 5 mL of a 0.2% copper(II) chloride dihydrate solution, dissolve it by ultrasonic wave and stir evenly, then add 5 mL of a 0.08% sodium hydroxide solution, stir evenly by magnetic force, and then slowly drop 10 μL of 30% hydrogen peroxide solution, stir for 20 min. Carry out ultra-high-speed centrifugation on the reaction mixture, the centrifugation speed is 6000 rpm, and the centrifugation time is 10 min. Collect the precipitate, wash it 5 times with ultrapure water, and freeze-dry. Metal peroxides can react with excessive hydrogen ions in osteoporotic bone tissue, and at the same time, the released copper ions help to induce and stimulate osteoblasts, which is beneficial to bone mineralization and bone regeneration, and obtain cupric peroxide nanoparticles;

[0083] (4) Disperse 1.0 g of sodium hydroxide in 40 mL of N-methylpyrrolidone, ultrasonically treat for 30 min, then add black phosphorus powder, and the addition amount of black phosphorus powder is 10.0 mg. The biodegradation product of black phosphorus can be transformed into calcium phosphate nanoparticles through self-mineralization, which has natural affinity with hydroxyapatite in bone tissue, thereby promoting the enrichment of black phosphorus in bone tissue, showing a certain bone-targeting effect. Then ultrasonically treat for 8 h in an ice bath environment, centrifuge the suspension 3 times, collect the supernatant, continue to centrifuge 4 times, collect the precipitate, and freeze-dry to obtain black phosphorus nanosheets. Disperse the cuprous oxide nanoparticles described in step (3) in 10 mL of ultrapure water, then add black phosphorus nanosheets, and magnetically stir for 2 h. Using electrostatic interaction, the positively charged cuprous oxide nanoparticles are adsorbed onto the surface of the negatively charged black phosphorus nanosheets to form a composite material with anti-inflammatory and osteogenic promotion effects. In the inflammatory and slightly acidic environment of osteoporosis, the cuprous oxide nanoparticles are decomposed into Cu 2+ and hydroxyl radicals, where Cu 2+ can produce a certain degree of osteogenic promotion effect on osteoblasts, and the hydroxyl radicals can accelerate the degradation of black phosphorus nanosheets in the light-free environment in vivo, thereby undergoing self-mineralization to produce calcium phosphate nanoparticles. Collect the solid substance to obtain cuprous oxide-black phosphorus composite particles;

[0084] (5) Disperse the ZIF-8 nanoparticles loaded with bone collagen peptide described in step (2) in 20 mL of ultrapure water, add the cuprous oxide-black phosphorus composite particles described in step (4), magnetically stir for 3 h, collect the precipitate, and attach the cuprous oxide-black phosphorus composite particles to the surface of the ZIF-8 nanoparticles loaded with bone collagen peptide to form a protective layer, which increases the stability of the ZIF-8 nanoparticles loaded with bone collagen peptide, improves the biocompatibility and bone-targeting performance of the nanoparticles, further enhances the effective release of bone collagen peptide, and is beneficial to improving the treatment effect of osteoporosis, to obtain targeted metal composite nanoparticles loaded with bone collagen peptide.

[0085] This example provides a preparation method for an osteoporosis repair-type bone peptide composition, which specifically includes the following steps:

[0086] S1. Dissolve 100.0 mg of chitosan in 5 mL of a 30% (mass fraction) aqueous dimethyl sulfoxide solution, adjust the pH to 5.0, and stir until completely dissolved for later use. Chitosan can enhance the activity of alkaline phosphatase, accelerate bone formation, inhibit the activity of osteoclasts, reduce bone resorption, and also enhance bone density by promoting calcium absorption. Then, dissolve chlorogenic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide in 5 mL of dimethyl sulfoxide and stir for activation for 1 h. The addition amount of chlorogenic acid is 178.0 mg, the addition amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 0.1 g, and the addition amount of N-hydroxysuccinimide is 80.0 mg. Chlorogenic acid is not only used as a cross-linking agent but also can maintain bone metabolism balance by regulating bone metabolism-related hormones (such as estrogen and parathyroid hormone), which helps prevent and treat osteoporosis. Then, mix the activated chitosan solution and chlorogenic acid solution, stir overnight, transfer to a dialysis bag, first dialyze with an acidic buffer solution of pH 5.0, change the buffer solution every 12 h, and on the third day, change to ultrapure water of pH 7.0 for dialysis for 3 h. Collect the reaction product and perform freeze-drying. The carboxyl group of chlorogenic acid reacts with the amino group of chitosan to synthesize a non-toxic biocrosslinker with a catechol structure, effectively improving the disadvantages of chlorogenic acid being unstable and having poor water solubility. Among them, the catechol structure can mimic the action of estrogen, which is beneficial to maintaining bone density and can also show high reactivity in the cross-linking reaction to obtain a biocrosslinker;

[0087] S2. Dissolve 2.0 g of sodium alginate in 100 mL of ultrapure water, stir evenly, add 0.1 g of gold nanorods. The addition of gold nanorods can form a large, coherent network structure with a high degree of cross-linking, which can accurately deliver active substances to bone tissue, improve the curative effect and reduce side effects. Stir magnetically for 1 h, then add the biocrosslinker described in step S1, and mix evenly by vibration. Under the action of the biocrosslinker and gold nanorods, the loading capacity and stability of the delivery matrix are improved, and it also has an auxiliary effect of promoting bone formation and inhibiting bone resorption, further improving the stability, release effectiveness, and therapeutic effect of a single active substance to obtain an injectable gel delivery matrix;

[0088] S3. Add the targeted metal composite nanoparticles loaded with bone collagen peptides into the injectable gel delivery matrix described in step S2, ultrasonically treat for 2 h, then spray it into 100 mL of a calcium chloride solution with a mass fraction of 2% using a portable electrospray device, let it stand for 6 h, and then rinse the gel product 5 times with ultrapure water. Through this process, a tightly complex porous microsphere structure is prepared. The pores of the microspheres adsorb the targeted metal composite nanoparticles loaded with bone collagen peptides, effectively protecting the stability of bone collagen peptides and reducing the inactivation risk caused by the external environment. It also significantly improves the bioavailability of bone collagen peptides in a slow-release and targeted-release mode of action. At the same time, by synergistically acting with other components, it plays a therapeutic role in osteoporosis from multiple aspects and obtains excellent therapeutic effects, resulting in an osteopeptide composition for osteoporosis repair.

[0089] Comparative Example 1

[0090] This comparative example provides an osteopeptide composition for osteoporosis repair. The difference from Example 1 is that the targeted metal composite nanoparticles loaded with bone collagen peptides do not contain cupric peroxide black phosphorus composite particles; the preparation method of the targeted metal composite nanoparticles loaded with bone collagen peptides does not include steps (3), (4), and (5); the preparation method of the osteopeptide composition for osteoporosis repair is the same as that of Example 1.

[0091] Comparative Example 2

[0092] This comparative example provides an osteopeptide composition for osteoporosis repair. The difference from Example 1 is that the injectable gel delivery matrix does not contain a biological crosslinking agent and gold nanorods; the preparation method of the targeted metal composite nanoparticles loaded with bone collagen peptides is the same as that of Example 1; the preparation method of the osteopeptide composition for osteoporosis repair does not include step S1 and gold nanorods are not added in step S2.

[0093] Comparative Example 3

[0094] This comparative example provides an osteopeptide composition for osteoporosis repair. The difference from Example 1 is that the osteopeptide composition for osteoporosis repair does not contain cupric peroxide black phosphorus composite particles, biological crosslinking agent, and gold nanorods; the preparation method of the targeted metal composite nanoparticles loaded with bone collagen peptides does not include steps (3), (4), and (5); the preparation method of the osteopeptide composition for osteoporosis repair does not include step S1 and gold nanorods are not added in step S2.

[0095] Experimental Example 1

[0096] Encapsulation efficiency experiment

[0097] Test samples: The osteopeptide compositions for osteoporosis repair prepared in Examples 1-4 and Comparative Examples 1-3.

[0098] Test method: Weigh 20 mg of the test sample, dissolve it in 2 mL of methanol to break the emulsion, add 10 mL of deionized water, centrifuge at 5000 rpm for 3 min, and detect the concentration of tuna bone collagen peptide in the supernatant using a MicroBCA protein concentration assay kit. The encapsulation efficiency formula for tuna bone collagen peptide is as follows:

[0099] Encapsulation efficiency (%) = (Total amount of tuna bone collagen peptide - Free amount of tuna bone collagen peptide) / Total amount of tuna bone collagen peptide × 100%

[0100] Figure 2 It is a graph of the encapsulation efficiency results for Examples 1-4 and Comparative Examples 1-3; as shown in the figure, the encapsulation efficiency of Examples 1-4 is 82.1 - 85.8%, indicating a relatively high encapsulation efficiency, effectively reducing the influence of external inactivation and degradation; the encapsulation efficiency of Comparative Examples 1-3 is 58.5 - 71.8%, indicating a relatively low encapsulation efficiency, unable to effectively reduce the influence of external inactivation and degradation; the targeted metal composite nanoparticles loaded with bone collagen peptide in Comparative Example 1 do not contain cupric oxide black phosphorus composite particles and cannot attach to the surface of the ZIF-8 nanoparticles loaded with bone collagen peptide to form a protective layer, resulting in a low encapsulation efficiency and being unable to effectively reduce the influence of external inactivation and degradation; the injectable gel delivery matrix in Comparative Example 2 does not contain a biocrosslinking agent and gold nanorods and cannot form a highly crosslinked large and tightly coherent network structure, which is not conducive to improving the encapsulation amount and encapsulation stability of the targeted metal composite nanoparticles loaded with bone collagen peptide by the matrix, resulting in a low encapsulation efficiency and being unable to effectively reduce the influence of external inactivation and degradation; the osteoporotic repair-type bone peptide composition in Comparative Example 3 does not contain cupric oxide black phosphorus composite particles, a biocrosslinking agent, and gold nanorods, only has the encapsulation effect of ZIF-8 nanoparticles, and at the same time, the injectable gel delivery matrix cannot adsorb the ZIF-8 nanoparticles loaded with bone collagen peptide more and more stably, resulting in a low encapsulation efficiency and being unable to effectively reduce the influence of external inactivation and degradation.

[0101] Experimental Example 2

[0102] Allergy reaction experiment

[0103] Test samples: Osteoporotic repair-type bone peptide compositions prepared in Examples 1-4 and Comparative Examples 1-3.

[0104] Test method: Select 70 male SD rats, each with a body weight of 220 g (purchased from Chengdu Dashuo Animal Co., Ltd.), divide them into groups of 10 each, randomly divide them into 7 groups, raise the rats in separate cages, keep the cages clean and dry, at a temperature of 24 °C and a humidity of 50%, allow free diet and water, and conduct feeding; after 10 days of feeding, administer 4 mg of the test sample by injection once a day for 20 days, and then observe and record the allergy rate (%) of the rats.

[0105] Figure 3Graph of the allergy rate results for Examples 1-4 and Comparative Examples 1-3; As shown in the figure, the allergy rate of Examples 1-4 is 10-20%, indicating fewer adverse reactions; the allergy rate of Comparative Examples 1-3 is 60-90%, indicating more adverse reactions; the targeted metal composite nanoparticles loaded with bone collagen peptide in Comparative Example 1 do not contain cupric peroxide black phosphorus composite particles, and cannot form a protection on the surface of the ZIF-8 nanoparticles loaded with bone collagen peptide, which is not conducive to improving its encapsulation effect and biocompatibility, resulting in more adverse reactions; the injectable gel delivery matrix in Comparative Example 2 does not contain a biocrosslinking agent and gold nanorods, and the delivery matrix cannot form a highly crosslinked, tightly coherent network structure, which is not conducive to the slow release of the targeted metal composite nanoparticles loaded with bone collagen peptide in terms of structure, nor is it conducive to its encapsulation stability, resulting in more adverse reactions; the osteoporotic repair type bone peptide composition in Comparative Example 3 does not contain cupric peroxide black phosphorus composite particles, a biocrosslinking agent and gold nanorods, which is neither conducive to the encapsulation effect of the active substance bone collagen peptide, nor can it reduce the irritation of bone collagen peptide in a slow release mode of action, resulting in more adverse reactions.

[0106] Experimental Example 3

[0107] Therapeutic effect experiment

[0108] Test samples: Osteoporotic repair type bone peptide compositions prepared in Examples 1-4 and Comparative Examples 1-3.

[0109] Test method: Select 140 patients with osteoporosis of different degrees, specifically manifested as symptoms such as easy fatigue, bone pain, difficulty breathing, loose teeth, hunchback and easy fractures, aged 50-75 years. The random number table method is used to determine the enrollment of patients, and they are divided into 7 groups, with 20 patients in each group. Each group uses the corresponding test sample, and the usage method is to inject the test sample once a day. Observe the effect after 7 days. At the same time, judge the therapeutic effect according to the following criteria, and calculate the treatment effective rate (%), specifically:

[0110] Effective: The symptoms of osteoporosis completely disappear, and the symptoms of osteoporosis change from severe to moderate, from moderate to mild, and from mild to normal;

[0111] Invalid: The symptoms of osteoporosis have not improved significantly.

[0112] The calculation formula for the treatment effective rate is as follows:

[0113] Treatment effective rate (%) = Number of effective people / 20 × 100%

[0114] Figure 4Graph of the treatment effective rates of Examples 1-4 and Comparative Examples 1-3; as shown in the figure, the treatment effective rates of Examples 1-4 are 85-95%, indicating better treatment effects; the treatment effective rates of Comparative Examples 1-3 are 55-70%, indicating poor treatment effects; the targeted metal composite nanoparticles loaded with bone collagen peptide in Comparative Example 1 do not contain cupric oxide black phosphorus composite particles and cannot form a protection on the surface of the ZIF-8 nanoparticles loaded with bone collagen peptide, which is not conducive to the stability of bone collagen peptide. Only relying on the targeting effect of the injectable gel delivery matrix reduces the precision of delivering active substances, resulting in poor treatment effects; the injectable gel delivery matrix in Comparative Example 2 does not contain a biocrosslinker and gold nanorods and cannot encapsulate more and more stably the targeted metal composite nanoparticles loaded with bone collagen peptide to achieve precise targeted delivery in bone tissue, resulting in poor treatment effects; the osteoporosis repair type bone peptide composition in Comparative Example 3 does not contain cupric oxide black phosphorus composite particles, a biocrosslinker and gold nanorods, does not have a targeting effect, cannot achieve targeted delivery of active substances, and is not conducive to maintaining the stability of bone collagen peptide, resulting in poor treatment effects.

[0115] The above experimental results show that the stability, adverse reactions and treatment effects of Examples 1-4 of the present invention are significantly better than those of the samples of Comparative Examples 1-3. Among them, Example 1 using the targeted metal composite nanoparticles loaded with bone collagen peptide and the injectable gel delivery matrix has better stability, fewer adverse reactions and better treatment effects. The tuna bone collagen peptide is encapsulated in the ZIF-8 nanoparticles, and a protective layer is formed by adsorbing cupric oxide black phosphorus composite particles on the surface, which enhances the encapsulation effect, reduces the inactivation and degradation of tuna bone collagen peptide by the external environment, and at the same time endows bone targeting and good biocompatibility. Then, the targeted metal composite nanoparticles loaded with bone collagen peptide are attached to the complex network of the injectable gel delivery matrix to form a microsphere structure, effectively protecting the stability of tuna bone collagen peptide. In the form of slow release and targeted release, the bioavailability of tuna bone collagen peptide is significantly improved, reducing both the adverse reactions caused by multiple administrations and obtaining an efficient treatment effect.

[0116] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made in these embodiments without departing from the principles and spirit of the present invention.

[0117] The present invention and its embodiments have been described above. Such description is not restrictive. What is shown in the drawings is only one of the embodiments of the present invention, and the actual application is not limited thereto. Generally speaking, if those of ordinary skill in the art are inspired by it and design in a non-creative way a similar way and embodiments to this technical solution without departing from the purpose of the present invention, they shall fall within the protection scope of the present invention.

Claims

1. A osteopeptide composition for repairing osteoporosis, characterized in that: The osteoporosis repairing bone peptide composition comprises the following components in parts by weight: 50-60 parts of targeted metal composite nanoparticles loaded with collagen peptide, 20-30 parts of injectable gel delivery matrix, and 10-20 parts of calcium chloride; the targeted metal composite nanoparticles loaded with collagen peptide comprise the following components in parts by weight: 20-30 parts of tuna collagen peptide, 10-20 parts of ZIF-8 nanoparticles, and 8-10 parts of copper peroxide black phosphorus composite particles; the injectable gel delivery matrix comprises the following components in parts by weight: 6-10 parts of a biocrosslinking agent, 1-5 parts of gold nanorods, and 10-20 parts of sodium alginate.

2. A method for preparing the osteoporosis repairing bone peptide composition according to claim 1, characterized in that: The specific steps include: S1. Dissolve 100.0 mg chitosan in 5 mL of a 30% aqueous solution of dimethyl sulfoxide by mass, adjust the pH to 5.0, stir until completely dissolved, and set aside. Dissolve chlorogenic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide in 5 mL of dimethyl sulfoxide, stir and activate for 1-2 hours, then mix the activated chitosan solution and chlorogenic acid solution, stir overnight, transfer to a dialysis bag, and dialyze with an acidic buffer solution of pH 5.0 first. Replace the buffer solution every 12 hours. On the third day, replace it with ultrapure water of pH 7.0 for dialysis for 3-4 hours. Collect the reaction product and freeze-dry it to obtain a biocrosslinking agent. S2, dissolving 1.0-2.0 g of sodium alginate in 100 mL of ultrapure water, stirring evenly, adding gold nanorods, magnetically stirring for 1-2 h, and then adding the biocrosslinking agent described in step S1, vibrating and mixing to obtain an injectable gel delivery matrix; S3. Add the targeted metal composite nanoparticles loaded with collagen peptides to the injectable gel delivery matrix described in step S2, ultrasonically treat for 2-3 hours, and then use a portable electrospray device to spray it into 100 mL of a 1-2% calcium chloride solution, let it stand for 6-8 hours, and then rinse the gel product with ultrapure water for 3-5 times to obtain a bone peptide composition for repairing osteoporosis.

3. The method for preparing the osteoporosis repairing bone peptide composition according to claim 2, characterized in that: In step S1, the amount of chlorogenic acid added is 170.0-178.0 mg, the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide added is 0.1 g, and the amount of N-hydroxysuccinimide added is 80.0 mg.

4. The method for preparing the osteoporosis repairing bone peptide composition according to claim 3, characterized in that: In step S2, the amount of gold nanorods added is 0.1-0.5 g.

5. The method for preparing the osteoporosis repairing bone peptide composition according to claim 4, characterized in that: The method for preparing the targeted metal composite nanoparticles loaded with collagen peptide specifically comprises the following steps: (1) Add 8.0-9.0 g of tuna bone powder to 100 mL of water, add animal protease, perform enzymolysis at pH 7.0 and temperature 50-55° C. for 6-8 hours, inactivate the enzyme at 90° C. for 30 minutes, and then perform refrigerated centrifugation at 6000-8000 rpm for 10-15 minutes, take the supernatant, intercept the enzymolysis solution by membrane separation, collect collagen peptides with a molecular weight of 250-1000 Da, and freeze-dry to obtain tuna collagen peptides; (2) Add 2.0 g of zinc nitrate hexahydrate to 20 mL of deionized water to obtain a zinc nitrate hexahydrate solution for standby use, then add 8.0 g of 2-methylimidazole to 40 mL of deionized water, then add the tuna collagen peptide described in step (1), stir at 60-80 rpm for 5-10 min, quickly drop the zinc nitrate hexahydrate solution into the 2-methylimidazole solution containing tuna collagen peptide, stir at 600-800 rpm for 20-30 min, collect the suspension by centrifugation, and wash the precipitate with deionized water for 3-5 times to obtain ZIF-8 nanoparticles loaded with collagen peptide; (3) Add 0.5 g of polyvinyl pyrrolidone to 5 mL of 0.1-0.2% copper chloride dihydrate solution, dissolve and stir evenly by ultrasonication, then add 5 mL of 0.08% sodium hydroxide solution, stir evenly by magnetic force, then slowly drip 10 μL of 30% hydrogen peroxide, stir for 20-30 min, subject the reaction mixture to ultra-high speed centrifugation at a speed of 5000-6000 rpm for 10-20 min, collect the precipitate, wash with ultrapure water for 3-5 times, and freeze-dry to obtain copper peroxide nanoparticles; (4) Dispersing 1.0 g of sodium hydroxide in 40 mL of N-methylpyrrolidone, ultrasonically treating for 30 min, then adding black phosphorus powder, and ultrasonically treating in an ice bath for 8-10 h, centrifuging the suspension 2-3 times, collecting the supernatant, continuing to centrifuge for 3-4 times, collecting the precipitate, and freeze-drying to obtain black phosphorus nanosheets, dispersing the copper peroxide nanoparticles described in step (3) in 10 mL of ultrapure water, then adding the black phosphorus nanosheets, magnetically stirring for 2-3 h, and collecting the solid matter to obtain copper peroxide black phosphorus composite particles; (5) Disperse the ZIF-8 nanoparticles loaded with collagen peptide described in step (2) in 20 mL of ultrapure water, add the copper peroxide black phosphorus composite particles described in step (4), stir magnetically for 3-5 hours, collect the precipitate, and obtain targeted metal composite nanoparticles loaded with collagen peptide.

6. The method for preparing the osteoporosis repairing bone peptide composition according to claim 5, characterized in that: In step (1), the amount of animal protease added is 1.5-2.5% of the amount of tuna bone powder added, and the enzyme activity is 300,000 U / g.

7. The method for preparing the osteoporosis repairing bone peptide composition according to claim 6, characterized in that: In step (4), the amount of black phosphorus powder added is 10.0-20.0 mg.

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