Bone-targeted nano-composite carrier, bone-targeted nano-composite drug and preparation method and application of bone-targeted nano-composite carrier

Abstract

The invention provides a bone targeting nano composite carrier, a bone targeting nano composite drug and a preparation method and application of the bone targeting nano composite carrier and the bone targeting nano composite drug, and belongs to the technical field of biological medicine, the bone targeting nano composite carrier comprises epigallocatechin gallate and E7 targeting peptide, and the mass ratio of the epigallocatechin gallate to the E7 targeting peptide is 1: (1.5-2.5); the epigallocatechin gallate and the E7 targeting peptide are connected through a condensation reaction. According to the invention, the bone-targeted nano composite carrier is used for entrapping an osteogenic induction component, and a bone-targeted nano composite drug is obtained through self-assembly. According to the invention, triple functions of osteogenesis promoting drugs, anti-inflammatory / anti-oxidation components and active targeting are integrated, and the bone-targeting nano composite drug not only can directly stimulate bone formation, but also can fundamentally improve the crucial pathological microenvironment in diabetic osteoporosis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a bone-targeting nano-composite carrier, a bone-targeting nano-composite drug, and a preparation method and application thereof. BACKGROUND

[0002] Globally, the incidence of diabetes is increasing year by year, and various complications caused by diabetes have gradually attracted widespread attention from the medical community, one of which is diabetic osteoporosis. Diabetic osteoporosis, as a secondary osteoporosis occurring in the background of metabolic disorders in diabetes, seriously threatens the bone health of patients. High glucose microenvironment can affect the skeletal system, significantly inhibit osteoblast function, exacerbate bone metabolism imbalance, and thus greatly increase the risk of bone fracture. The incidence of osteoporosis in diabetic patients is much higher than that in the general population, which brings great physical pain to patients and seriously affects the quality of life of patients. Current anti-osteoporosis drugs have problems such as poor long-term efficacy and obvious side effects, so it is of great significance to develop new treatment strategies for bone metabolism abnormalities in the context of diabetes.

[0003] Current treatment strategies are single and difficult to cope with complex pathological environments: existing solutions focus on simply promoting osteogenesis (such as using a single osteogenic inducer) or resisting absorption, which seriously ignores the two key pathological links of chronic inflammation and oxidative stress that persist in diabetic osteoporosis. This single mechanism treatment strategy is difficult to fundamentally reverse the imbalance of the bone microenvironment in the context of diabetes, resulting in poor treatment effect.

[0004] Current treatment strategies lack targeting and have high risk of systemic side effects: conventional drug administration distributes widely in the body and is difficult to effectively enrich in the bone lesion site. In order to achieve effective treatment, it is often necessary to increase the dosage, which not only reduces the utilization efficiency of the drug, but also increases the potential risk of adverse reactions caused by systemic exposure.

[0005] Current drug carrier materials have single function, and insufficient biocompatibility and functionality: although various nano-carriers have been used to improve delivery efficiency, most of the carriers themselves are only inert film materials and do not have biological activity. The commonly used synthetic carriers also have cytotoxicity problems. They can only solve the problem of "delivery", but cannot participate in the process of "treatment", and cannot coordinate the regulation of the disease microenvironment. SUMMARY

[0006] Therefore, the purpose of the present application is to provide a bone-targeting nano-composite carrier, a bone-targeting nano-composite drug, and a preparation method and application thereof.

[0007] The bone-targeting nano-composite carrier provided by the application is obtained by condensation of epigallocatechin gallate and E7 targeting peptide molecules, and has good biocompatibility; the E7 peptide can specifically recognize and target bone marrow mesenchymal stem cells, and improve the enrichment of drugs in bone tissue sites; as a natural polyphenol compound, EGCG not only participates in carrier assembly as a structural unit, but also plays the functions of antioxidation, anti-inflammation and promotion of osteogenic differentiation, and cooperatively improves the impaired osteogenic microenvironment under the condition of diabetes.

[0008] The bone-targeting nano-composite carrier is used to encapsulate osteogenic induction active ingredients to obtain a bone-targeting nano-composite drug, which integrates the triple functions of pro-osteogenic drugs, anti-inflammatory / antioxidant ingredients (EGCG) and active targeting (E7 peptide) into one. The bone-targeting nano-composite drug can not only directly stimulate bone formation, but also fundamentally improve the pathological microenvironment which is crucial for diabetic osteoporosis.

[0009] The application provides a bone-targeting nano-composite carrier, which comprises epigallocatechin gallate and E7 targeting peptide, and the mass ratio of the epigallocatechin gallate to the E7 targeting peptide is 1: (1.5-2.5); and the epigallocatechin gallate and the E7 targeting peptide are connected through a condensation reaction.

[0010] The application provides a preparation method of the bone-targeting nano-composite carrier, which comprises the following steps: 1) mixing epigallocatechin gallate, E7 targeting peptide and water to obtain a mixed solution through dissolution; 2) adding a polyformaldehyde solution dropwise into the mixed solution to perform a condensation reaction, and collecting colloidal particles to obtain the bone-targeting nano-composite carrier; The concentration of the epigallocatechin gallate in the mixed solution is 0.5-1.5 mg / ml. The concentration of the E7 targeting peptide in the mixed solution is 1-3 mg / ml. The volume ratio of the polyformaldehyde solution to the mixed solution is 1:40-60.

[0011] Preferably, the dissolution process in step 1) is accompanied by stirring, and the stirring speed is 550-650 rpm.

[0012] Preferably, the concentration of the polyformaldehyde solution in step 2) is 8-12% (w / v).

[0013] Preferably, the condensation reaction in step 2) is performed for 1.5-2.5 h, and the condensation reaction process is accompanied by stirring, and the stirring speed is 550-650 rpm.

[0014] The application provides a bone-targeting nano-composite drug, wherein an osteoinductive component is loaded in the bone-targeting nano-composite carrier to obtain a self-assembly.

[0015] Preferably, the osteoinductive component comprises sodium beta-glycerophosphate.

[0016] The application provides a preparation method of the bone-targeting nano-composite drug, comprising the following steps: S1) dissolving the bone-targeting nano-composite carrier in water to obtain a colloidal particle solution; S2) mixing the osteoinductive component with the colloidal particle solution, adjusting the pH to 3.8-4.2, and performing self-assembly to obtain the bone-targeting nano-composite drug.

[0017] Preferably, the concentration of the bone-targeting nano-composite carrier in the colloidal particle solution is 20-50 mg / ml; the concentration of the osteoinductive component is 30-60 mg / ml; and the volume ratio of the osteoinductive component to the colloidal particle aqueous solution is 1: (0.8-1.2).

[0018] The application provides the bone-targeting nano-composite carrier, the bone-targeting nano-composite drug prepared by the preparation method, and application of the bone-targeting nano-composite carrier and the bone-targeting nano-composite drug prepared by the preparation method in preparation of a drug for treating diabetic osteoporosis.

[0019] Compared with the prior art, the application has the following beneficial effects: The bone-targeting nano-composite carrier provided by the application is a copolymer formed by chemical condensation reaction of a natural active molecule epigallocatechin gallate (EGCG) and E7 peptide capable of specifically targeting bone marrow mesenchymal stem cells, and has good biocompatibility. The E7 peptide can specifically recognize and target bone marrow mesenchymal stem cells, and improve the enrichment of the drug in bone tissue. Through the specific targeting effect of the E7 peptide on bone marrow mesenchymal stem cells (BMMSCs), the drug can be "navigated" to the active site most in need of repair in the bone tissue. This can greatly improve the effective concentration of the drug at the bone lesion, avoid the invalid dissipation of the drug in non-target tissues, and thus achieve a stronger local treatment effect with a lower systemic drug dose. As a natural polyphenolic compound, EGCG not only participates in the assembly of the carrier as a structural unit, but also plays the functions of antioxidant, anti-inflammatory and pro-osteogenic differentiation, and cooperatively improves the impaired osteogenic microenvironment under the diabetic state.

[0020] The application utilizes the bone-targeting nano-composite carrier to encapsulate the bone-inducing active ingredients, to obtain a bone-targeting nano-composite drug, and the application integrates the bone formation promoting active ingredients, anti-inflammatory / anti-oxidation and active targeting into one; the bone-targeting nano-composite drug can not only directly stimulate bone formation, but also fundamentally improve the pathological microenvironment (chronic inflammation + oxidative stress) which is crucial in diabetic osteoporosis, breaks the vicious cycle of "high glucose→inflammation / oxidative stress→bone formation inhibition→bone loss", and is expected to achieve more thorough and lasting effects. The bone-targeting nano-composite drug provided by the application simultaneously realizes bone-targeting delivery, bone-inducing and microenvironment regulation, and provides a new idea and experimental basis for the comprehensive treatment of diabetic osteoporosis. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 For performance detection of the bone-targeting nano-composite drug, a: the Tyndall effect of the β-GP@EGCG-E7 solution; b: the transmission electron microscope image of the β-GP@EGCG-E7 (scale = 100 nm); c: the particle size distribution curve of the β-GP@EGCG-E7; Figure 2 For detection of the cytotoxicity of the bone-targeting nano-composite drug β-GP@EGCG-E7; Figure 3 For detection of the relative expression levels of the antioxidant-related genes Sod2, Nrf2 and Gpx1 by qRT-PCR; statistical analysis adopts one-way ANOVA, p < 0.05 indicates that the difference has statistical significance; Figure 4 For detection of the relative expression levels of the inflammation and macrophage polarization-related genes Tnf-α, IL-1β, iNOS and Arg1 by qRT-PCR; statistical analysis adopts one-way ANOVA, p < 0.05 indicates that the difference has statistical significance; Figure 5 For detection of the alkaline phosphatase (ALP) activity and calcium nodule formation of the osteogenic differentiation marker in the osteoblasts treated in different manners; wherein a: the alkaline phosphatase (ALP) staining image (scale = 200 μm); b: the alizarin red S (ARS) staining image (scale = 200 μm); Figure 6 For target evaluation of the bone-targeting nano-composite drug β-GP@EGCG-E7, the representative fluorescence images of the femurs of the two groups of mice at 6, 12 and 24 hours after intravenous injection of the drug; Figure 7Evaluation of the osteogenic effect of beta-GP@EGCG-E7 in diabetic osteoporotic mice, wherein a: induction and treatment time chart of diabetic mouse model; b: micro-CT imaging of mouse femur, including two-dimensional images of coronal and transverse sections, and three-dimensional reconstruction images of transverse sections; c: proximal femur bone mass and bone density analysis of mice, to evaluate bone trabecula neogenesis, including parameters such as Tb.Th, Tb.N, Tb.PF and BV / TV; statistical analysis uses one-way ANOVA, p < 0.05 indicates that the difference is statistically significant. DETAILED DESCRIPTION

[0022] The application provides a bone-targeting nano composite carrier, which comprises epigallocatechin gallate and E7 targeting peptide, and the mass ratio of the epigallocatechin gallate and the E7 targeting peptide is 1: (1.5-2.5); the epigallocatechin gallate and the E7 targeting peptide are connected through a condensation reaction.

[0023] In the application, the mass ratio of the epigallocatechin gallate and the E7 targeting peptide is preferably 1: (1.8-2.2), and more preferably 1:2; the E7 targeting peptide is EPLQLKM. In the application, the condensation of the epigallocatechin gallate and the E7 targeting peptide is preferably achieved through a condensing agent, and the condensing agent is preferably a paraformaldehyde solution.

[0024] The application provides a preparation method of the bone-targeting nano composite carrier, which comprises the following steps: 1) mixing epigallocatechin gallate, E7 targeting peptide and water to obtain a mixed solution; 2) adding a paraformaldehyde solution dropwise into the mixed solution to perform a condensation reaction, and collecting colloidal particles to obtain the bone-targeting nano composite carrier.

[0025] In the application, the epigallocatechin gallate, the E7 targeting peptide and water are mixed to obtain a mixed solution. In the application, the water is preferably deionized water; the concentration of the epigallocatechin gallate in the mixed solution is preferably 0.5-1.5 mg / ml, more preferably 0.7-1.3 mg / ml, and most preferably 0.9-1.1 mg / ml; the concentration of the E7 targeting peptide in the mixed solution is preferably 1-3 mg / ml, more preferably 1.5-2.5 mg / ml, and most preferably 1.8-2.2 mg / ml. In the application, the dissolution process is accompanied by stirring, and the stirring speed is preferably 550-650 rpm, more preferably 580-620 rpm, and most preferably 600 rpm; in the application, the dissolution time is preferably 30-60 min.

[0026] In this invention, after obtaining the mixed solution, a paraformaldehyde solution is added dropwise to the mixed solution to initiate a condensation reaction. The collected colloidal particles are the bone-targeting nanocomposite carrier. In this invention, the volume ratio of the paraformaldehyde solution to the mixed solution is preferably 1:40~60, more preferably 1:45~55, and most preferably 1:48~52. In this invention, the concentration of the paraformaldehyde solution is preferably 8~12% (w / v), more preferably 9~11% (w / v), and most preferably 10% (w / v). In this invention, the condensation reaction time is preferably 1.5~2.5 h, more preferably 1.8~2.2 h, and most preferably 2 h; the condensation reaction is accompanied by stirring, and the stirring speed is preferably 550~650 rpm, more preferably 580~620 rpm, and most preferably 600 rpm. After the condensation reaction is completed, the colloidal particles are collected. Preferred method of collection is centrifugation, with a centrifugal force preferably of 6500-7500 g, more preferably 6800-7200 g, and most preferably 7000 g. The centrifugation time is preferably 8-12 min, more preferably 9-11 min, and most preferably 10 min. After obtaining the colloidal particles, they are preferably washed with deionized water, preferably 2-4 times, more preferably 3 times. The colloidal particles are preferably stored at 3-5°C.

[0027] The present invention also provides a bone-targeting nanocomposite drug, which is obtained by self-assembly of osteogenic inducing components encapsulated in the bone-targeting nanocomposite carrier.

[0028] In this invention, the osteogenic inducing component preferably includes sodium β-glycerophosphate or zoledronic acid. The concentration of the osteogenic inducing component in the bone-targeting nanocomposite drug is preferably 10-40 mg / ml, more preferably 15-30 mg / ml, and most preferably 20-25 mg / ml.

[0029] The present invention also provides a method for preparing the bone-targeting nanocomposite drug, comprising the following steps: S1) dissolving the bone-targeting nanocomposite carrier in water to obtain a colloidal particle solution; S2) mixing the osteogenic inducing component with the colloidal particle solution, adjusting the pH to 4.2~4.7, and performing self-assembly to obtain the bone-targeting nanocomposite drug.

[0030] In this invention, the bone-targeting nanocomposite carrier is dissolved in water to obtain a colloidal particle solution; the concentration of the bone-targeting nanocomposite carrier in the colloidal particle solution is preferably 20-50 mg / ml, more preferably 30-45 mg / ml, and most preferably 40 mg / ml; the osteogenic inducing component is preferably used in solution form, and the concentration of the osteogenic inducing component is preferably 30-60 mg / ml, more preferably 35-45 mg / ml, and most preferably 40 mg / ml.

[0031] In this invention, osteogenic inducing components are mixed with a colloidal particle solution, and the pH is adjusted to 3.8-4.2, followed by self-assembly to obtain a bone-targeting nanocomposite drug. The preferred volume ratio of the osteogenic inducing components to the colloidal particle aqueous solution is 1:(0.8-1.2), more preferably 1:(0.9-1.1), and most preferably 1:1. After mixing, hydrochloric acid is preferably used to adjust the pH, more preferably to 4.2-4.7, and most preferably 4.5. The purpose of adjusting the pH is to prevent oxidation of the EGCG colloid due to pH changes caused by the addition of sodium β-glycerophosphate. The osteogenic inducing components and colloidal particles self-assemble under the above conditions to obtain a bone-targeting nanocomposite drug.

[0032] This invention provides the bone-targeting nanocomposite carrier, the bone-targeting nanocomposite carrier prepared by the aforementioned method, the bone-targeting nanocomposite drug, and the application of the bone-targeting nanocomposite drug prepared by the aforementioned method in the preparation of a drug for treating diabetic osteoporosis. The bone-targeting nanocomposite drug of this invention, when applied to diabetic osteoporosis model mice, significantly improved key trabecular bone parameters such as bone volume fraction (BV / TV), trabecular bone number (Tb.N), and trabecular bone thickness (Tb.Th), while simultaneously significantly reducing the trabecular bone pattern factor (Tb.Pf), indicating that β-GP@EGCG-E7 can effectively increase bone mass and improve bone microstructure.

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1

[0035] Preparation of bone-targeting nanocomposite carriers

[0036] Synthesis of EGCG colloid: 10 mg EGCG and 20 mg E7 peptide were dissolved in 10 ml of deionized water and stirred at 600 rpm until completely dissolved. Then, 200 μL of 10% paraformaldehyde was slowly added dropwise, and the mixture was stirred at 600 rpm for 2 hours at room temperature. The colloidal particles were collected by centrifugation at 7000g for 10 minutes and washed three times with deionized water to obtain the bone-targeting nanocomposite carrier EGCG-E7, which was stored at 4°C for later use.

[0037] Preparation of bone-targeting nanocomposite drugs

[0038] 1) Dissolve the bone-targeting nanocomposite carrier EGCG-E7 (colloidal particles) in deionized water to prepare a colloidal solution with a concentration of 40 mg / mL; 2) Then, β-GP was dissolved in water to prepare a β-GP aqueous solution with a concentration of 40 mg / mL; 3) Add 100 µL of β-GP aqueous solution to 100 µL of colloidal solution and mix thoroughly. Adjust the pH to 4.5 with HCl to induce the self-assembly of the nanomedicine and prepare the bone-targeting nanocomposite drug β-GP@EGCG-E7 with a drug concentration of 20 mg / mL.

[0039] 1. Observations on colloid stability and the Tyndall effect

[0040] Method: The prepared aqueous solution of β-GP@EGCG-E7 nanoparticles was placed in a transparent glass sample bottle.

[0041] Instrumentation: A red laser pointer with a wavelength of 650 nm is used as the light source.

[0042] Procedure: In a dark environment, pass the laser beam perpendicularly through the sample solution and observe whether a clear light path appears from the direction perpendicular to the beam.

[0043] Conclusion determination: For example Figure 1 As shown in a, a significant Tyndall effect was observed, proving that the system is a colloidal dispersion, and the nanoparticles are uniformly and stably distributed in the solution without significant sedimentation or aggregation.

[0044] 2. Observation by transmission electron microscopy

[0045] Instrument: JEM-1400PLUS transmission electron microscope (JEOL).

[0046] Accelerating voltage: 120 kV

[0047] Sample preparation (negative staining): The β-GP@EGCG-E7 nanoparticle solution was appropriately diluted with ultrapure water.

[0048] Take a drop (about 5~10 μL) of the diluted solution and drop it onto the copper grid supported by the ultrathin carbon film.

[0049] After letting it stand for a few minutes, use filter paper to absorb the excess liquid from the edges.

[0050] Then add one drop of 2% (w / v) phosphotungstic acid (PTA) solution for negative staining. After standing for several tens of seconds, use filter paper to absorb the excess staining solution again.

[0051] Place the copper mesh in a petri dish and allow it to air dry at room temperature.

[0052] Imaging and Analysis: Observe the particle morphology and acquire images at appropriate magnification.

[0053] Transmission electron microscopy further confirmed that the nanoparticles are regular spheres with a diameter of approximately 100 nm. Figure 1 (b) After drug loading, the particle size of the nanoparticles increased significantly, providing direct morphological evidence for successful drug loading. Figure 1 (c in the text)

[0054] Example 2

[0055] Bioactivity assay of bone-targeted nanocomposite drugs

[0056] Cytotoxicity assay: MC3T3-E1 cells (purchased from Wuhan Pronosei Biotechnology Co., Ltd.) were co-cultured at different concentrations of β-GP@EGCG-E7 for 24 h, 72 h, and 120 h, respectively. Cell viability was then assessed using a CCK-8 assay kit. The results are as follows: Figure 2 As shown in the figure. The cytotoxicity assessment results showed that after co-culturing cells with β-GP@EGCG-E7 nanoparticles at different concentrations for 1, 3 and 5 days, respectively, no significant inhibition of cell viability was observed, proving that it had no obvious cytotoxicity within this time range.

[0057] Anti-inflammatory and antioxidant assays: For antioxidant studies, MC3T3-E1 cells were pre-cultured with different concentrations of β-GP@EGCG-E7 for 5 hours, and then replaced with freshly prepared high-purity β-GP@EGCG-E7. Samples were collected 2-6 hours after inducing oxidative stress in a culture medium with a final concentration of 100 μmol / L. For inflammation studies, Raw264.7 cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) were cultured for 24 hours with lipopolysaccharide (LPS, final concentration 1 μg / mL) added simultaneously with β-GP@EGCG-E7 to establish an inflammation model. Cells were collected and RNA extracted. The expression levels of antioxidant and inflammation-related genes were detected by qPCR. The results are as follows: Figure 3 and Figure 4As shown.

[0058] MC3T3-E1: G1-G4 represent: Control, H2O2 (100 μmol / L), EGCG (100 μg / mL), β-GP@EGCG-E7 (100 μg / mL), respectively; Raw264.7: L1-L4 represent: Control, LPS (1μg / mL), EGCG (100μg / mL), β-GP@EGCG-E7 (100μg / mL), respectively.

[0059] Experimental results showed that β-GP@EGCG-E7 treatment significantly regulated antioxidant and anti-inflammatory pathways. In MC3T3-E1 cells, it effectively upregulated the expression of key antioxidant genes (Sod2, Nrf2, and Gpx1). Figure 3 Simultaneously, in Raw264.7 cells, this drug significantly inhibited the expression of pro-inflammatory genes (TNF-α, IL-1β, and iNOS) and promoted the upregulation of the anti-inflammatory gene Arg-1, demonstrating its bidirectional regulatory effect. Figure 4 ).

[0060] Osteogenesis testing:

[0061] MC3T3-E1 osteoblasts were co-cultured with 100 μg / mL β-GP@EGCG-E7 for 7 days and 21 days, respectively, and the activity of alkaline phosphatase (ALP), a marker of osteogenic differentiation, and the formation of calcium nodules were detected.

[0062] The control group set up in this experiment included: OM group (basal α-MEM medium + 10% FBS + 1% antibiotics, and supplemented with 10 mM β-GP + 10 mM vitamin C + 1 mM dexamethasone). The β-GP group, EGCG group, and FPBE group were all supplemented with 100 μg / mL β-GP, 100 μg / mL EGCG, and 100 μg / mL β-GP@EGCG-E7, respectively, on a high-glucose, high-inflammatory OM medium (containing 35 mmol / L glucose + 1 μg / mL LPS).

[0063] The experimental results are summarized in Figure 5 The results showed that ALP staining in the β-GP@EGCG-E7 treatment group ( Figure 5 (a, scale bar = 200 μm) and Alizarin Red staining ( Figure 5 The depth of b in the model (scale bar = 1000 μm) was significantly greater than that in the control group, indicating enhanced ALP activity and calcium nodule deposition.

[0064] Targeting evaluation of β-GP@EGCG-E7: β-GP (100 μg / mL) with Cy5 fluorescence and β-GP@EGCG-E7 (100 μg / mL) with Cy5 fluorescence were injected into mice via the tail vein in 200 μL. In vivo imaging of the small animals was performed at 6 h, 12 h, and 24 h.

[0065] β-GP with Cy5 fluorescence (100 μg / mL) was synthesized by Xi’an Ruixi Biotechnology Co., Ltd. The preparation method of β-GP@EGCG-E7 with Cy5 fluorescence is described in Example 1, except that the raw material β-GP is replaced with fluorescent β-GP.

[0066] The results showed that the femoral fluorescence intensity in the β-GP@EGCG-E7 group was significantly increased compared with that in the β-GP group. Figure 6 ).

[0067] Example 3

[0068] Evaluation of the osteogenic effect of β-GP@EGCG-E7 prepared in Example 1 in diabetic osteoporotic mice

[0069] Eight-week-old male C57BL / 6J mice were selected. To induce a diabetic osteoporosis model, streptozotocin (STZ) was first used to establish the diabetes model. STZ was prepared fresh in 0.1M citrate buffer (pH 4.5) and stored on ice protected from light. Mice were randomly divided into groups and injected intraperitoneally with STZ (180 mg / kg, single dose), while the control group received an equal volume of 0.1M citrate buffer. After injection, mice were given 5% glucose solution for 24 hours to prevent acute hypoglycemia. From day 7 post-injection, fasting blood glucose levels were monitored daily. Two consecutive fasting blood glucose levels ≥16.7 mmol / L were considered successful in inducing the diabetes model. Drug administration began in week 4 after diabetes stabilized. The experiment consisted of five groups: The control group was the control group that did not receive any treatment; The STZ group consisted of mice that were injected with saline to establish a diabetic osteoporosis model. The β-GP group consisted of mice with a diabetic osteoporosis model who were injected with β-GP (50 mg / kg). The EGCG group consisted of mice with a diabetic osteoporosis model who were injected with EGCG (50 mg / kg). The β-GP@EGCG-E7 group was a diabetic osteoporosis model that was injected with a small amount (50 mg / kg) of the β-GP@EGCG-E7 complex.

[0070] All drug dosages were calculated based on body weight and uniformly dissolved in 200 μL of normal saline before intraperitoneal injection. Intraperitoneal injection was administered every other day for 4 weeks.

[0071] To assess the femoral bone microstructure, the left femur of mice was dissected after the experiment. After fixation, the femoral metaphysis was scanned using a high-resolution Micro-CT scanner. Key scanning parameters were set as follows: resolution 6 μm, voltage 50 kV, current 200 μA, and a 0.25 mm aluminum filter. After 3D reconstruction, a cubic region of 100 consecutive slices (approximately 0.6 mm thick) below the femoral metaphysis was selected as the region of interest for bone morphometric analysis. Analytical parameters included bone volume fraction (BV / TV), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), and trabecular bone pattern factor (Tb.Pf). All data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups. p < 0.05 is considered statistically significant.

[0072] The results showed that the bone mineral density, as well as the number and thickness of trabeculae, were significantly increased in the β-GP@EGCG-E7 treatment group mice. (p < 0.05) Figure 7 Micro-CT analysis showed that, compared with the control group, the STZ group mice had significantly reduced bone mass and degenerated trabecular bone structure, confirming the successful establishment of the diabetic osteopenia model. Compared with the STZ group, the β-GP@EGCG-E7 treatment group showed significantly improved key trabecular bone parameters such as bone volume fraction (BV / TV), trabecular bone number (Tb.N), and trabecular bone thickness (Tb.Th), while the trabecular bone pattern factor (Tb.Pf) was significantly reduced, indicating that β-GP@EGCG-E7 can effectively increase bone mass and improve bone microstructure. Figure 7 (B and C in the text).

[0073] As can be seen from the above embodiments, the bone-targeting nanocomposite drug provided by the present invention integrates the triple functions of bone drug, anti-inflammatory / antioxidant components and active targeting into one, which can not only directly stimulate bone formation, but also fundamentally improve the pathological microenvironment that is crucial in diabetic osteoporosis; providing new ideas and experimental evidence for the comprehensive treatment of diabetic osteoporosis.

[0074] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A bone-targeting nanocomposite carrier, characterized in that, It includes epigallocatechin gallate and E7 targeting peptide, wherein the mass ratio of epigallocatechin gallate and E7 targeting peptide is 1:(1.5~2.5); the epigallocatechin gallate and E7 targeting peptide are linked by a condensation reaction.

2. The method for preparing the bone-targeting nanocomposite carrier according to claim 1, characterized in that, Includes the following steps: 1) Mix epigallocatechin gallate, E7 targeting peptide with water to dissolve and obtain a mixed solution; 2) Add paraformaldehyde solution dropwise to the mixed solution to carry out a condensation reaction, and collect the colloidal particles to obtain the bone-targeting nanocomposite carrier; The concentration of epigallocatechin gallate in the mixed solution is 0.5~1.5 mg / ml; The concentration of the E7-targeting peptide in the mixed solution is 1-3 mg / ml; The volume ratio of the paraformaldehyde solution to the mixed solution is 1:40~60.

3. The preparation method according to claim 2, characterized in that, Step 1) The dissolution process is accompanied by stirring, and the stirring speed is 550~650 rpm.

4. The preparation method according to claim 2, characterized in that, Step 2) The concentration of the paraformaldehyde solution is 8~12% (w / v).

5. The preparation method according to claim 4, characterized in that, Step 2) The condensation reaction takes 1.5 to 2.5 hours, and the condensation reaction is accompanied by stirring at a speed of 550 to 650 rpm.

6. A bone-targeting nanocomposite drug, characterized in that, Osteogenic inducing components are encapsulated in the bone-targeting nanocomposite carrier according to claim 1 and obtained by self-assembly, wherein the concentration of the osteogenic inducing components is 10~40 mg / ml.

7. The bone-targeting nanocomposite drug according to claim 6, characterized in that, The osteogenic inducing component includes sodium β-glycerophosphate.

8. The method for preparing the bone-targeting nanocomposite drug according to claim 6 or 7, characterized in that, Includes the following steps: S1) Dissolve the bone-targeting nanocomposite carrier in water to obtain a colloidal particle solution; S2) The osteogenic inducing component is mixed with the colloidal particle solution, the pH is adjusted to 4.2-4.7, and self-assembly is performed to obtain bone-targeting nanocomposite drugs.

9. The preparation method according to claim 8, characterized in that, The concentration of the bone-targeting nanocomposite carrier in the colloidal particle solution is 20-50 mg / ml; the concentration of the osteogenic inducing component is 30-60 mg / ml; and the volume ratio of the osteogenic inducing component to the colloidal particle aqueous solution is 1:(0.8-1.2).

10. The use of the bone-targeting nanocomposite carrier according to claim 1, the bone-targeting nanocomposite carrier prepared by the preparation method according to any one of claims 2 to 5, the bone-targeting nanocomposite drug according to claim 6, and the bone-targeting nanocomposite drug prepared by the preparation method according to any one of claims 7 to 9 in the preparation of a drug for treating diabetic osteoporosis.

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