A kind of osteopeptide composition for repairing osteoporosis and preparation method thereof

Abstract

The invention discloses an osteoporosis-repairing bone peptide composition and a preparation method thereof, relating to the technical field of biological preparations. The osteoporosis-repairing bone peptide composition comprises targeted metal composite nanoparticles loaded with bone collagen peptide, an injectable gel delivery matrix, and calcium chloride. Tuna collagen peptide is encapsulated in ZIF-8 nanoparticles, and copper peroxide-black phosphorus composite particles are adsorbed on the surface to enhance the encapsulation effect, reduce the inactivation and degradation effects of the external environment on the tuna collagen peptide, and also impart bone targeting and good biocompatibility. The targeted metal composite nanoparticles loaded with bone collagen peptide are then attached to the injectable gel delivery matrix to form a microsphere structure, effectively protecting the stability of the tuna collagen peptide. The slow release and targeted release modes of action reduce adverse reactions and improve the therapeutic effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological preparations, and specifically relates to an osteoporosis repairing osteopeptide composition and a preparation method thereof. Background Art

[0002] Osteoporosis is a metabolic bone disease that is common in the elderly and menopausal women. It is mainly manifested by decreased bone mass, bone density and quality, resulting in damage to bone microstructure, increased bone brittleness, increased risk of fractures, and in severe cases, bone necrosis. It is generally believed that osteoporosis is caused by an imbalance in bone metabolism due to decreased bone formation and increased bone resorption. Osteoblasts participate in the dynamic process of bone formation and are the most critical cells in the bone reconstruction process. They can secrete a variety of 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 reconstruction are the key to preventing osteoporosis and promoting bone health.

[0003] Conventional drug treatments inhibit osteoclastogenesis or promote osteogenesis through antiresorptive agents, anabolic agents, and selective estrogen receptor modulators to maintain bone homeostasis. However, long-term use of antiresorptive drugs can inhibit bone turnover, impair natural bone repair, produce significant adverse reactions, and affect compliance. Therefore, the search for safe and effective bioactive peptides that can regulate bone metabolism has become a research hotspot.

[0004] The existing technology currently has 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 prone to cause adverse reactions. Summary of the Invention

[0006] In view of the above situation, in order to overcome the defects of the prior art, the present invention proposes a bone peptide composition for repairing osteoporosis, comprising the following components by weight: 50-60 parts of targeted metal composite nanoparticles loaded with collagen peptides, 20-30 parts of injectable gel delivery matrix, and 10-20 parts of calcium chloride.

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

[0008] 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.

[0009] The method for preparing the targeted metal composite nanoparticles loaded with collagen peptide specifically comprises the following steps:

[0010] (1) 8.0-9.0 g of tuna bone powder was added to 100 mL of water, and animal protease was added. The mixture was enzymolyzed at pH 7.0 and 50-55 ° C for 6-8 h. After the enzymolysis was completed, the enzyme was inactivated at 90 ° C for 30 min, and then the mixture was refrigerated and centrifuged at 6000-8000 rpm for 10-15 min. The supernatant was collected and the enzymolysis solution was intercepted by membrane separation. Bone collagen peptides with a molecular weight of 250-1000 Da were collected and freeze-dried. The low molecular weight tuna collagen peptides can significantly increase the activity of alkaline phosphatase and osteocalcin in osteoblasts, thereby promoting the differentiation of osteoblasts, and can promote the secretion of collagen type I, the main component of bone organic matrix, thereby contributing to the formation of bone matrix. Therefore, the prepared tuna collagen peptide has a good ability to promote osteoblast proliferation, differentiation and mineralization, and tuna collagen peptides are obtained;

[0011] (2) Add 2.0 g of zinc nitrate hexahydrate to 20 mL of deionized water to obtain a zinc nitrate hexahydrate solution, which is set aside. Then, add 8.0 g of 2-methylimidazole to 40 mL of deionized water, and 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. 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 collagen peptides, reduce the risk of inactivation or degradation in the external environment, and achieve sustained release of drugs in a weakly acidic environment, thereby improving the bioavailability of tuna collagen peptides. In addition, ZIF-8 is stable under physiological conditions and has low toxicity to cells and tissues. It can also inhibit the damage of inflammatory factors to bone tissue by releasing zinc ions, thereby obtaining ZIF-8 nanoparticles loaded with collagen peptides.

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

[0013] (4) 1.0 g of sodium hydroxide was dispersed in 40 mL of N-methylpyrrolidone and ultrasonically treated for 30 min. Black phosphorus powder was then added and ultrasonically treated in an ice bath for 8-10 h. The suspension was centrifuged 2-3 times, the supernatant was collected, and the centrifugation was continued for 3-4 times. The precipitate was collected and freeze-dried to obtain black phosphorus nanosheets. The copper peroxide nanoparticles described in step (3) were dispersed in 10 mL of ultrapure water, and then black phosphorus nanosheets were added and magnetically stirred for 2-3 h. The positively charged copper peroxide nanoparticles were adsorbed onto the negatively charged surface of the black phosphorus nanosheets by electrostatic interaction, forming a composite material with anti-inflammatory and osteogenic effects. In the inflammatory and slightly acidic environment of osteoporosis, the copper peroxide nanoparticles were decomposed into Cu 2+ With hydroxyl radicals, Cu 2+ It can produce a certain degree of osteogenic effect on osteoblasts, and hydroxyl radicals can accelerate the degradation of black phosphorus nanosheets in the body under a lightless environment, thereby causing self-mineralization to produce calcium phosphate nanoparticles, and collecting solid substances to obtain copper peroxide black phosphorus composite particles;

[0014] (5) The ZIF-8 nanoparticles loaded with collagen peptide described in step (2) are dispersed in 20 mL of ultrapure water, and the copper peroxide black phosphorus composite particles described in step (4) are added, and magnetic stirring is carried out for 3-5 hours. The precipitate is collected, and the copper peroxide black phosphorus composite particles are attached to the surface of the ZIF-8 nanoparticles loaded with collagen peptide to form a protective layer, thereby increasing the stability of the ZIF-8 nanoparticles loaded with collagen peptide, and also improving the biocompatibility and bone targeting performance of the nanoparticles, further enhancing the effective release of collagen peptide, and being beneficial to improving the therapeutic effect of osteoporosis, thereby obtaining targeted metal composite nanoparticles loaded with collagen peptide;

[0015] Preferably, 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. Animal protease can decompose macromolecular proteins such as collagen in bone tissue into small molecular peptides and amino acids, thereby improving the extraction rate and bioavailability of bone peptides;

[0016] Preferably, in step (4), the amount of black phosphorus powder added is 10.0-20.0 mg. The biodegradation products of black phosphorus can be converted into calcium phosphate nanoparticles through self-mineralization, which have a 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 method for preparing an osteoporosis repairing osteopeptide composition, which specifically comprises the following steps:

[0018] S1. Dissolve 100.0 mg of chitosan in 5 mL of a 30% aqueous solution of dimethyl sulfoxide, adjust the pH to 5.0, stir until completely dissolved, and set aside. Chitosan can increase the activity of alkaline phosphatase, accelerate bone formation, inhibit osteoclast activity, reduce bone resorption, and enhance bone density by promoting calcium absorption. Dissolve chlorogenic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide in 5 mL of dimethyl sulfoxide, stir and activate for 1-2 hours, and then mix the activated chitosan solution with the chlorogenic acid solution. , stirred overnight, transferred to a dialysis bag, first dialyzed with an acidic buffer solution of pH 5.0, and the buffer solution was replaced every 12 hours. On the third day, it was replaced with ultrapure water of pH 7.0 for dialysis for 3-4 hours. The reaction product was collected and freeze-dried. The carboxyl group of chlorogenic acid reacted with the amino group of chitosan to synthesize a non-toxic bio-crosslinking agent with a catechol structure, which effectively improved the shortcomings of chlorogenic acid such as instability and poor water solubility. Among them, the catechol structure can simulate the effect of estrogen, which is beneficial to maintaining bone density, and can also show high reactivity in the cross-linking reaction to obtain a bio-crosslinking agent;

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

[0020] 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 spray it into 100 mL of a 1-2% calcium chloride solution using a portable electrospray device. Let it stand for 6-8 hours, and then rinse the gel product with ultrapure water 3-5 times. A compact and complex porous microsphere structure is prepared by this process. The targeted metal composite nanoparticles loaded with collagen peptides are adsorbed in the pores of the microspheres, effectively protecting the stability of the collagen peptides and reducing the risk of inactivation caused by the external environment. The slow release and targeted release modes of action significantly improve the bioavailability of the collagen peptides. At the same time, the composition synergizes with other components to achieve a multi-pronged therapeutic effect on osteoporosis, thereby achieving excellent therapeutic effects and obtaining a bone peptide composition for osteoporosis repair.

[0021] Preferably, 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. 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), thereby helping to prevent and treat osteoporosis.

[0022] Preferably, in step S2, the amount of gold nanorods added is 0.1-0.5 g. 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 efficacy and reduce side effects.

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

[0024] The present invention encapsulates tuna collagen peptide in ZIF-8 nanoparticles, and then adsorbs copper peroxide black phosphorus composite particles on the surface to form a protective layer, which further enhances the encapsulation effect, reduces the inactivation and degradation of the tuna collagen peptide by the external environment, and also gives it bone targeting and good biocompatibility. Then, the targeted metal composite nanoparticles loaded with collagen peptide are attached to the complex network of the injectable gel delivery matrix to form a microsphere structure, which effectively protects the stability of tuna collagen peptide, and significantly improves the effect of slow release and targeted release on tuna bone. The bioavailability of collagen peptides, multiple substances are enriched and act on the diseased parts of osteoporosis in a multi-pronged manner, which not only reduces the adverse reactions caused by multiple administrations, but also obtains efficient therapeutic effects; in the targeted metal composite nanoparticles loaded with collagen peptides, ZIF-8 nanoparticles are used as carriers to encapsulate the active substance tuna collagen peptide, thereby protecting the stability of tuna collagen peptides and achieving sustained release of drugs in a weakly acidic environment, and then copper peroxide black phosphorus composite particles are attached to the surface of ZIF-8 nanoparticles loaded with collagen peptides to protect the stability of tuna collagen peptides in an encapsulated manner. , reducing the inactivation effect of the external environment, reducing the adverse stimulation to the human body, and also having a certain bone targeting effect, thereby increasing the enrichment of tuna collagen peptides in osteoporotic areas, thereby increasing bioavailability and improving the effectiveness of treating osteoporosis; in the injectable gel delivery matrix, under the action of the biocrosslinking agent and the gold nanorods, a highly cross-linked large-scale tightly connected network structure is formed, which can slowly release and accurately deliver active substances to bone tissue, further increasing the encapsulation amount and encapsulation stability of the targeted metal composite nanoparticles loaded with collagen peptides, and reducing The invention reduces the adverse stimulation of tuna collagen peptide and improves the bioavailability and therapeutic effect of tuna collagen peptide. Among them, the biocrosslinker and gold nanorods also have the auxiliary effect of promoting bone formation and inhibiting bone resorption, which helps to improve the therapeutic effect. The invention uses targeted metal composite nanoparticles loaded with collagen peptide, an injectable gel delivery matrix and calcium chloride to prepare an osteoporosis repair-type bone peptide composition, which can slowly release tuna collagen peptide in a targeted manner, improve the stability and bioavailability of the active substance, reduce the occurrence of adverse reactions, and have a highly effective therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0026] Figure 2 The encapsulation efficiency results of Examples 1-4 and Comparative Examples 1-3 of the present invention are shown in FIG.

[0027] Figure 3 The allergy rate results of Examples 1-4 and Comparative Examples 1-3 of the present invention are shown in FIG.

[0028] Figure 4 This is a graph showing the therapeutic efficacy results of Examples 1-4 of the present invention and Comparative Examples 1-3. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only 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 ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0031] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0032] Example 1

[0033] This embodiment proposes a bone peptide composition for repairing osteoporosis, comprising the following components in parts by weight: 60 parts of targeted metal composite nanoparticles loaded with collagen peptides, 30 parts of injectable gel delivery matrix, and 20 parts of calcium chloride.

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

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

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

[0037] (1) Add 9.0g tuna bone powder to 100mL water, add animal protease, the amount of animal protease added is 2.5% of the amount of tuna bone powder added, the enzyme activity is 300,000 U / g, animal protease can decompose large molecular proteins such as collagen in bone tissue into small molecular peptides and amino acids, improve the extraction rate and bioavailability of bone peptides, enzymatic hydrolysis at pH 7.0 and temperature 55℃ for 8h, after the enzymatic hydrolysis is completed, inactivate the enzyme at 90℃ for 30min, and then centrifuge at 8000rpm for 15min, and take The supernatant is intercepted by a membrane separation method to intercept the enzymatic hydrolyzate, and the collagen peptides with a molecular weight of 250-1000 Da are collected and freeze-dried. The low-molecular-weight tuna collagen peptides can significantly increase the activity of alkaline phosphatase and osteocalcin in osteoblasts, thereby facilitating the differentiation of osteoblasts and promoting the secretion of collagen type I, the main component of the bone organic matrix, thereby contributing to the formation of bone matrix. Therefore, the prepared tuna collagen peptides have a good ability to promote the proliferation, differentiation and mineralization of osteoblasts, thereby obtaining tuna collagen peptides;

[0038] (2) 2.0 g of zinc nitrate hexahydrate was added to 20 mL of deionized water to obtain a zinc nitrate hexahydrate solution for standby use. 8.0 g of 2-methylimidazole was added to 40 mL of deionized water, and then the tuna collagen peptide described in step (1) was added. The mixture was stirred at 80 rpm for 10 min. The zinc nitrate hexahydrate solution was quickly dripped into the 2-methylimidazole solution containing tuna collagen peptide. The mixture was stirred at 800 rpm for 30 min. The suspension was collected and centrifuged. The precipitate was washed 5 times with deionized water. The porous structure and pH sensitivity of ZIF-8 make it an ideal drug delivery carrier. The active substance can be encapsulated through its pore structure, the stability of tuna collagen peptide can be protected, the risk of inactivation or degradation in the external environment can be reduced, and the sustained release of the drug can be achieved in a weakly acidic environment, thereby improving the bioavailability of tuna collagen peptide. In addition, ZIF-8 is stable under physiological conditions and has low toxicity to cells and tissues. It can also inhibit the damage of inflammatory factors to bone tissue by releasing zinc ions, thereby obtaining ZIF-8 nanoparticles loaded with collagen peptide.

[0039] (3) Add 0.5 g of polyvinyl pyrrolidone to 5 mL of 0.2% copper chloride dihydrate solution, ultrasonically dissolve and stir evenly, then add 5 mL of 0.08% sodium hydroxide solution, magnetically stir evenly, then slowly drop 10 μL of 30% hydrogen peroxide, stir for 30 minutes, and subject the reaction mixture to ultrahigh-speed centrifugation at a speed of 6000 rpm for 20 minutes. Collect the precipitate, wash it with ultrapure water 5 times, and freeze-dry it. The metal peroxide can react with excess hydrogen ions in osteoporotic bone tissue. The released copper ions help to induce and stimulate osteoblasts, which is beneficial to bone mineralization and bone regeneration, thereby obtaining copper peroxide nanoparticles.

[0040] (4) 1.0 g of sodium hydroxide was dispersed in 40 mL of N-methylpyrrolidone and ultrasonically treated for 30 min. Then, black phosphorus powder was added. The amount of black phosphorus powder added was 20.0 mg. The biodegradation products of black phosphorus can be converted into calcium phosphate nanoparticles through self-mineralization, which have a natural affinity with hydroxyapatite in bone tissue, thereby promoting the enrichment of black phosphorus in bone tissue and showing a certain bone targeting effect. The suspension was ultrasonically treated for 10 h in an ice bath environment. The suspension was centrifuged 3 times, the supernatant was collected, and the centrifugation was continued for 4 times. The precipitate was collected and freeze-dried to obtain black phosphorus nanosheets. The copper peroxide nanoparticles described in step (3) were dispersed in 10 mL of ultrapure water, and then black phosphorus nanosheets were added. The mixture was magnetically stirred for 3 h. The positively charged copper peroxide nanoparticles were adsorbed onto the surface of the negatively charged black phosphorus nanosheets by electrostatic interaction, forming a composite material with anti-inflammatory and osteopromoting effects. In the inflammatory and slightly acidic environment of osteoporosis, the copper peroxide nanoparticles were decomposed into Cu 2+ With hydroxyl radicals, Cu 2+ It can produce a certain degree of osteogenic effect on osteoblasts, and hydroxyl radicals can accelerate the degradation of black phosphorus nanosheets in the body under a lightless environment, thereby causing self-mineralization to produce calcium phosphate nanoparticles, and collecting solid substances to obtain copper peroxide black phosphorus composite particles;

[0041] (5) The ZIF-8 nanoparticles loaded with collagen peptide described in step (2) are dispersed in 20 mL of ultrapure water, and the copper peroxide black phosphorus composite particles described in step (4) are added. The mixture is magnetically stirred for 5 h, and the precipitate is collected. The copper peroxide black phosphorus composite particles are attached to the surface of the ZIF-8 nanoparticles loaded with collagen peptide to form a protective layer, thereby increasing the stability of the ZIF-8 nanoparticles loaded with collagen peptide, and also improving the biocompatibility and bone targeting performance of the nanoparticles, further enhancing the effective release of collagen peptide, and being beneficial to improving the therapeutic effect of osteoporosis, thereby obtaining targeted metal composite nanoparticles loaded with collagen peptide.

[0042] This embodiment provides a method for preparing an osteoporosis repairing osteopeptide composition, which specifically comprises the following steps:

[0043] S1. Dissolve 100.0 mg of chitosan in 5 mL of a 30% aqueous solution of dimethyl sulfoxide, adjust the pH to 5.0, stir until completely dissolved, and set aside. Chitosan can increase the activity of alkaline phosphatase, accelerate bone formation, inhibit osteoclast activity, reduce bone resorption, and enhance bone density by promoting calcium absorption. Dissolve chlorogenic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide in 5 mL of dimethyl sulfoxide, stir and activate for 2 hours. The amount of chlorogenic acid added is 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. Chlorogenic acid is not only used as a cross-linking agent, but can also be used as a By regulating bone metabolism-related hormones (such as estrogen and parathyroid hormone), maintaining bone metabolism balance, and helping to prevent and treat osteoporosis, the activated chitosan solution and chlorogenic acid solution are mixed, stirred overnight, and transferred to a dialysis bag. Dialysis is first performed using a pH 5.0 acidic buffer solution, and the buffer solution is replaced every 12 hours. On the third day, it is replaced with ultrapure water at pH 7.0 for 4 hours. The reaction product is collected and freeze-dried. The carboxyl group of chlorogenic acid reacts with the amino group of chitosan to synthesize a non-toxic bio-crosslinking agent with a catechol structure, which effectively improves the shortcomings of chlorogenic acid, such as instability and poor water solubility. Among them, the catechol structure can simulate the effects of estrogen, which is beneficial to maintaining bone density, and can also show high reactivity in the cross-linking reaction to obtain a bio-crosslinking agent;

[0044] S2. Dissolve 2.0 g of sodium alginate in 100 mL of ultrapure water, stir evenly, and 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 efficacy and reduce side effects. Magnetic stirring is carried out for 2 hours, and then the biocrosslinker described in step S1 is added and vibrated to mix. Under the action of the biocrosslinker and gold nanorods, the loading capacity and stability of the delivery matrix are increased, 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 the single active substance, and obtaining an injectable gel delivery matrix;

[0045] S3. Add the targeted metal composite nanoparticles loaded with collagen peptides to the injectable gel delivery matrix described in step S2, ultrasonically treat for 3 hours, and then spray it into 100 mL of a 2% calcium chloride solution using a portable electrospray device. Let it stand for 8 hours, and then rinse the gel product 5 times with ultrapure water. A compact and complex porous microsphere structure is prepared by this process. The targeted metal composite nanoparticles loaded with collagen peptides are adsorbed in the pores of the microspheres, which effectively protects the stability of the collagen peptides and reduces the risk of inactivation caused by the external environment. It also significantly improves the bioavailability of the collagen peptides through slow release and targeted release. At the same time, it synergizes with other components to exert a multi-pronged therapeutic effect on osteoporosis, achieving excellent therapeutic effects and obtaining a bone peptide composition for osteoporosis repair.

[0046] In this example, the prepared osteoporosis repairing osteopeptide composition was subjected to scanning electron microscopy to observe its microscopic morphology. Figure 1 This is a 1000-fold magnified SEM image of the osteoporosis repairing osteopeptide composition prepared in Example 1. Figure 1 The osteoporosis repairing osteopeptide composition prepared in this example presents a microsphere structure with a highly cross-linked surface.

[0047] Example 2

[0048] This embodiment proposes a bone peptide composition for repairing osteoporosis, comprising the following components in parts by weight: 50 parts of targeted metal composite nanoparticles loaded with collagen peptides, 20 parts of injectable gel delivery matrix, and 10 parts of calcium chloride.

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

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

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

[0052] (1) Add 8.0g of tuna bone powder to 100mL of water, add animal protease, the amount of animal protease added is 1.5% of the amount of tuna bone powder added, the enzyme activity is 300,000 U / g, animal protease can decompose large molecular proteins such as collagen in bone tissue into small molecular peptides and amino acids, improve the extraction rate and bioavailability of bone peptides, enzymatic hydrolysis at pH 7.0 and temperature 50℃ for 6h, after the enzymatic hydrolysis is completed, inactivate the enzyme at 90℃ for 30min, and then centrifuge at 6000rpm for 10min, and take The supernatant is intercepted by a membrane separation method to intercept the enzymatic hydrolyzate, and the collagen peptides with a molecular weight of 250-1000 Da are collected and freeze-dried. The low-molecular-weight tuna collagen peptides can significantly increase the activity of alkaline phosphatase and osteocalcin in osteoblasts, thereby facilitating the differentiation of osteoblasts and promoting the secretion of collagen type I, the main component of the bone organic matrix, thereby contributing to the formation of bone matrix. Therefore, the prepared tuna collagen peptides have a good ability to promote the proliferation, differentiation and mineralization of osteoblasts, thereby obtaining tuna collagen peptides;

[0053] (2) 2.0 g of zinc nitrate hexahydrate was added to 20 mL of deionized water to obtain a zinc nitrate hexahydrate solution for standby use. 8.0 g of 2-methylimidazole was added to 40 mL of deionized water, and then the tuna collagen peptide described in step (1) was added. The mixture was stirred at 60 rpm for 5 min. The zinc nitrate hexahydrate solution was quickly dripped into the 2-methylimidazole solution containing tuna collagen peptide. The mixture was stirred at 600 rpm for 20 min. The suspension was collected and centrifuged. The precipitate was washed 3 times with deionized water. The porous structure and pH sensitivity of ZIF-8 make it an ideal drug delivery carrier. The active substance can be encapsulated through its pore structure, the stability of tuna collagen peptide can be protected, the risk of inactivation or degradation in the external environment can be reduced, and the sustained release of the drug can be achieved in a weakly acidic environment, thereby improving the bioavailability of tuna collagen peptide. In addition, ZIF-8 is stable under physiological conditions and has low toxicity to cells and tissues. It can also inhibit the damage of inflammatory factors to bone tissue by releasing zinc ions, thereby obtaining ZIF-8 nanoparticles loaded with collagen peptide.

[0054] (3) Add 0.5 g of polyvinyl pyrrolidone to 5 mL of 0.1% copper chloride dihydrate solution, ultrasonically dissolve and stir evenly, then add 5 mL of 0.08% sodium hydroxide solution, magnetically stir evenly, then slowly drop 10 μL of 30% hydrogen peroxide, stir for 20 minutes, and subject the reaction mixture to ultrahigh-speed centrifugation at a speed of 5000 rpm for 10 minutes. Collect the precipitate, wash it three times with ultrapure water, and freeze-dry it. The metal peroxide can react with excess hydrogen ions in osteoporotic bone tissue. The released copper ions help to induce and stimulate osteoblasts, which is beneficial to bone mineralization and bone regeneration, thereby obtaining copper peroxide nanoparticles.

[0055] (4) 1.0 g of sodium hydroxide was dispersed in 40 mL of N-methylpyrrolidone and ultrasonically treated for 30 min. Then, black phosphorus powder was added. The amount of black phosphorus powder added was 10.0 mg. The biodegradation products of black phosphorus can be converted into calcium phosphate nanoparticles through self-mineralization, which have a natural affinity with hydroxyapatite in bone tissue, thereby promoting the enrichment of black phosphorus in bone tissue and showing a certain bone targeting effect. The suspension was ultrasonically treated for 8 h in an ice bath environment. The suspension was centrifuged twice, the supernatant was collected, and the centrifugation was continued for 3 times. The precipitate was collected and freeze-dried to obtain black phosphorus nanosheets. The copper peroxide nanoparticles described in step (3) were dispersed in 10 mL of ultrapure water, and then black phosphorus nanosheets were added. The mixture was magnetically stirred for 2 h. The positively charged copper peroxide nanoparticles were adsorbed onto the surface of the negatively charged black phosphorus nanosheets by electrostatic interaction, forming a composite material with anti-inflammatory and osteopromoting effects. In the inflammatory and slightly acidic environment of osteoporosis, the copper peroxide nanoparticles were decomposed into Cu 2+ With hydroxyl radicals, Cu 2+ It can produce a certain degree of osteogenic effect on osteoblasts, and hydroxyl radicals can accelerate the degradation of black phosphorus nanosheets in the body under a lightless environment, thereby causing self-mineralization to produce calcium phosphate nanoparticles, and collecting solid substances to obtain copper peroxide black phosphorus composite particles;

[0056] (5) The ZIF-8 nanoparticles loaded with collagen peptide described in step (2) were dispersed in 20 mL of ultrapure water, and the copper peroxide black phosphorus composite particles described in step (4) were added. The mixture was magnetically stirred for 3 h, and the precipitate was collected. The copper peroxide black phosphorus composite particles were attached to the surface of the ZIF-8 nanoparticles loaded with collagen peptide to form a protective layer, thereby increasing the stability of the ZIF-8 nanoparticles loaded with collagen peptide, and also improving the biocompatibility and bone targeting performance of the nanoparticles, further enhancing the effective release of collagen peptide, and being beneficial to improving the therapeutic effect of osteoporosis, thereby obtaining targeted metal composite nanoparticles loaded with collagen peptide.

[0057] This embodiment provides a method for preparing an osteoporosis repairing osteopeptide composition, which specifically comprises the following steps:

[0058] S1. Dissolve 100.0 mg of chitosan in 5 mL of a 30% aqueous solution of dimethyl sulfoxide, adjust the pH to 5.0, stir until completely dissolved, and set aside. Chitosan can increase the activity of alkaline phosphatase, accelerate bone formation, inhibit osteoclast activity, reduce bone resorption, and enhance bone density by promoting calcium absorption. Dissolve chlorogenic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide in 5 mL of dimethyl sulfoxide, stir and activate for 1 hour. The amount of chlorogenic acid added is 170.0 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. Chlorogenic acid is not only used as a cross-linking agent, but can also be used as a By regulating bone metabolism-related hormones (such as estrogen and parathyroid hormone), maintaining bone metabolism balance, and helping to prevent and treat osteoporosis, the activated chitosan solution and chlorogenic acid solution are mixed, stirred overnight, and transferred to a dialysis bag. Dialysis is first performed using a pH 5.0 acidic buffer solution, and the buffer solution is replaced every 12 hours. On the third day, it is replaced with ultrapure water at pH 7.0 for 3 hours. The reaction product is collected and freeze-dried. The carboxyl group of chlorogenic acid reacts with the amino group of chitosan to synthesize a non-toxic bio-crosslinking agent with a catechol structure, which effectively improves the shortcomings of chlorogenic acid, such as instability and poor water solubility. Among them, the catechol structure can simulate the effects of estrogen, which is beneficial to maintaining bone density, and can also show high reactivity in the cross-linking reaction to obtain a bio-crosslinking agent;

[0059] S2. Dissolve 1.0 g of sodium alginate in 100 mL of ultrapure water, stir evenly, and 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 efficacy and reduce side effects. Magnetic stirring is carried out for 1 hour, and then the biocrosslinking agent described in step S1 is added and vibrated to mix. Under the action of the biocrosslinking agent and gold nanorods, the loading capacity and stability of the delivery matrix are increased, 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 the single active substance, and obtaining an injectable gel delivery matrix;

[0060] 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 hours, and then use a portable electrospray device to spray it into 100 mL of 1% calcium chloride solution, let it stand for 6 hours, and then rinse the gel product 3 times with ultrapure water. A compact and complex porous microsphere structure is prepared by this process. The targeted metal composite nanoparticles loaded with collagen peptides are adsorbed in the pores of the microspheres, which effectively protects the stability of the collagen peptides and reduces the risk of inactivation caused by the external environment. It also significantly improves the bioavailability of the collagen peptides through slow release and targeted release. At the same time, it synergizes with other components to exert a multi-pronged therapeutic effect on osteoporosis, achieving excellent therapeutic effects and obtaining a bone peptide composition for osteoporosis repair.

[0061] Example 3

[0062] This embodiment proposes a bone peptide composition for repairing osteoporosis, comprising the following components in parts by weight: 55 parts of targeted metal composite nanoparticles loaded with collagen peptides, 25 parts of injectable gel delivery matrix, and 15 parts of calcium chloride.

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

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

[0065] The preparation method of targeted metal composite nanoparticles loaded with collagen peptide specifically comprises the following steps:

[0066] (1) Add 8.5 g of tuna bone powder to 100 mL of water, and add animal protease. The amount of animal protease added is 2.0% of the amount of tuna bone powder added, and the enzyme activity is 300,000 U / g. Animal protease can decompose large molecular proteins such as collagen in bone tissue into small molecular peptides and amino acids, thereby improving the extraction rate and bioavailability of bone peptides. The enzyme was hydrolyzed at pH 7.0 and temperature 52.5 ° C for 7 h. After the enzymatic hydrolysis, the enzyme was inactivated at 90 ° C for 30 min, and then refrigerated centrifuged at 7000 rpm for 12.5 min. , taking the supernatant, intercepting the enzymatic hydrolysate by membrane separation, collecting the collagen peptides with a molecular weight of 250-1000Da, and freeze-drying. The low-molecular-weight tuna collagen peptides can significantly increase the activity of alkaline phosphatase and osteocalcin in osteoblasts, thereby facilitating the differentiation of osteoblasts, and can promote the secretion of collagen type I, the main component of the bone organic matrix, thereby contributing to the formation of bone matrix. Therefore, the prepared tuna collagen peptide has a good ability to promote osteoblast proliferation, differentiation and mineralization, thereby obtaining tuna collagen peptide;

[0067] (2) 2.0 g of zinc nitrate hexahydrate was added to 20 mL of deionized water to obtain a zinc nitrate hexahydrate solution for standby use. 8.0 g of 2-methylimidazole was added to 40 mL of deionized water, and then the tuna collagen peptide described in step (1) was added. The mixture was stirred at 70 rpm for 7.5 min. The zinc nitrate hexahydrate solution was quickly added dropwise to the 2-methylimidazole solution containing tuna collagen peptide. The mixture was stirred at 700 rpm for 25 min. The suspension was collected and centrifuged. The precipitate was washed 4 times with deionized water. The porous structure and pH sensitivity of ZIF-8 make it an ideal drug delivery carrier. The active substance can be encapsulated through its pore structure, the stability of tuna collagen peptide can be protected, the risk of inactivation or degradation in the external environment can be reduced, and the sustained release of the drug can be achieved in a weakly acidic environment, thereby improving the bioavailability of tuna collagen peptide. In addition, ZIF-8 is stable under physiological conditions and has low toxicity to cells and tissues. It can also inhibit the damage of inflammatory factors to bone tissue by releasing zinc ions, thereby obtaining ZIF-8 nanoparticles loaded with collagen peptide.

[0068] (3) Add 0.5 g of polyvinyl pyrrolidone to 5 mL of 0.15% copper chloride dihydrate solution, ultrasonically dissolve and stir evenly, then add 5 mL of 0.08% sodium hydroxide solution, magnetically stir evenly, then slowly drop 10 μL of 30% hydrogen peroxide, stir for 25 minutes, and subject the reaction mixture to ultrahigh-speed centrifugation at a speed of 5500 rpm for 15 minutes. Collect the precipitate, wash it 4 times with ultrapure water, and freeze-dry it. The metal peroxide can react with excess hydrogen ions in osteoporotic bone tissue. The released copper ions help to induce and stimulate osteoblasts, which is beneficial to bone mineralization and bone regeneration, thereby obtaining copper peroxide nanoparticles.

[0069] (4) 1.0 g of sodium hydroxide was dispersed in 40 mL of N-methylpyrrolidone and ultrasonically treated for 30 min. Then, black phosphorus powder was added. The amount of black phosphorus powder added was 15.0 mg. The biodegradation products of black phosphorus can be converted into calcium phosphate nanoparticles through self-mineralization, which have a natural affinity with hydroxyapatite in bone tissue, thereby promoting the enrichment of black phosphorus in bone tissue and showing a certain bone targeting effect. The suspension was ultrasonically treated for 9 h in an ice bath environment. The suspension was centrifuged twice, the supernatant was collected, and the centrifugation was continued for 3 times. The precipitate was collected and freeze-dried to obtain black phosphorus nanosheets. The copper peroxide nanoparticles described in step (3) were dispersed in 10 mL of ultrapure water, and then black phosphorus nanosheets were added. The mixture was magnetically stirred for 2.5 h. The positively charged copper peroxide nanoparticles were adsorbed onto the surface of the negatively charged black phosphorus nanosheets by electrostatic interaction, forming a composite material with anti-inflammatory and osteopromoting effects. In the inflammatory and slightly acidic environment of osteoporosis, the copper peroxide nanoparticles were decomposed into Cu 2+ With hydroxyl radicals, Cu 2+ It can produce a certain degree of osteogenic effect on osteoblasts, and hydroxyl radicals can accelerate the degradation of black phosphorus nanosheets in the body under a lightless environment, thereby causing self-mineralization to produce calcium phosphate nanoparticles, and collecting solid substances to obtain copper peroxide black phosphorus composite particles;

[0070] (5) The ZIF-8 nanoparticles loaded with collagen peptide described in step (2) are dispersed in 20 mL of ultrapure water, and the copper peroxide black phosphorus composite particles described in step (4) are added. The mixture is magnetically stirred for 4 h, and the precipitate is collected. The copper peroxide black phosphorus composite particles are attached to the surface of the ZIF-8 nanoparticles loaded with collagen peptide to form a protective layer, thereby increasing the stability of the ZIF-8 nanoparticles loaded with collagen peptide, and also improving the biocompatibility and bone targeting performance of the nanoparticles, further enhancing the effective release of collagen peptide, and being beneficial to improving the therapeutic effect of osteoporosis, thereby obtaining targeted metal composite nanoparticles loaded with collagen peptide.

[0071] This embodiment provides a method for preparing an osteoporosis repairing osteopeptide composition, which specifically comprises the following steps:

[0072] S1. Dissolve 100.0 mg of chitosan in 5 mL of a 30% aqueous solution of dimethyl sulfoxide, adjust the pH to 5.0, stir until completely dissolved, and set aside. Chitosan can increase the activity of alkaline phosphatase, accelerate bone formation, inhibit osteoclast activity, reduce bone resorption, and enhance bone density by promoting calcium absorption. Dissolve chlorogenic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide in 5 mL of dimethyl sulfoxide, stir and activate for 1.5 hours. The amount of chlorogenic acid added is 174.0 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. Chlorogenic acid is not only used as a cross-linking agent, but can also be used as a By regulating bone metabolism-related hormones (such as estrogen and parathyroid hormone), maintaining bone metabolism balance, and helping to prevent and treat osteoporosis, the activated chitosan solution and chlorogenic acid solution are mixed, stirred overnight, and transferred to a dialysis bag. Dialysis is first performed using a pH 5.0 acidic buffer solution, and the buffer solution is replaced every 12 hours. On the third day, it is replaced with ultrapure water with pH 7.0 for dialysis for 3.5 hours. The reaction product is collected and freeze-dried. The carboxyl group of chlorogenic acid reacts with the amino group of chitosan to synthesize a non-toxic bio-crosslinking agent with a catechol structure, which effectively improves the shortcomings of chlorogenic acid, such as instability and poor water solubility. Among them, the catechol structure can simulate the effects of estrogen, which is beneficial to maintaining bone density, and can also show high reactivity in the cross-linking reaction to obtain a bio-crosslinking agent;

[0073] S2. Dissolve 1.5 g of sodium alginate in 100 mL of ultrapure water, stir evenly, and 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 efficacy and reduce side effects. Magnetic stirring is carried out for 1.5 hours, and then the biocrosslinking agent described in step S1 is added and vibrated to mix. Under the action of the biocrosslinking agent and gold nanorods, the loading capacity and stability of the delivery matrix are increased, 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 the single active substance, and obtaining an injectable gel delivery matrix;

[0074] S3. Add the targeted metal composite nanoparticles loaded with collagen peptides to the injectable gel delivery matrix described in step S2, ultrasonically treat for 2.5 hours, and then use a portable electrospray device to spray it into 100 mL of a 1.5% calcium chloride solution. Let it stand for 7 hours, and then rinse the gel product 4 times with ultrapure water. A compact and complex porous microsphere structure is prepared by this process. The targeted metal composite nanoparticles loaded with collagen peptides are adsorbed in the pores of the microspheres, which effectively protects the stability of the collagen peptides and reduces the risk of inactivation caused by the external environment. It also significantly improves the bioavailability of the collagen peptides through slow release and targeted release. At the same time, it synergizes with other components to exert a multi-pronged therapeutic effect on osteoporosis, achieving excellent therapeutic effects and obtaining a bone peptide composition for osteoporosis repair.

[0075] Example 4

[0076] This embodiment proposes a bone peptide composition for repairing osteoporosis, comprising the following components in parts by weight: 60 parts of targeted metal composite nanoparticles loaded with collagen peptides, 20 parts of injectable gel delivery matrix, and 20 parts of calcium chloride.

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

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

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

[0080] (1) Add 9.0g tuna bone powder to 100mL water, add animal protease, the amount of animal protease added is 2.5% of the amount of tuna bone powder added, the enzyme activity is 300,000 U / g, animal protease can decompose large molecular proteins such as collagen in bone tissue into small molecular peptides and amino acids, improve the extraction rate and bioavailability of bone peptides, enzymatic hydrolysis at pH 7.0 and temperature 55℃ for 6h, after the enzymatic hydrolysis is completed, inactivate the enzyme at 90℃ for 30min, and then centrifuge at 8000rpm for 10min, and take The supernatant is intercepted by a membrane separation method to intercept the enzymatic hydrolyzate, and the collagen peptides with a molecular weight of 250-1000 Da are collected and freeze-dried. The low-molecular-weight tuna collagen peptides can significantly increase the activity of alkaline phosphatase and osteocalcin in osteoblasts, thereby facilitating the differentiation of osteoblasts and promoting the secretion of collagen type I, the main component of the bone organic matrix, thereby contributing to the formation of bone matrix. Therefore, the prepared tuna collagen peptides have a good ability to promote the proliferation, differentiation and mineralization of osteoblasts, thereby obtaining tuna collagen peptides;

[0081] (2) 2.0 g of zinc nitrate hexahydrate was added to 20 mL of deionized water to obtain a zinc nitrate hexahydrate solution, which was set aside. 8.0 g of 2-methylimidazole was added to 40 mL of deionized water, and then the tuna collagen peptide described in step (1) was added. The mixture was stirred at 80 rpm for 5 min. The zinc nitrate hexahydrate solution was quickly dripped into the 2-methylimidazole solution containing tuna collagen peptide. The mixture was stirred at 800 rpm for 20 min. The suspension was collected and centrifuged. The precipitate was washed 5 times with deionized water. The porous structure and pH sensitivity of ZIF-8 make it an ideal drug delivery carrier. The active substance can be encapsulated through its pore structure, the stability of tuna collagen peptide can be protected, the risk of inactivation or degradation in the external environment can be reduced, and the sustained release of the drug can be achieved in a weakly acidic environment, thereby improving the bioavailability of tuna collagen peptide. In addition, ZIF-8 is stable under physiological conditions and has low toxicity to cells and tissues. It can also inhibit the damage of inflammatory factors to bone tissue by releasing zinc ions, thereby obtaining ZIF-8 nanoparticles loaded with collagen peptide.

[0082] (3) Add 0.5 g of polyvinyl pyrrolidone to 5 mL of 0.2% copper chloride dihydrate solution, ultrasonically dissolve and stir evenly, then add 5 mL of 0.08% sodium hydroxide solution, magnetically stir evenly, then slowly drop 10 μL of 30% hydrogen peroxide, stir for 20 minutes, and subject the reaction mixture to ultrahigh-speed centrifugation at a speed of 6000 rpm for 10 minutes. Collect the precipitate, wash it 5 times with ultrapure water, and freeze-dry it. The metal peroxide can react with excess hydrogen ions in osteoporotic bone tissue. The released copper ions help to induce and stimulate osteoblasts, which is beneficial to bone mineralization and bone regeneration, and obtain copper peroxide nanoparticles.

[0083] (4) 1.0 g of sodium hydroxide was dispersed in 40 mL of N-methylpyrrolidone and ultrasonically treated for 30 min. Then, black phosphorus powder was added. The amount of black phosphorus powder added was 10.0 mg. The biodegradation products of black phosphorus can be converted into calcium phosphate nanoparticles through self-mineralization, which have a natural affinity with hydroxyapatite in bone tissue, thereby promoting the enrichment of black phosphorus in bone tissue and showing a certain bone targeting effect. The suspension was ultrasonically treated for 8 h in an ice bath environment. The suspension was centrifuged 3 times, the supernatant was collected, and the centrifugation was continued for 4 times. The precipitate was collected and freeze-dried to obtain black phosphorus nanosheets. The copper peroxide nanoparticles described in step (3) were dispersed in 10 mL of ultrapure water, and then black phosphorus nanosheets were added. The mixture was magnetically stirred for 2 h. The positively charged copper peroxide nanoparticles were adsorbed onto the surface of the negatively charged black phosphorus nanosheets by electrostatic interaction, forming a composite material with anti-inflammatory and osteopromoting effects. In the inflammatory and slightly acidic environment of osteoporosis, the copper peroxide nanoparticles were decomposed into Cu 2+ With hydroxyl radicals, Cu 2+ It can produce a certain degree of osteogenic effect on osteoblasts, and hydroxyl radicals can accelerate the degradation of black phosphorus nanosheets in the body under a lightless environment, thereby causing self-mineralization to produce calcium phosphate nanoparticles, and collecting solid substances to obtain copper peroxide black phosphorus composite particles;

[0084] (5) The ZIF-8 nanoparticles loaded with collagen peptide described in step (2) were dispersed in 20 mL of ultrapure water, and the copper peroxide black phosphorus composite particles described in step (4) were added. The mixture was magnetically stirred for 3 h, and the precipitate was collected. The copper peroxide black phosphorus composite particles were attached to the surface of the ZIF-8 nanoparticles loaded with collagen peptide to form a protective layer, thereby increasing the stability of the ZIF-8 nanoparticles loaded with collagen peptide, and also improving the biocompatibility and bone targeting performance of the nanoparticles, further enhancing the effective release of collagen peptide, and being beneficial to improving the therapeutic effect of osteoporosis, thereby obtaining targeted metal composite nanoparticles loaded with collagen peptide.

[0085] This embodiment provides a method for preparing an osteoporosis repairing osteopeptide composition, which specifically comprises the following steps:

[0086] S1. Dissolve 100.0 mg of chitosan in 5 mL of a 30% aqueous solution of dimethyl sulfoxide, adjust the pH to 5.0, stir until completely dissolved, and set aside. Chitosan can increase the activity of alkaline phosphatase, accelerate bone formation, inhibit osteoclast activity, reduce bone resorption, and enhance bone density by promoting calcium absorption. Dissolve chlorogenic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide in 5 mL of dimethyl sulfoxide, stir and activate for 1 hour. The amount of chlorogenic acid added is 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. Chlorogenic acid is not only used as a cross-linking agent, but can also be used as a By regulating bone metabolism-related hormones (such as estrogen and parathyroid hormone), maintaining bone metabolism balance, and helping to prevent and treat osteoporosis, the activated chitosan solution and chlorogenic acid solution are mixed, stirred overnight, and transferred to a dialysis bag. Dialysis is first performed using a pH 5.0 acidic buffer solution, and the buffer solution is replaced every 12 hours. On the third day, it is replaced with ultrapure water at pH 7.0 for 3 hours. The reaction product is collected and freeze-dried. The carboxyl group of chlorogenic acid reacts with the amino group of chitosan to synthesize a non-toxic bio-crosslinking agent with a catechol structure, which effectively improves the shortcomings of chlorogenic acid, such as instability and poor water solubility. Among them, the catechol structure can simulate the effects of estrogen, which is beneficial to maintaining bone density, and can also show high reactivity in the cross-linking reaction to obtain a bio-crosslinking agent;

[0087] S2. Dissolve 2.0 g of sodium alginate in 100 mL of ultrapure water, stir evenly, and 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 efficacy and reduce side effects. Magnetic stirring is carried out for 1 hour, and then the biocrosslinker described in step S1 is added and vibrated to mix. Under the action of the biocrosslinker and gold nanorods, the loading capacity and stability of the delivery matrix are increased, 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 the single active substance, and obtaining an injectable gel delivery matrix;

[0088] 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 hours, and then spray it into 100 mL of 2% calcium chloride solution using a portable electrospray device. Let it stand for 6 hours, and then rinse the gel product with ultrapure water 5 times. A compact and complex porous microsphere structure is prepared by this process. The targeted metal composite nanoparticles loaded with collagen peptides are adsorbed in the pores of the microspheres, which effectively protects the stability of the collagen peptides and reduces the risk of inactivation caused by the external environment. It also significantly improves the bioavailability of the collagen peptides through slow release and targeted release. At the same time, it synergizes with other components to exert a multi-pronged therapeutic effect on osteoporosis, achieving excellent therapeutic effects and obtaining a bone peptide composition for osteoporosis repair.

[0089] Comparative Example 1

[0090] This comparative example provides a bone peptide composition for repairing osteoporosis, which differs from Example 1 in that the targeted metal composite nanoparticles loaded with collagen peptide do not contain copper peroxide black phosphorus composite particles; the preparation method of the targeted metal composite nanoparticles loaded with collagen peptide does not include steps (3), (4), and (5); the preparation method of the bone peptide composition for repairing osteoporosis is the same as that of Example 1.

[0091] Comparative Example 2

[0092] This comparative example provides an osteoporosis-repairing osteopeptide composition, which differs from Example 1 in that the injectable gel delivery matrix does not contain a biocrosslinker or gold nanorods; the preparation method of the targeted metal composite nanoparticles loaded with collagen peptides is the same as that of Example 1; the preparation method of the osteoporosis-repairing osteopeptide composition does not include step S1 and gold nanorods are not added in step S2.

[0093] Comparative Example 3

[0094] This comparative example provides a osteoporosis repairing osteopeptide composition, which differs from Example 1 in that the osteoporosis repairing osteopeptide composition does not contain copper peroxide black phosphorus composite particles, biocrosslinking agent and gold nanorods; the preparation method of targeted metal composite nanoparticles loaded with collagen peptide does not include steps (3), (4) and (5); the preparation method of the osteoporosis repairing osteopeptide composition does not include step S1 and gold nanorods are not added in step S2.

[0095] Experimental Example 1

[0096] Encapsulation efficiency experiment

[0097] Test sample: osteoporosis repairing osteopeptide composition prepared in Examples 1-4 and Comparative Examples 1-3.

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

[0099] Encapsulation efficiency (%) = (total amount of tuna collagen peptide - free amount of tuna collagen peptide) / total amount of tuna collagen peptide × 100%

[0100] Figure 2 The encapsulation efficiency results of Examples 1-4 and Comparative Examples 1-3 are shown in FIG; As shown in the figure, the encapsulation efficiency of Examples 1-4 is 82.1-85.8%, indicating that the encapsulation efficiency is high, effectively reducing the inactivation and degradation effects of the outside world; the encapsulation efficiency of Comparative Examples 1-3 is 58.5-71.8%, indicating that the encapsulation efficiency is low, and the inactivation and degradation effects of the outside world cannot be effectively reduced; the targeted metal composite nanoparticles loaded with collagen peptides in Comparative Example 1 do not contain copper peroxide black phosphorus composite particles, and cannot be attached to the surface of the ZIF-8 nanoparticles loaded with collagen peptides to form a protective layer, resulting in a low encapsulation efficiency and an inability to effectively reduce the inactivation and degradation effects of the outside world; the injectable gel delivery matrix of Comparative Example 2 The matrix does not contain biocrosslinking agents or gold nanorods, and cannot form a highly crosslinked, large, tightly connected network structure, which is not conducive to improving the encapsulation amount and encapsulation stability of the matrix for the targeted metal composite nanoparticles loaded with collagen peptides, resulting in a low encapsulation rate and inability to effectively reduce the inactivation and degradation effects of the outside world; the osteoporosis repair-type bone peptide composition of Comparative Example 3 does not contain copper peroxide black phosphorus composite particles, biocrosslinking agents and gold nanorods, and only has the encapsulation effect of ZIF-8 nanoparticles. At the same time, the injectable gel delivery matrix cannot more stably adsorb the ZIF-8 nanoparticles loaded with collagen peptides, resulting in a low encapsulation rate and inability to effectively reduce the inactivation and degradation effects of the outside world.

[0101] Experimental Example 2

[0102] Allergic reaction test

[0103] Test sample: osteoporosis repairing osteopeptide composition prepared in Examples 1-4 and Comparative Examples 1-3.

[0104] Test method: 70 SD male rats, each weighing 220g (purchased from Chengdu Dashuo Animal Co., Ltd.), were selected and randomly divided into 7 groups of 10 rats in each group. The rats were housed in separate cages, which were kept clean and dry. The temperature was 24℃ and the humidity was 50%. The rats had free access to food and water. After feeding for 10 days, 4mg of the test sample was injected once a day for 20 days. The allergic rate (%) of the rats was observed and recorded.

[0105] Figure 3The allergy rate results of Examples 1-4 and Comparative Examples 1-3 are shown in the figure. As shown in the figure, the allergy rate of Examples 1-4 is 10-20%, indicating that there are fewer adverse reactions; the allergy rate of Comparative Examples 1-3 is 60-90%, indicating that there are more adverse reactions; the targeted metal composite nanoparticles loaded with collagen peptides in Comparative Example 1 do not contain copper peroxide black phosphorus composite particles, which cannot form protection on the surface of the ZIF-8 nanoparticles loaded with collagen peptides, 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 Without biocrosslinking agents and gold nanorods, the delivery matrix cannot form a highly crosslinked, tightly connected network structure, which is not conducive to the slow release of the targeted metal composite nanoparticles loaded with collagen peptides on the structure, nor is it conducive to the encapsulation stability thereof, resulting in more adverse reactions; the osteoporosis repair-type bone peptide composition of Comparative Example 3 does not contain copper peroxide black phosphorus composite particles, biocrosslinking agents and gold nanorods, which is not conducive to the encapsulation effect of the active substance collagen peptide, nor can it reduce the irritation of the collagen peptide by a slow release mode of action, resulting in more adverse reactions.

[0106] Experimental Example 3

[0107] Treatment effect experiments

[0108] Test sample: osteoporosis repairing osteopeptide composition prepared in Examples 1-4 and Comparative Examples 1-3.

[0109] Test method: 140 patients with varying degrees of osteoporosis, with symptoms such as fatigue, bone pain, difficulty breathing, loose teeth, hunchback, and easy fractures, aged between 50 and 75 years old, were selected. A random number table was used to determine the patients' enrollment. They were divided into 7 groups, with 20 patients in each group. Each group received the corresponding test sample. The test sample was injected once a day, and the effect was observed after 7 days. The treatment effect was determined according to the following criteria, and the treatment efficiency (%) was calculated, specifically:

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

[0111] Ineffective: There was no significant improvement in osteoporosis symptoms.

[0112] The formula for calculating the treatment effectiveness is as follows:

[0113] Treatment effectiveness (%) = effective number of people / 20×100%

[0114] Figure 4The therapeutic efficacy results of Examples 1-4 and Comparative Examples 1-3 are shown in FIG. As shown in the figure, the therapeutic efficacy of Examples 1-4 is 85-95%, indicating that the therapeutic effect is better; the therapeutic efficacy of Comparative Examples 1-3 is 55-70%, indicating that the therapeutic effect is poor; the targeted metal composite nanoparticles loaded with collagen peptide in Comparative Example 1 do not contain copper peroxide black phosphorus composite particles, and cannot form protection on the surface of the ZIF-8 nanoparticles loaded with collagen peptide, which is not conducive to the stability of the collagen peptide, and only relies on the targeting effect of the injectable gel delivery matrix, which reduces the delivery activity. The accuracy of the active substance is affected, resulting in poor treatment effect; the injectable gel delivery matrix of Comparative Example 2 does not contain biocrosslinking agents and gold nanorods, and cannot more stably encapsulate the targeted metal composite nanoparticles loaded with collagen peptides to achieve precise targeted delivery in bone tissue, resulting in poor treatment effect; the osteoporosis repair-type bone peptide composition of Comparative Example 3 does not contain copper peroxide black phosphorus composite particles, biocrosslinking agents and gold nanorods, has no targeting effect, cannot achieve targeted delivery of active substances, and is not conducive to maintaining the stability of collagen peptides, resulting in poor treatment effect.

[0115] The above experimental results show that the stability, adverse reactions and therapeutic 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 targeted metal composite nanoparticles loaded with collagen peptides and an injectable gel delivery matrix has better stability, fewer adverse reactions and better therapeutic effects. The tuna 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 enhances the encapsulation effect and reduces the inactivation and degradation of the tuna collagen peptide by the external environment. At the same time, it also imparts bone targeting and good biocompatibility. The targeted metal composite nanoparticles loaded with collagen peptides are then attached to the complex network of the injectable gel delivery matrix to form a microsphere structure, which effectively protects the stability of the tuna collagen peptide. The bioavailability of the tuna collagen peptide is significantly improved by slow release and targeted release, which not only reduces the adverse reactions caused by multiple administrations, but also achieves efficient therapeutic effects.

[0116] While the embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that various changes, modifications, substitutions, and alterations can be made to the embodiments without departing from the principles and spirit of the invention.

[0117] The present invention and its embodiments are described above. Such description is not restrictive. The drawings show only one embodiment of the present invention, and actual applications are not limited thereto. In short, if a person skilled in the art is inspired by the above, and does not deviate from the purpose of the present invention, any method and embodiment similar to the technical solution without creative design shall fall within the scope of protection 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; The preparation method of the biocrosslinking agent specifically comprises the following steps: Dissolve 100.0 mg of chitosan in 5 mL of a 30% by mass dimethyl sulfoxide aqueous solution, adjust the pH to 5.0, stir until completely dissolved, and set aside. Then, dissolve chlorogenic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide in 5 mL of dimethyl sulfoxide and stir to activate for 1-2 hours. Then, mix the activated chitosan solution and chlorogenic acid solution, stir overnight, and transfer to a dialysis bag. First, dialyze with an acidic buffer solution of pH 5.

0. Change the buffer 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 biocross-linking agent.

2. A method for preparing the osteoporosis repairing osteopeptide composition according to claim 1, characterized in that: The specific steps include: S1. Dissolve 100.0 mg of chitosan in 5 mL of a 30% by mass dimethyl sulfoxide aqueous solution, adjust the pH to 5.0, stir until completely dissolved, and set aside. Then, 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 a pH 5.0 acidic buffer solution. Change the buffer solution every 12 hours. On the third day, change to ultrapure water with a pH of 7.0 and dialyze for 3-4 hours. Collect the reaction product and freeze-dry it to obtain a biocrosslinker. S2. Dissolve 1.0-2.0 g of sodium alginate in 100 mL of ultrapure water, stir evenly, add gold nanorods, magnetically stir for 1-2 h, then add the biocrosslinker described in step S1, and vibrate to mix to obtain an injectable gel delivery matrix; S3. Add the targeted metal composite nanoparticles loaded with collagen peptide to the injectable gel delivery matrix described in step S2, ultrasonically treat for 2-3 hours, and then spray it into 100 mL of a 1-2% calcium chloride solution using a portable electrospray device. Let it stand for 6-8 hours, and then rinse the gel product with ultrapure water 3-5 times to obtain a bone peptide composition for repairing osteoporosis.

3. The method for preparing the osteoporosis repairing osteopeptide composition according to claim 2, wherein: 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 osteopeptide composition according to claim 3, wherein: In step S2, the amount of gold nanorods added is 0.1-0.5 g.

5. The method for preparing the osteoporosis repairing osteopeptide 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) 8.0-9.0 g of tuna bone powder was added to 100 mL of water, and animal protease was added. The mixture was enzymatically hydrolyzed at pH 7.0 and 50-55°C for 6-8 h. After the enzymatic hydrolysis, the enzyme was inactivated at 90°C for 30 min. The mixture was then refrigerated and centrifuged at 6000-8000 rpm for 10-15 min. The supernatant was collected and the enzymatic hydrolyzate was intercepted by membrane separation. The collagen peptide with a molecular weight of 250-1000 Da was collected and freeze-dried to obtain tuna collagen peptide. (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 it by ultrasonication and stir it evenly, then add 5 mL of 0.08% sodium hydroxide solution, stir it evenly by magnetic stirring, then slowly drop 10 μL of 30% hydrogen peroxide solution, stir it for 20-30 min, and subject the reaction mixture to ultrahigh-speed centrifugation at a speed of 5000-6000 rpm for 10-20 min. Collect the precipitate, wash it with ultrapure water 3-5 times, and freeze-dry it 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 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 h, collect the precipitate, and obtain targeted metal composite nanoparticles loaded with collagen peptide.

6. The method for preparing the osteoporosis repairing osteopeptide 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 osteopeptide 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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