Chrysin PEG-PCL nanomicelles modified with alendronic acid and their application in the preparation of drugs for treating osteoporosis

Abstract

The invention discloses an alendronic acid (ALN)-modified Chrysin PEG-PCL (ALN@PEG-PCL@Chrysin) nano-micelle and its application in the preparation of a drug for treating osteoporosis, belonging to the technical field of pharmaceutical preparations. The invention aims to improve the water solubility, stability and bioavailability of Chrysin, while enhancing its bone targeting and anti-osteoporosis effect. The nano-micelle uses PEG-PCL as a carrier, encapsulates the hydrophobic drug Chrysin through a self-assembly process, and modifies alendronic acid on the surface of the micelle to enhance the bone targeting effect. The nano-micelle can not only effectively prevent the sudden release of the drug, but also realize the sustained release and dosage control of the drug through material encapsulation. The research results show that the nano-micelle has good particle size distribution, drug encapsulation rate and stability, and exhibits a significant sustained release effect during the in vitro release process. In vitro and in vivo experiments further confirm the potential application value of the nano-micelle in the treatment of osteoporosis, and provide new ideas and methods for the clinical application of Chrysin and other similar water-insoluble compounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to an alendronic acid (ALN)-modified ChrysinPEG-PCL (ALN@PEG-PCL@Chrysin) nano-micelle and an application thereof in the preparation of a drug for treating osteoporosis. Background Art

[0002] Osteoporosis is a metabolic bone disease characterized by osteopenia and bone microarchitecture degradation, which seriously affects a large number of middle-aged and elderly people around the world, especially postmenopausal women. Existing treatments for osteoporosis mainly include calcium supplements, vitamin D, hormone replacement therapy and bisphosphonates (such as alendronic acid, ALN, etc.). Among them, bisphosphonates have become an important choice for the treatment of osteoporosis due to their high targeting to bone tissue and obvious effects in inhibiting osteoclast activity. However, bisphosphonates may cause some side effects during treatment, such as gastrointestinal discomfort, hypocalcemia, and osteosclerosis caused by long-term use, which limits their application.

[0003] At the same time, Chrysin is a flavonoid compound. In previous studies by the inventor's research group, it was found that it is an important natural compound with multiple biological activities such as antioxidant, anti-inflammatory, and anti-apoptotic. Especially in inhibiting osteoclast activity and differentiation and promoting osteoblast differentiation, the inventor's previous studies have proved that Chrysin has significant therapeutic potential, and relevant patents have been applied for. However, Chrysin faces some challenges in drug applications, mainly due to its poor water solubility, low bioavailability, and rapid metabolism and excretion in the body. These problems limit the application of Chrysin in the treatment of osteoporosis.

[0004] In order to solve the problem of poor water solubility and low bioavailability of Chrysin, nano drug delivery systems have attracted widespread attention in recent years. Nano micelles can effectively encapsulate hydrophobic drugs due to their unique core-shell structure, improve the stability and sustained-release properties of drugs, and improve the targeted delivery effect of drugs by modifying specific targeting ligands on the surface. However, how to construct a nano micelle system with both bone targeting and sustained-release functions has become a key challenge in current drug delivery technology.

[0005] In summary, the prior art has the following defects and deficiencies:

[0006] (1) Chrysin has poor water solubility and low bioavailability

[0007] Chrysin is a potential anti-osteoporosis drug, and its therapeutic effect has been verified in in vitro studies. However, due to its poor water solubility, the drug absorption rate is low when used in vivo, making it difficult to achieve an effective concentration. In addition, Chrysin is rapidly metabolized in the body and has a short half-life, which further reduces its bioavailability and limits its application as an effective drug.

[0008] (2) Side effects of existing osteoporosis drugs

[0009] Although bisphosphonates such as alendronic acid have a good therapeutic effect on osteoporosis, their long-term use may cause side effects such as digestive discomfort and hypocalcemia, and may even lead to bone sclerosis. Existing treatment options cannot take into account bone targeting, effectiveness, and the need to reduce side effects at the same time.

[0010] (3) Difficulty in drug release control

[0011] Although the existing nano-micelle technology can improve the water solubility and stability of drugs to a certain extent, it faces great challenges in controlling drug release, and is prone to rapid drug release (burst release), resulting in premature disappearance of drug efficacy or increased toxic side effects. How to control the sustained release of drugs through effective carrier design to achieve long-term drug efficacy maintenance is another major shortcoming of the existing technology.

[0012] (4) Poor bone targeting

[0013] Traditional nano drug delivery systems lack the function of specifically targeting bone tissue, and the drug is widely distributed in the body, which easily leads to drug accumulation in non-targeted sites and increases toxic side effects. Although the concept of targeted delivery has been proposed in some studies, there is still much room for improvement in the specific targeted delivery of bone tissue.

[0014] Based on the above reasons, this application is filed. Summary of the invention

[0015] Based on the above reasons, in view of the problems or defects existing in the prior art, the purpose of the present invention is to provide an alendronic acid modified Chrysin PEG-PCL (ALN@PEG-PCL@Chrysin) nano-micelle and its use in the preparation of anti-osteoporosis drugs, so as to solve or at least partially solve the above technical defects existing in the prior art: The present invention adopts nano-micelle carrier technology to improve the water solubility and stability of Chrysin, enhance its bioavailability in the body, so as to better play its role in the treatment of osteoporosis.

[0016] In order to achieve the above first object of the present invention, the technical solution adopted by the present invention is as follows:

[0017] A method for preparing alendronic acid-modified Chrysin PEG-PCL (ALN@PEG-PCL@Chrysin) nanomicelles, the method specifically comprising the following steps:

[0018] Step 1, dissolving chrysin in an organic solvent 1 according to a ratio to obtain a chrysin solution; dissolving carboxyl polyethylene glycol polycaprolactone (HOOC-PEG-PCL) in an organic solvent 2 to obtain a HOOC-PEG-PCL solution;

[0019] Step 2, slowly adding the Chrysin solution to the HOOC-PEG-PCL solution according to the ratio and mixing; then slowly dropping the obtained mixed solution into ultrapure water under ultrasound for emulsification for 3-5 minutes to form an emulsion; finally, the obtained emulsion is subjected to rotary evaporation, dialysis, concentration and filtration in sequence to obtain COOH-PEG-PCL@Chrysin nano-micelles;

[0020] Step 3, at room temperature, N-hydroxysuccinimide (NHS) and (1-ethyl-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (EDCI) are added to the organic solvent 3 in order according to the ratio, and mixed; then, COOH-PEG-PCL@Chrysin and triethylamine (TEA) described in step 2 are added to the obtained mixed solution, and the obtained reaction solution is placed in a light-proof condition to react for 7-9 hours; after the reaction is completed, an activated NHS ester intermediate is formed;

[0021] Step 4, dissolving alendronic acid (ALN) in deionized water according to a ratio to obtain an alendronic acid solution; then simultaneously adding the alendronic acid solution and the NaOH solution dropwise to the activated NHS ester intermediate in step S3, respectively, and controlling the pH value of the reaction solution to be neutral; after the addition is completed, reacting the obtained reaction solution at room temperature for 1-3 hours; after the reaction is completed, continue to dropwise add the NaOH solution to the reaction system, adjust the pH value of the obtained product to 8.5-9.5, and continue the reaction for 8-12 hours;

[0022] Step 5: After the reaction is completed, the obtained product is dialyzed and freeze-dried to obtain the ALN@PEG-PCL@Chrysin nanomicelles.

[0023] Furthermore, in the above technical solution, the organic solvent 1 in step 1 is a highly polar organic solvent, such as any one of dimethyl sulfoxide (DMSO), ethanol (Ethanol), methanol (Methanol), etc.

[0024] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the organic solvent 2 in step 1 is chloroform.

[0025] Furthermore, in the above technical solution, the amount of the organic solvent 1 in step 1 may not be specifically limited, as long as the complete and uniform dissolution of chrysin can be achieved. For example, in a preferred embodiment of the present invention, the amount ratio of chrysin to the organic solvent 1 is 15 parts by mass: 1 part by volume; wherein: the mass parts and the volume parts are based on mg: mL.

[0026] Further, in the above technical solution, the amount of the organic solvent 2 in step 1 may not be specifically limited, as long as the carboxyl polyethylene glycol polycaprolactone can be completely and evenly dissolved. For example, in a preferred embodiment of the present invention, the amount ratio of the carboxyl polyethylene glycol polycaprolactone to the organic solvent 2 is 25 parts by mass: 1 part by volume; wherein: the mass parts and the volume parts are based on mg: mL.

[0027] Furthermore, in the above technical solution, the mass ratio of chrysin to carboxyl polyethylene glycol polycaprolactone in the mixed solution of step 2 is (10-20):(70-150), and the preferred mass ratio is 3:20.

[0028] Furthermore, in the above technical solution, the volume ratio of the mixed liquid to ultrapure water in step 2 is 1:1 to 1:3.

[0029] Specifically, in the above technical solution, the emulsification process in step 2 is carried out at room temperature.

[0030] Furthermore, in the above technical solution, the rotary evaporation temperature in step 2 is controlled at 35-40° C. The purpose of the rotary evaporation is to completely remove the organic solvent 2 (such as chloroform) in the emulsion, so the time of the rotary evaporation is based on the complete removal of the organic solvent 2 (such as chloroform).

[0031] Specifically, in the above technical solution, the purpose of the dialysis in step 2 is to completely remove the organic solvent 1 (such as dimethyl sulfoxide) and other low molecular weight impurities in the emulsion, so the number of dialysis is based on the complete removal of the organic solvent 1 (such as dimethyl sulfoxide) and other low molecular weight impurities.

[0032] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the molecular cutoff of the dialysis bag used for dialysis in step 2 is 14000Da.

[0033] Furthermore, in the above technical solution, the mass ratio of NHS to EDCI in step 3 is (4-6): (5-10).

[0034] Furthermore, in the above technical solution, the organic solvent 3 in step 3 is any one of 1,4-dioxane, dichloromethane, tetrahydrofuran, etc.

[0035] Furthermore, in the above technical solution, the amount of the organic solvent 3 in step 3 may not be specifically limited, as long as the complete and uniform dissolution of NHS and EDCI can be achieved. For example, in a preferred embodiment of the present invention, the amount ratio of EDCI to organic solvent 3 is (5-10) parts by mass: (10-20) parts by volume; wherein: the mass parts and volume parts are based on mg:mL.

[0036] Furthermore, in the above technical solution, the mass ratio of EDCI to COOH-PEG-PCL@Chrysin in step 3 is (5-10): (100-130).

[0037] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the volume ratio of the organic solvent 3 to TEA in step 3 is 3 mL:4 μL.

[0038] Further, in the above technical solution, the amount of deionized water in step 4 may not be specifically limited, as long as the ALN can be completely and evenly dissolved. For example, in a preferred embodiment of the present invention, the amount ratio of ALN to deionized water is 4 parts by mass: 5 parts by volume; wherein: the mass parts and volume parts are based on mg: mL.

[0039] Furthermore, in the above technical solution, the mass ratio of ALN to NHS in step 4 is 6:(2-3).

[0040] Furthermore, in the above technical solution, the concentration of the NaOH solution in step 4 is 0.1-0.5M.

[0041] Furthermore, in the above technical solution, the molecular retention capacity of the dialysis membrane used in the dialysis in step 5 is 3.5 kDa.

[0042] The second object of the present invention is to provide ALN@PEG-PCL@Chrysin nanomicelles prepared by the above method.

[0043] The third object of the present invention is to provide the use of ALN@PEG-PCL@Chrysin nano-micelles prepared by the above method in the preparation of anti-osteoporosis drugs.

[0044] The present invention also provides a drug for treating osteoporosis, comprising the ALN@PEG-PCL@Chrysin nano-micelles prepared by the method described above.

[0045] The fourth object of the present invention is to provide the use of the ALN@PEG-PCL@Chrysin nanomicelles prepared by the above method in the preparation of osteoclast activity inhibitors and / or osteoblast differentiation promoters.

[0046] The present invention also provides an osteoclast activity inhibitor and / or osteoblast differentiation promoter, comprising the ALN@PEG-PCL@Chrysin nanomicelles prepared by the above-mentioned method of the present invention.

[0047] The main raw materials used in the present invention play the following roles:

[0048] 1. The role of EDCI is: EDCI of the present invention promotes the condensation reaction between carboxyl and amino groups to form a stable amide bond. In the present invention, EDCI is used to activate the carboxyl group in COOH-PEG-PCL@Chrysin so that it can react with NHS to generate NHS ester.

[0049] 2. The role of NHS is: the present invention activates the carboxyl group in COOH-PEG-PCL@Chrysin through the synergistic effect of NHS and EDCI to form NHS ester, thereby improving the reaction efficiency between the carboxyl group and the amino group and making the formation of amide bonds more efficient.

[0050] 3. The role of ALN is: In the present invention, ALN is modified on the surface of COOH-PEG-PCL@Chrysin nano-micelles through amide bonds, making it bone-targeted, which helps to deliver the encapsulated Chrysin to the bone in a targeted manner and enhance the accuracy of drug delivery.

[0051] 4. The function of Chrysin is: the present invention uses nano-micelle technology to encapsulate Chrysin to improve its water solubility and bioavailability, especially for the treatment of osteoporosis, showing good bone formation and anti-bone resorption effects.

[0052] 5. The role of HOOC-PEG-PCL is: PEG-PCL is an amphiphilic block copolymer, the PEG part is hydrophilic, and the PCL part is hydrophobic. In the present invention, PEG-PCL forms nano-micelles by self-assembly, the PEG shell increases the water solubility, and the PCL core wraps the hydrophobic drug Chrysin, which plays a role in sustained release and stabilization of the drug. Through carboxyl modification, it can further bind to ALN.

[0053] 6. The role of organic solvent 1 is: Organic solvent 1 not only serves as a reaction solvent, but also helps to improve the solubility of hydrophobic drugs such as Chrysin, making it easier to be subsequently encapsulated into nano-micelles. During the reaction process, organic solvent 1 provides a uniform dissolution environment, helps the reaction proceed smoothly, and ensures that the drug molecules can be effectively dispersed in the reaction system.

[0054] 7. The role of TEA is: In the present invention, TEA acts as an alkaline catalyst to neutralize the by-products generated by the reaction during the formation of the amide bond, stabilize the pH value of the reaction, and promote the reaction.

[0055] 8. The role of NaOH is: NaOH is used to adjust the pH value of the reaction solution to keep it in a suitable reaction environment and ensure that ALN can smoothly combine with PEG-PCL to form a stable amide bond.

[0056] The reaction mechanism involved in the invention is as follows:

[0057] 1. Preparation reaction mechanism of COOH-PEG-PCL@Chrysin

[0058] In the process of preparing COOH-PEG-PCL@Chrysin nanomicelles, Chrysin was first dissolved in organic solvent 1. This step is critical because the hydrophobic nature of Chrysin makes it very low in solubility in water, while organic solvent 1, as a class of highly polar organic solvents, can effectively dissolve Chrysin, providing the necessary premise for the subsequent embedding process. Then, the formed Chrysin solution was slowly added to the HOOC-PEG-PCL solution. HOOC-PEG-PCL is a polyethylene glycol polycaprolactone copolymer with a carboxyl end group, in which the PEG part of the structure is hydrophilic and the PCL part is hydrophobic. This amphiphilic copolymer can form nanoparticles through self-assembly, in which the hydrophobic core of PCL can encapsulate the hydrophobic Chrysin, while the hydrophilic shell of PEG helps to increase the stability and biocompatibility of the nanoparticles in an aqueous environment. After the Chrysin solution was mixed with the HOOC-PEG-PCL solution, the system was treated by ultrasound to promote better mixing and emulsification. The effect of ultrasound not only helps disperse the polymer and drug, but also destroys the solvent-solvent interaction of organic solvent 1 and organic solvent 2 through local high temperature and pressure, further promoting the migration of Chrysin to the PCL core. Subsequently, the mixture is slowly added dropwise to ultrapure water. Due to the difference in polarity between organic solvent 1 and organic solvent 2, this step forces the polymer to rearrange and form a nanomicelle structure with PCL as the core and PEG as the shell. Organic solvent 2 is then removed by rotary evaporation. This step is necessary because the volatile nature of organic solvent 2 means that it can be completely removed under mild conditions, avoiding possible organic solvent residue problems. Finally, in order to remove organic solvent 1 and other possible low molecular weight impurities used in the preparation process, the emulsion is dialyzed to retain larger nanomicelles and remove small molecules. The dialyzed sample is further washed and concentrated by ultrafiltration and filtration steps to ensure the purity and size distribution of the final product.

[0059] 2. Reaction mechanism of ALN connecting with COOH-PEG-PCL@Chrysin through amide bond:

[0060] In the process of preparing ALN@COOH-PEG-PCL@Chrysin nanomicelles, the key step is to connect alendronic acid (ALN) with COOH-PEG-PCL@Chrysin through an amide bond. This process is to activate the carboxyl moiety in COOH-PEG-PCL@Chrysin by EDCI and NHS, so that it can react with the amino group in alendronic acid to form a stable amide bond. First, EDCI reacts with the carboxyl group of PEG-PCL@Chrysin to form an O-acylisourea intermediate, and then NHS reacts with O-acylisourea to form NHS ester, which is an activated ester that is more reactive with amine groups. Subsequently, the amine groups of ALN are reacted with these activated carboxyl groups. Since the amine groups in ALN ​​can act as nucleophiles, they can attack the activated ester carbon atoms, thereby promoting the formation of amide bonds. It is advantageous to carry out the reaction in a neutral to weakly alkaline environment because the amine groups are more active under such pH conditions. During the reaction, TEA is added as an alkaline buffer to help neutralize carboxylic acids or other acidic byproducts that may be released during the process, thereby maintaining a stable pH in the reaction environment. Maintaining an appropriate pH is essential for promoting effective amide bond formation and helps prevent the micelle structure from being unstable due to pH fluctuations. Finally, the pH of the reaction solution is adjusted by controlling the addition of NaOH solution to ensure that the formation of amide bonds is both efficient and selective throughout the process. Fine control of pH is also a key factor in ensuring that the reaction proceeds along the expected path. As the reaction proceeds, the pH is gradually increased to 8.5-9.5 and the reaction is allowed to proceed for 8-12 hours to ensure that all reactions are fully carried out and to maximize the yield and purity of the product. Dialysis and freeze drying are key steps to ensure product purity and stability. Dialysis is used to remove unreacted starting materials, small molecule byproducts, and any unbound ALN to ensure the purity of the final product. Freeze drying is mainly used to remove moisture from the system and convert the nanomicelles into a dry powder form to enhance their storage stability and facilitate transportation, and to prevent the nanomicelles from disintegrating or aggregating in an aqueous environment. Through this series of finely regulated chemical steps, the present invention successfully covalently links ALN on the surface of COOH-PEG-PCL@Chrysin nanomicelles, which not only enhances the bioavailability of the nanomicelles but also improves their potential effects in orthopedic applications through the bone-targeting properties of ALN.

[0061] In a preferred embodiment of the present invention, the reaction equation involved is shown in the following formula 1:

[0062]

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] 1. The present invention effectively improves the water solubility and stability of Chrysin and increases its bioavailability in the body through the self-assembly technology of PEG-PCL nano-micelles.

[0065] 2. The present invention utilizes the bone targeting property of alendronic acid to enhance the aggregation of nano-micelles in bone tissue, thereby improving the bone tissue specificity of the drug, reducing the accumulation of the drug in non-target tissues, and reducing side effects.

[0066] 3. The present invention realizes the sustained release control of the drug through the core-shell structure of PEG-PCL, avoids the sudden release of Chrysin, and prolongs the drug effect time.

[0067] 4. The present invention encapsulates Chrysin in the hydrophobic core of HOOC-PEG-PCL by a physical embedding method to enhance its water solubility and bioavailability. The final product is stored at 4°C to maintain stability and extend the shelf life. Alendronate is then modified on the micelle surface through an amide bond to form bone-targeted nano-micelles.

[0068] 5. The present invention modifies alendronic acid on the surface of nanomicelles to achieve bone-targeted delivery, and at the same time verifies the potential application value of the nanomicelles in the treatment of osteoporosis through in vivo and in vitro experiments, providing a new anti-osteoporosis treatment solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0070] Figure 1 This is a schematic diagram of the process flow for preparing ALN@PEG-PCL@Chrysin nanomicelles according to Example 1 of the present invention.

[0071] Figure 2 FTIR spectra of the ALN@PEG-PCL@Chrysin nanomicelles prepared in Example 1 of the present invention, and the raw materials ALN and HOOC-PEG-PCL used.

[0072] Figure 3 TEM, particle size and potential comparison diagram of ALN@PEG-PCL@Chrysin nanomicelles and ALN@PEG-PCL nanomicelles prepared in Example 1 of the present invention and Comparative Example 1, respectively.

[0073] Figure 4 The left figure is a comparison of the encapsulation efficiency of the ALN@PEG-PCL@Chrysin nanomicelles prepared in Examples 1-3 of the present invention; the right figure is a test result of the drug loading rate of the ALN@PEG-PCL@Chrysin nanomicelles prepared in Example 1 of the present invention.

[0074] Figure 5 This is a comparison chart of the drug release curves of ALN@PEG-PCL@Chrysin nanomicelles and Chrysin prepared in Example 1 of the present invention from 0 to 96 hours.

[0075] Figure 6 The figure is a relationship diagram between the absorbance peak value and the drug content obtained by ultraviolet-visible absorption detection of ALN@PEG-PCL@Chrysin nanomicelles prepared in Example 1 of the present invention at different concentrations.

[0076] Figure 7 It is a comparison diagram of the UV-visible absorption spectra of ALN@PEG-PCL@Chrysin nanomicelle suspensions of different concentrations in the present invention.

[0077] Figure 8 Treatment scheme and in vivo experimental flow chart of ALN@PEG-PCL@Chrysin nanomicelles in the ovariectomized mouse model.

[0078] Fig. 9 This is a diagram showing the repairing effect of the ALN@PEG-PCL@Chrysin nano-micelles prepared in Example 1 of the present invention on the femoral microstructure of ovariectomized mice - Micro-CT analysis results.

[0079] Fig.10 The improvement of trabecular bone microstructure indicators of ovariectomized mice by ALN@PEG-PCL@Chrysin nano-micelles prepared in Example 1 of the present invention - quantitative analysis results of BV / TV, BS / TV, Tb.N and Tb.Th. *p<0.05, **p<0.01, ***p<0.001. Normal group: no treatment and only injected with saline; Sham group: OVX surgery, injection of saline; ALN@PEG-PCL@Chrysin group: OVX surgery, injection of ALN@PEG-PCL@Chrysin.

[0080] Fig.11 This is a graph showing the repairing effect of the ALN@PEG-PCL@Chrysin nanomicelles prepared in Example 1 of the present invention on the femoral trabecular structure of ovariectomized mice - HE staining analysis results.

[0081] Fig.12This is the effect of the ALN@PEG-PCL@Chrysin nano-micelles prepared in Example 1 of the present invention on the expression of osteogenic markers ALP, OCN, Osterix and Runx2 in ovariectomized mice - immunohistochemical staining analysis results.

[0082] Fig.13 This is a diagram showing the quantitative analysis results of the expression of osteogenic markers ALP, OCN, Osterix and Runx2 by the ALN@PEG-PCL@Chrysin nanomicelles prepared in Example 1 of the present invention.

[0083] Fig.14 This is a diagram showing the inhibitory effect of the ALN@PEG-PCL@Chrysin nanomicelles prepared in Example 1 of the present invention on the osteoclast activity of ovariectomized mice - TRAP staining analysis results.

[0084] Fig.15 This is a diagram showing the regulatory effect of ALN@PEG-PCL@Chrysin nanomicelles prepared in Example 1 of the present invention on the serum CTX-1 level in ovariectomized mice.

[0085] Fig.16 The inhibitory effect of ALN@PEG-PCL@Chrysin nanomicelles prepared in Example 1 of the present invention on RANKL-induced osteoclast activity - CCK-8 detection.

[0086] Fig.17 This is a graph showing the inhibitory effect of the ALN@PEG-PCL@Chrysin nanomicelles prepared in Example 1 of the present invention on the expression of RANKL-induced osteoclast-specific genes CTSK, c-FOS, MMP-9 and NFATc1 - qPCR analysis results.

[0087] Fig.18 This is a graph showing the inhibitory effect of the ALN@PEG-PCL@Chrysin nanomicelles prepared in Example 1 of the present invention on the expression of RANKL-induced osteoclast-specific proteins - Western Blot analysis results.

[0088] Fig.19 The promoting effect of ALN@PEG-PCL@Chrysin nanomicelles prepared in Example 1 of the present invention on the expression of osteoblast differentiation markers ALP, RUNX2, OCN and OPN - qPCR analysis results. *p<0.05, **p<0.01, ***p<0.001, ****p<0.001. DETAILED DESCRIPTION

[0089] The invention discloses an alendronic acid-modified Chrysin PEG-PCL nano-micelle and its application in the preparation of an anti-osteoporosis drug, aiming to provide a novel nano-micelle system to improve the water solubility, stability and bioavailability of Chrysin, and enhance its bone targeting and anti-osteoporosis effect. The nano-micelle uses PEG-PCL as a carrier, encapsulates the hydrophobic drug Chrysin through a self-assembly process, and modifies alendronic acid on the surface of the micelle to enhance the bone targeting effect. The nano-micelle can not only effectively prevent the sudden release of the drug, but also realize the sustained release and dosage control of the drug through material encapsulation. The research results show that the nano-micelle has good particle size distribution, drug encapsulation rate and stability, and exhibits a significant sustained release effect in the in vitro release process. In vitro and in vivo experiments further confirm the potential application value of the nano-micelle in the treatment of osteoporosis, and provide new ideas and methods for the clinical application of Chrysin and other similar water-insoluble compounds.

[0090] The present invention is further described in detail below through implementation cases.

[0091] According to the information contained in this application, various changes can be easily made to the precise description of the present invention for those skilled in the art.It should be understood that the scope of the present invention is not limited to defined processes, properties or components, because these embodiments and other descriptions are only for illustrating specific aspects of the present invention.

[0092] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers used in this application to express the amount, percentage, and other numerical values ​​should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods.

[0093] The equipment and raw materials used in the present invention can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0094] Except Chrysin, all reagents of the present invention were purchased from Sigma-Aldrich; among them: the product number of HOOC-PEG-PCL is: 901702, and the molecular weight is 5000; the CAS number of NHS is: 6066-82-6; the CAS number of EDCI is: 25952-53-8; and the CAS number of TEA is: 121-44-8.

[0095] Chrysin (99.85% purity) used in the present invention was purchased from Med Chem Express (HY-14589) with a product number of 480-40-0.

[0096] All chemical reagents used in the present invention are of analytical grade and no further purification is required.

[0097] Example 1

[0098] The present embodiment provides a method for preparing alendronic acid-modified Chrysin PEG-PCL (ALN@PEG-PCL@Chrysin) nanomicelles, the method comprising the following steps:

[0099] Step S1, weighing 15 mg of Chrysin and dissolving it in 1 mL of DMSO to obtain a DMSO solution of Chrysin; in addition, dissolving 100 mg of HOOC-PEG-PCL in 4 mL of chloroform to obtain a chloroform solution containing HOOC-PEG-PCL; wherein: the molar ratio of the HOOC-PEG-PCL to Chrysin is 1:3.

[0100] Step 2, slowly add the DMSO solution of Chrysin described in step S1 to the chloroform solution containing HOOC-PEG-PCL and mix well. Then, slowly drop the obtained mixture into 20 mL of ultrapure water under the action of ultrasound, emulsify at room temperature for 4 minutes to form an emulsion, and then use a rotary evaporator to remove the chloroform in the emulsion at 35-40° C. until the chloroform is completely removed.

[0101] The chloroform-free emulsion was then transferred to a dialysis bag with a molecular cutoff of 14,000 Da and dialyzed in pure water for more than 24 hours. The dialysis water was changed at least three times during the dialysis process to completely remove DMSO and other low-molecular impurities. Finally, the emulsion was further concentrated using an ultrafiltration system and filtered using a 0.22 μm aqueous syringe filter to remove any aggregates, ensuring that the prepared nanoparticles had an appropriate size and purity to obtain COOH-PEG-PCL@Chrysin nanomicelles.

[0102] Step 3, at room temperature, add 4.6 mg of NHS and 7.8 mg of EDCI to 15 mL of 1,4-dioxane solvent, add 110 mg of COOH-PEG-PCL@Chrysin nanomicelles prepared in step 2 and 20 μL of TEA, mix well, and react the resulting reaction solution in a dark environment for 8 hours to complete the activation reaction and form an activated NHS ester intermediate.

[0103] Step 4, dissolve 12 mg of ALN in 15 mL of deionized water, slowly add it to the activated NHS ester intermediate described in the above step 3, and at the same time, control the pH value of the reaction solution to be stable at about 7.0 by adding 0.2 M NaOH solution, and react for 2 hours; after the reaction is carried out for 2 hours, continue to add 0.2 M NaOH solution, adjust the pH value to 9.0, and react overnight.

[0104] Step 5: After the reaction, the unreacted substances (such as free ALN and unbound PEG-PCL) were removed by dialysis using a MWCO 3.5 kDa dialysis membrane. After dialysis, freeze-drying was performed to finally obtain ALN@PEG-PCL@Chrysin nanomicelles.

[0105] Example 2

[0106] The preparation method of alendronic acid modified Chrysin PEG-PCL (ALN@PEG-PCL@Chrysin) nanomicelles in this example is basically the same as that in Example 1, except that: the mass of Chrysin added in this example is 5 mg; that is, the molar ratio of HOOC-PEG-PCL to Chrysin is 1:1.

[0107] Example 3

[0108] The preparation method of alendronic acid modified Chrysin PEG-PCL (ALN@PEG-PCL@Chrysin) nanomicelles in this embodiment is basically the same as that in Example 1, except that the mass of Chrysin added in this embodiment is 25 mg, that is, the molar ratio of HOOC-PEG-PCL to Chrysin is 1:5.

[0109] Comparative Example 1

[0110] The comparative example is an ALN@PEG-PCL nanomicelle without Chrysin encapsulation. The preparation method of the nanomicelle is basically the same as that of Example 1, except that Chrysin is not used in step S1 of the comparative example, and the other steps are the same.

[0111] The final products prepared in Example 1 and Comparative Example 1 were subjected to structural analysis by infrared spectroscopy (IR), transmission electron microscopy (TEM), particle size and Zeta potential determination, etc., to verify the synthesis of the target compound. The specific structural characterization test method is as follows:

[0112] (I) ALN@PEG-PCL@Chrysin morphology observation

[0113] Observe the morphology of micelles using a transmission electron microscope: prepare a micelle solution of about 0.5 mg / mL, filter it with a 0.45 μm filter membrane, drop it on a copper mesh membrane, and when the solvent is almost dry, add a drop of phosphotungstic acid solution for negative staining. After drying, observe it with a transmission electron microscope.

[0114] (II) Determination of ALN@PEG-PCL@Chrysin particle size and Zeta potential

[0115] Weigh an appropriate amount of drug-loaded micelles and dissolve them in distilled water to prepare a micelle solution with a concentration of about 1 mg / mL. Filter through a 0.45 μm filter membrane and measure the particle size and Zeta potential using a Zeta potential and nanoparticle size analyzer. Repeat the measurement three times.

[0116] (III) Determination of drug loading and encapsulation efficiency of ALN@PEG-PCL@Chrysin

[0117] Step 1: Determine the background fluorescence of blank micelles

[0118] A certain amount of blank PEG-PCL micelles was weighed, dissolved in 4M dilute hydrochloric acid solution, and heated in a 60°C oil bath for 2 hours to ensure complete dissolution. Subsequently, an equal amount of 4M sodium hydroxide solution was added for neutralization to obtain a neutral solution. 100 μL of the neutral solution was taken out, added to 3mL of DMSO and mixed thoroughly, and its fluorescence intensity was measured to obtain the background fluorescence value. Each sample was measured three times and the average value was taken.

[0119] Step 2: Determination of drug content in drug-loaded micelles

[0120] Weigh a certain amount of Chrysin-loaded PEG-PCL micelles, dissolve them in 4M dilute hydrochloric acid solution, and heat them in a 60°C oil bath for 2 hours to ensure that the drug in the micelles is completely released. Then, add an equal amount of 4M sodium hydroxide solution for neutralization to obtain a neutral solution. Take out 100μL of the neutral solution, add it to 3mL of DMSO and mix it thoroughly. Measure its fluorescence intensity to determine the actual Chrysin content in the drug-loaded micelles. Each sample is measured three times and the average value is taken.

[0121] The drug loading (DL) and encapsulation efficiency (EF) were calculated by the following formula:

[0122]

[0123]

[0124] (IV) Drug release test method:

[0125] The present invention uses dialysis to evaluate the drug release characteristics of Chrysin and ALN@PEG-PCL@Chrysin nanomicelles. A suspension of Chrysin or ALN@PEG-PCL@Chrysin nanomicelles with a known concentration of 100 μg / mL was added to a dialysis bag (molecular cutoff 3.5 kDa), and the dialysis bag was placed in a release medium containing 100 mL of pH 7.4 PBS buffer, maintained at a constant temperature of 37°C, and stirred at 100 rpm. In the experiment, 1 mL of sample was taken out from the release medium every 6 hours to determine the release concentration of Chrysin. After sampling, an equal amount of fresh PBS buffer was immediately added to maintain the consistency of the release medium volume and release conditions. The Chrysin concentration of the sample was measured at 340 nm by a UV-visible spectrophotometer, and the release amount at each time point was calculated according to the standard curve.

[0126] The calculation formula of the cumulative drug release rate is:

[0127]

[0128] Among them, C i represents the drug concentration of the ith sampling, V s represents the sampling volume, C n Indicates the concentration of the remaining drug in the system at the current time point, V t represents the total volume of release medium.

[0129] (V) Establishment of Chrysin Standard Curve

[0130] Chrysin concentration was determined by fluorescence spectrophotometry. DMSO was used as a solvent to prepare Chrysin standard stock solution, which was then diluted to 2, 4, 6, 8, and 10 ppm (i.e., 2 mg / L, 4 mg / L, 6 mg / L, 8 mg / L, and 10 mg / L). Fluorescence intensity was measured by a fluorescence spectrophotometer. The concentration was linearly fitted with fluorescence intensity, and the regression equation for the detection of DOX standard curve was A=0.0793C+0.17324, R2=0.95. When the DOX concentration was within the range of 4-10 ppm, the fluorescence intensity showed a good linear relationship with the concentration.

[0131] (VI) UV-Vis absorption spectrum test method:

[0132] The present invention uses a UV-visible spectrophotometer to test the absorption spectra of ALN@PEG-PCL@Chrysin suspensions of different concentrations to evaluate their optical properties and stability. First, ALN@PEG-PCL@Chrysin suspensions with concentrations of 2ppm, 4ppm, 6ppm, 8ppm and 10ppm were prepared respectively. The UV-visible absorption spectrum of the sample was measured in the range of 200-325nm. The test conditions were controlled to a constant temperature of 25°C to ensure the consistency of the experimental environment. The absorbance data of each concentration sample was measured three times at each wavelength, and the average value was taken to draw the spectral curve.

[0133] The structural characterization results are as follows:

[0134] In view of the advantages of nano-micelles in the treatment of osteoporosis, such as easy absorption and good dispersibility, the present invention uses amphiphilic PEG-PCL micelles to encapsulate Chrysin, wherein the hydrophobic core composed of PCL encapsulates Chrysin, and PEG forms a hydrophilic shell, which enhances the water solubility, stability and bioavailability of Chrysin. Moreover, ALN is modified on the surface of the micelles to further enhance the bone targeting effect. The preparation process of the self-assembled ALN@PEG-PCL@Chrysin is as follows: Figure 1 As shown. The present invention successfully modified ALN on the surface of PEG-PCL micelles ( Figure 2 ) and took TEM images ( Figure 3 ). The surface Zeta potential of ALN@PEG-PCL@Chrysin is 8.74±0.44mv and the particle size is 165.28±4.27nm, which is similar to the surface potential and particle size of ALN@PEG-PCL, indicating that Chrysin is completely encapsulated in the hydrophobic cavity of PEG-PCL.

[0135] Depend on Figure 4 It can be seen that the drug loading rate of ALN@PEG-PCL@Chrysin nanomicelles prepared in Example 1 of the present invention is 11.91±0.43%, and the encapsulation rate is 88.67±3.65%. Figure 4 It can also be seen from the left figure that the encapsulation rate of the ALN@PEG-PCL@Chrysin nanomicelles prepared in Example 1 of the present invention is significantly higher than the encapsulation rate of the ALN@PEG-PCL@Chrysin nanomicelles prepared in Example 1 and Example 3. It can be seen that the encapsulation effect is best when the molar ratio of HOOC-PEG-PCL to Chrysin is 1:3.

[0136] The present invention also compares the cumulative release of free Chrysin and the ALN@PEG-PCL@Chrysin nanomicelles prepared in Example 1 within 96 hours to evaluate the sustained release effect of the nanomicelles on Chrysin. Figure 5 It shows that the release rate of free Chrysin is significantly higher than that of Chrysin encapsulated in nanomicelles. Within the first 24 hours, free Chrysin is rapidly released to about 90%, and is almost completely released within 48 hours, with a cumulative release rate close to 100%. In contrast, the release rate of Chrysin in ALN@PEG-PCL@Chrysin nanomicelles is significantly lower, showing the characteristics of sustained release. Within the first 12 hours, the cumulative release rate of Chrysin encapsulated in nanomicelles is only about less than 20%, and gradually released to less than 30% in 24 hours. Thereafter, the release rate of Chrysin in the nanomicelles remains stable, and the cumulative release rates reach about 40% and 45% at 48 hours and 96 hours, respectively. This significant difference in release rate indicates that the ALN@PEG-PCL@Chrysin nanomicelles prepared by the present invention effectively delay the release process of Chrysin, form a stable sustained release curve, and reflect good drug loading and sustained release characteristics. Compared with the rapid release of free Chrysin, nano-micelles significantly prolonged the release time of Chrysin, which may prolong the drug's action time in vivo, reduce the frequency of administration, and improve the bioavailability of Chrysin. This sustained-release behavior verifies the effectiveness of ALN@PEG-PCL as a drug delivery carrier, provides technical support for the targeted bone delivery and sustained release of Chrysin, and has good application potential.

[0137] The UV-visible absorption of ALN@PEG-PCL@Chrysin nanomicelles prepared in Example 1 with different concentrations was detected ( Figure 6 ) and absorption intensity calculation ( Figure 7 ). Figure 7 The results showed that the characteristic peaks of ultraviolet absorption at different concentrations were mainly located near about 250nm. As the drug concentration increased from 2ppm to 10ppm, the intensity of the absorption peak gradually increased, and the absorption curves of each concentration remained consistent, indicating that ALN@PEG-PCL@Chrysin has good linear response characteristics in a wide concentration range. The absorption spectrum curves of ALN@PEG-PCL@Chrysin nanomicelles are consistent in shape over the entire wavelength range, indicating that the nanomicelles have good spectral stability at different concentrations. In addition, through repeated measurements of nanomicelles at various concentrations, it was found that there was no significant change in the height and position of the absorption peak at the same concentration (the standard deviation was less than 0.02), which further proved the optical stability of ALN@PEG-PCL@Chrysin nanomicelles at different concentrations.

[0138] In general, the above results indicate that ALN@PEG-PCL micelles exhibit sustained and stable release of Chrysin during degradation, providing a favorable method for intra-articular delivery of Chrysin and OP treatment.

[0139] The biological performance test methods are as follows:

[0140] (I) Castration model

[0141] All experimental mice were 6-8 weeks old C57BL / 6J female mice (weight 18-22g), purchased from Sbefor (Beijing) Biotechnology Co., Ltd. (SCXK (Beijing) 2019-0010), and were adaptively raised for 3 days. The mice were anesthetized by intraperitoneal injection of 1% pentobarbital, and bilateral ovariectomy (OVX) was performed to establish an osteoporosis model. ALN@PEG-PCL@Chrysin was treated on the day of modeling, and continued until the eighth week when the femur was taken after sacrifice. The experimental group was injected with 25uL of ALN@PEG-PCL@Chrysin nanomicelles prepared in Example 1 into the knee joint cavity. Normal group: no treatment, only injection of saline; Sham group: OVX surgery, injection of saline; ALN@PEG-PCL@Chrysin group: OVX surgery, injection of ALN@PEG-PCL@Chrysin. The animals were dosed between 9 am and 12 noon. All experiments related to these mice complied with the rules for the care and use of laboratory animals and obtained ethical approval from the 920th Hospital of the Joint Logistics Support Center.

[0142] (II) HE and immunohistochemistry

[0143] In the sample processing, the tissue was fixed with 4% paraformaldehyde solution, embedded in paraffin and sliced. Subsequently, the slices were baked in a 64°C incubator for 1 hour, and finally dewaxed and dehydrated. The steps of hematoxylin counterstaining, eosin staining, transparent and sealing remained unchanged. After processing, the whole slice was scanned.

[0144] The sample processing of immunohistochemistry experiment includes tissue fixation, paraffin embedding, sectioning, baking, dewaxing, antigen repair and antibody incubation. The primary antibodies used in the present invention are Ocn (DF12303, Affinity), Osterix (DF7731, Affinity), Runx2 (ET1612-47, BUABIO), ALP (DF6225, Affinity), and the secondary antibody is Rabbit IgG (S0001, Affinity). After the processing is completed, the whole piece is scanned.

[0145] (III) Micro CT

[0146] To evaluate the changes in trabecular bone microarchitecture of the femur of each group of mice, femur specimens were collected immediately after sacrifice and fixed in 4% paraformaldehyde solution. The femur was scanned using high-resolution micro-computed tomography (micro-CT, Skyscan1276, Bruker), and the parameters were set as follows: voltage 65kV, current 153μA, and image resolution 9μm. The region of interest (ROI) analysis was performed on the distal femur, and the area within 1.0mm from the upper edge of the femoral growth plate was selected for trabecular bone structure reconstruction and analysis. During the reconstruction and analysis process, NRecon software (Bruker) was used for image reconstruction, and CTAn software (Bruker) was used for quantitative analysis to obtain the following bone microarchitecture parameters: bone volume / total volume (BV / TV), bone surface area / total volume (BS / TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N).

[0147] (IV) TRAP staining to detect osteoclast count

[0148] Use a pipette to remove the liquid in the induction well plate and wash with PBS. Then, add 1 ml of fixative solution for 30 minutes at room temperature. Wash with PBS, add 1 mL of TRAP (P0332, Beyotime) staining solution to each well, incubate at 37°C in the dark for 1-2 hours, and wash with PBS.

[0149] (V) Cell proliferation detection (CCK8)

[0150] Osteoclast precursor cells were seeded in a 96-well plate, with 5,000 cells per well. After the cells adhered to the wall, different concentrations of ALN@PEG-PCL@Chrysin were added for treatment. After 24 hours, CCK8 reagent was used according to the instructions of the kit to detect cell viability. The absorbance was read at a wavelength of 450 nm on an ELISA reader.

[0151] (VI) qPCR detection

[0152] Total RNA was extracted from cells and reverse transcribed into cDNA using a reverse transcription kit. qPCR reactions were performed using SYBRGreen reagent on a real-time fluorescence quantitative PCR instrument to detect the mRNA expression of osteoclast-specific genes (CTSK, c-FOS, MMP-9, NFATc1) and osteoblast differentiation markers (ALP, RUNX2, OCN, OPN). The relative expression of each gene was calculated by the ΔΔCt method, using GAPDH as the internal reference gene.

[0153] (VII) Protein expression level detection

[0154] Total protein was extracted from the cell lysate, separated by SDS-PAGE and transferred to a PVDF membrane. Western blot was performed using specific antibodies against osteoclast-specific proteins. Protein bands were visualized by chemiluminescence, GAPDH was used as an internal reference protein, and the grayscale values ​​of the protein bands were quantitatively analyzed.

[0155] 8. Data Analysis

[0156] All statistical analyses were performed using SPSS 22.0 software, and data were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) or t-test was used to compare data among groups. P < 0.05 was considered statistically significant.

[0157] The biological performance test results are as follows:

[0158] The OVX-induced mouse osteoporosis model is widely used to simulate the human osteoporosis model, reflecting the typical clinical manifestations of osteoporosis, such as bone loss, bone microstructure degradation and increased bone fragility. The present invention uses the OVX mouse model to explore the therapeutic potential of ALN@PEG-PCL@Chrysin nano-micelles prepared in Example 1 for osteoporosis. Chrysin, as a natural flavonoid compound, is known to have antioxidant, anti-inflammatory and anti-apoptotic properties. Therefore, the inventors hypothesize that Chrysin may have multiple beneficial effects in the treatment of osteoporosis. To verify this hypothesis, the inventors used the ALN@PEG-PCL@Chrysin nano-micelles prepared in Example 1 as a preparation and conducted a systematic study in the OVX mouse model. In the experiment, the inventors divided the mice into three groups: a normal group (Normal), an ovariectomized control group (Sham), and an OVX mouse group (ALN@PEG-PCL@Chrysin) treated with the ALN@PEG-PCL@Chrysin nano-micelles prepared in Example 1. From the day of model establishment to the 56th day, the ALN@PEG-PCL@Chrysin group was injected with 2.5 mg / kg ALN@PEG-PCL@Chrysin suspension through the knee joint cavity, while the Sham group received an equal amount of saline. Figure 8 At the end of the experiment, the femoral bone mass changes were evaluated by micro-computed tomography (Micro-CT) ( Fig. 9 The results showed that compared with the Sham group, the mice in the ALN@PEG-PCL@Chrysin group had significant improvements in the bone volume / total volume (BV / TV), bone surface area / total volume (BS / TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N) of trabecular bone, showing a therapeutic effect such as Fig.10 shown.

[0159] Bone volume / total volume (BV / TV): The BV / TV of the ALN@PEG-PCL@Chrysin group reached about 45%, which was significantly higher than 20% of the Sham group (p<0.001), but lower than about 70% of the normal group ( Fig.10 A), indicating that the treatment has a significant improvement effect on BV / TV. Bone surface area / total volume (BS / TV): The BS / TV of the ALN@PEG-PCL@Chrysin group was about 15%, which was significantly higher than that of the Sham group (about 9%) (p<0.001) and close to that of the normal group (about 17%). Fig.10 B). This result indicates that ALN@PEG-PCL@Chrysin has a potential role in restoring the surface area of ​​trabecular bone. Trabecular number (Tb.N): The trabecular number of the ALN@PEG-PCL@Chrysin group reached about 4, which was significantly higher than 3 in the Sham group (p<0.001) and close to 4.6 in the normal group ( Fig.10 C), showing a significant improvement in the number of trabeculae in the treatment group. Trabecular thickness (Tb.Th): In terms of trabecular thickness, the Tb.Th value of the ALN@PEG-PCL@Chrysin group was about 0.08, which was significantly higher than 0.06 in the Sham group (p<0.001) and close to 0.09 in the normal group ( Fig.10 D), indicating that ALN@PEG-PCL@Chrysin helps to restore trabecular thickness. The above data show that ALN@PEG-PCL@Chrysin treatment has a significant improvement effect on the trabecular bone structure of mice, especially in key bone parameters such as BV / TV, BS / TV, Tb.N and Tb.Th, which are significantly better than the Sham group and close to the level of the normal group.

[0160] The above results were further verified by HE staining, and the trabecular bone area of ​​the ALN@PEG-PCL@Chrysin group was significantly increased ( Fig.11 ), which is consistent with the results of quantitative analysis. Further immunohistochemical analysis confirmed the therapeutic effect of ALN@PEG-PCL@Chrysin, and the expression levels of osteogenic markers ALP, Ocn, Osterix and Runx2 in the treated mice were significantly increased ( Fig.12 ).

[0161] Quantitative analysis showed that the expression of osteogenic markers in the ALN@PEG-PCL@Chrysin group was significantly higher than that in the Sham group, indicating a significant enhancement in osteoblast activity ( Fig.13). Alkaline phosphatase (ALP): The positive rate of ALP in the ALN@PEG-PCL@Chrysin group reached about 40%, which was significantly higher than 20% in the Sham group (p<0.001), but slightly lower than 50% in the normal group. This indicates that ALN@PEG-PCL@Chrysin has a significant promoting effect on early osteogenic activity. Osteocalcin (OCN): In terms of OCN expression, the positive rate of the ALN@PEG-PCL@Chrysin group was about 70%, which was significantly higher than 30% in the Sham group (p<0.001), and close to 80% in the normal group. This result indicates that the treatment significantly promoted the mid- and late-stage differentiation of osteoblasts. Osterix: The positive rate of Osterix in the ALN@PEG-PCL@Chrysin group reached about 50%, which was significantly increased compared with 10% in the Sham group (p<0.001), but slightly lower than 70% in the normal group. This further confirms the positive regulatory effect of the treatment on osteoblast differentiation. Runx2: In terms of Runx2 expression, the positive rate of the ALN@PEG-PCL@Chrysin group was about 40%, which was significantly higher than 20% in the Sham group (p<0.001) and lower than 70% in the normal group. The increase in Runx2 indicates that ALN@PEG-PCL@Chrysin has a positive effect in regulating osteogenic transcription factors. The above data show that ALN@PEG-PCL@Chrysin treatment can significantly increase the expression of osteogenic markers, especially the expression levels of ALP, OCN, Osterix and Runx2, indicating that the treatment group has significantly enhanced osteoblast activity and differentiation ability, and there is a significant statistical difference compared with the Sham group (p<0.001).

[0162] In addition, TRAP (tartrate-resistant acid phosphatase) staining results showed that the number of osteoclasts in the ALN@PEG-PCL@Chrysin group was significantly reduced, indicating the potential role of this treatment in inhibiting osteoclast activity and reducing bone resorption ( Fig.14 ).

[0163] In the quantitative analysis of serum CTX-1, the CTX-1 level of mice in the sham group was significantly increased, indicating active bone resorption, while the CTX-1 level of the ALN@PEG-PCL@Chrysin group was significantly decreased, further indicating that ALN@PEG-PCL@Chrysin can effectively inhibit bone resorption and improve the pathological manifestations of osteoporosis ( Fig.15). In the quantitative analysis of serum CTX-1, the results showed that the CTX-1 level of mice in the Sham group increased significantly, reaching about 420pg / ml, which was significantly higher than about 150pg / ml in the normal group (p<0.001), indicating that the bone resorption of mice in the Sham group was active. The CTX-1 level of mice in the ALN@PEG-PCL@Chrysin group was about 220pg / ml, which was significantly lower than that in the Sham group (p<0.001) and close to the level of the normal group. This result further shows that ALN@PEG-PCL@Chrysin can effectively inhibit bone resorption, thereby improving the pathological manifestations of osteoporosis. This data supports the therapeutic potential of ALN@PEG-PCL@Chrysin in reducing the level of CTX-1, a bone resorption marker.

[0164] In summary, ALN@PEG-PCL@Chrysin exhibits significant therapeutic potential in the treatment of osteoporosis by inhibiting osteoclast activity and enhancing osteoblast function.

[0165] In in vitro experiments, ALN@PEG-PCL@Chrysin significantly inhibited the activity and specific gene expression of osteoclasts, and played a positive role in promoting osteoblast differentiation. Specifically, the CCK8 test results ( Fig.16 ) showed that ALN@PEG-PCL@Chrysin significantly reduced the activity of osteoclasts.

[0166] In addition, qPCR analysis further revealed the significant inhibitory effect of the ALN@PEG-PCL@Chrysin treatment group on osteoclast-specific gene expression. Compared with the RANKL treatment group, ALN@PEG-PCL@Chrysin significantly reduced the mRNA expression levels of osteoclast marker genes. Among them, the relative expression level of CTSK in the ALN@PEG-PCL@Chrysin treatment group was approximately 0.5, which was significantly lower than that of the RANKL group (1.3 (p<0.001) ( Fig.17 A); The relative expression of c-FOS in the treatment group was about 0.3, which was significantly lower than 0.8 in the RANKL group (p<0.001) ( Fig.17 B); the expression of MMP-9 in the treatment group was about 0.2, while that in the RANKL group reached 0.7 (p<0.001) ( Fig.17 C); the expression of NFATc1 in the treatment group was about 0.4, which was significantly lower than 0.8 in the RANKL group (p<0.001) ( Fig.17 D). These results indicate that ALN@PEG-PCL@Chrysin can effectively inhibit the expression of osteoclast-specific genes.

[0167] In addition, Western Blot results showed that the protein levels of CTSK, c-FOS, MMP-9 and NFATc1 were also significantly decreased in the ALN@PEG-PCL@Chrysin treated group ( Fig.18 ). In the RANKL group, the expression levels of these proteins were high, but decreased significantly after the addition of ALN@PEG-PCL@Chrysin, further confirming the effectiveness of this nanomaterial in inhibiting osteoclast differentiation. These data strongly support the potential application of ALN@PEG-PCL@Chrysin in osteoclastogenesis inhibition.

[0168] At the same time, qPCR test results showed that ALN@PEG-PCL@Chrysin significantly promoted the differentiation of osteoblasts. Specifically, in terms of early osteogenic differentiation markers, the relative expression level of ALP mRNA was significantly increased to about 90 in the ALN@PEG-PCL@Chrysin treatment group, while it was only about 10 in the RANKL group (p<0.001)( Fig.19 A); the expression level of RUNX2 in the treatment group reached about 35, which was also significantly higher than 5 in the RANKL group (p<0.001) ( Fig.19 B). This indicates that ALN@PEG-PCL@Chrysin significantly promoted the early differentiation of osteoblasts. In terms of late osteogenic differentiation markers, the OCN expression level in the ALN@PEG-PCL@Chrysin-treated group reached about 4, while that in the RANKL group was only 1, showing a significant upregulation (p<0.001)( Fig.19 C); the expression level of OPN also increased from about 4 in the RANKL group to 8 in the treatment group (p<0.001) ( Fig.19 D). These data indicate that ALN@PEG-PCL@Chrysin also plays a significant role in promoting the late differentiation of osteoblasts. Overall, ALN@PEG-PCL@Chrysin treatment significantly upregulated the mRNA expression levels of early and late osteoblast differentiation markers (ALP, RUNX2, OCN, and OPN), which were significantly different from the RANKL group. These data further support the potential application value of ALN@PEG-PCL@Chrysin in promoting osteoblast differentiation.

[0169] In summary, the ALN@PEG-PCL@Chrysin nanomicelles prepared in the present invention showed significant therapeutic potential in the treatment of osteoporosis by inhibiting osteoclast activity and enhancing osteoblast function.

Claims

1. A method for preparing alendronic acid-modified Chrysin PEG-PCL nanomicelles, characterized in that: The method specifically comprises the following steps: Step 1, dissolving Chrysin in an organic solvent 1 according to a ratio to obtain a Chrysin solution; dissolving carboxyl polyethylene glycol polycaprolactone HOOC-PEG-PCL in an organic solvent 2 to obtain a HOOC-PEG-PCL solution; Step 2, slowly adding the Chrysin solution to the HOOC-PEG-PCL solution according to the ratio and mixing; then slowly dropping the obtained mixed solution into ultrapure water under ultrasound for emulsification for 3-5 minutes to form an emulsion; finally, the obtained emulsion is subjected to rotary evaporation, dialysis, concentration and filtration in sequence to obtain COOH-PEG-PCL@Chrysin nano-micelles; Step 3, at room temperature, N-hydroxysuccinimide NHS and (1-ethyl-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) EDCI are added to the organic solvent 3 in order according to the ratio, and mixed; then, COOH-PEG-PCL@Chrysin and triethylamine TEA described in step 2 are added to the obtained mixed solution, and the obtained reaction solution is placed in a light-proof condition to react for 7-9 hours; after the reaction is completed, an activated NHS ester intermediate is formed; Step 4, dissolving alendronic acid ALN in deionized water according to a ratio to obtain an alendronic acid solution; then simultaneously adding the alendronic acid solution and the NaOH solution dropwise to the activated NHS ester intermediate in step 3, respectively, and controlling the pH value of the reaction solution to be neutral; after the addition is completed, reacting the obtained reaction solution at room temperature for 1-3 hours; after the reaction is completed, continue to dropwise add the NaOH solution to the reaction system, adjust the pH value of the obtained product to 8.5-9.5, and continue to react for 8-12 hours; Step 5: After the reaction is completed, the obtained product is dialyzed and freeze-dried to obtain the ALN@PEG-PCL@Chrysin nanomicelles.

2. The preparation method according to claim 1, characterized in that: The mass ratio of chrysin to carboxyl polyethylene glycol polycaprolactone in the mixed solution of step 2 is (10-20): (70-150).

3. The preparation method according to claim 1, characterized in that: The mass ratio of NHS to EDCI in step 3 is (4-6):(5-10).

4. The preparation method according to claim 1, characterized in that: The mass ratio of EDCI to COOH-PEG-PCL@Chrysin in step 3 is (5-10):(100-130).

5. The preparation method according to claim 1, characterized in that: The mass ratio of ALN to NHS in step 4 is 6:(2-3).

6. ALN@PEG-PCL@Chrysin nanomicelles prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the ALN@PEG-PCL@Chrysin nanomicelles prepared by the preparation method according to any one of claims 1 to 5 in the preparation of drugs for treating osteoporosis.

8. A drug for treating osteoporosis, characterized in that: The invention comprises ALN@PEG-PCL@Chrysin nanomicelles prepared by the method according to any one of claims 1 to 5.

9. Use of the ALN@PEG-PCL@Chrysin nanomicelles prepared by the preparation method according to any one of claims 1 to 5 in the preparation of osteoclast activity inhibitors and / or osteoblast differentiation promoters.

10. An osteoclast activity inhibitor and / or osteoblast differentiation promoter, characterized in that: The invention comprises ALN@PEG-PCL@Chrysin nanomicelles prepared by the method according to any one of claims 1 to 5.

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