Metformin nanomaterial, and preparation method and application thereof

Abstract

The application belongs to the technical field of bioactive nanomaterials, and particularly relates to a metformin nanomaterial, a preparation method and application thereof. The average hydrodynamic diameter of the nanomaterial is less than 500 nm, and the nanomaterial is prepared by a preparation method comprising the following steps: self-assembly of metformin and polyphenols to obtain metformin-polyphenol nanoparticles; reaction of bisphosphonate and polyethylene glycol to obtain bisphosphonate-polyethylene glycol organic segments; and reaction of the metformin-polyphenol nanoparticles and the bisphosphonate-polyethylene glycol organic segments to obtain the nanomaterial. The metformin, polyphenol, polyethylene glycol and bisphosphonate are prepared into the metformin-loaded nanomaterial through specific reactions, the systemic delivery of metformin is realized, the efficacy and bioavailability of metformin on osteoporosis are improved, and the bone targeting of metformin is also improved.

Description

Technical Field

[0001] This invention belongs to the field of bioactive nanomaterials technology, specifically relating to a metformin nanomaterial, its preparation method, and its application. Background Technology

[0002] Osteoporosis (OP) is a systemic bone disease characterized by decreased bone mineral density and deterioration of bone microstructure, leading to increased bone fragility and fracture susceptibility. Its pathological basis is an imbalance in bone metabolism caused by bone resorption exceeding bone formation. Current treatments for osteoporosis utilize a combination of pharmacological and non-pharmacological interventions. Metformin's mechanism of action and efficacy in treating osteoporosis have been extensively studied. Metformin can activate AMPK and its downstream pathways, coordinating the balance between osteogenic and osteoclastogenesis, and inhibiting bone marrow steatosis and ferroptosis, thus exerting anti-osteoporotic effects on multiple targets, particularly showing potential osteoprotective effects in diabetic osteoporosis. However, the oral administration of metformin is limited by the complex physiological environment within the human body (acidic gastric environment, large amounts of digestive enzymes, etc.) and complex medication requirements (fasting in the morning, drinking plenty of water, etc.), often resulting in drug inactivation, low bioavailability, and gastrointestinal ulceration, which also affects systemic drug delivery and long-term efficacy.

[0003] Therefore, metformin nanomaterials have emerged as a promising alternative to metformin. Metformin nanomaterials can improve oral bioavailability, prolong half-life, and reduce gastrointestinal irritation. Furthermore, their morphology and size characteristics are very similar to natural bone tissue. Through surface modification, nanomaterials can be precisely delivered to bone tissue, thereby increasing local drug concentration while reducing toxicity to non-target tissues, making them highly suitable for targeted drug delivery. Currently, various nanoparticles, such as chitosan liposomes, PLGA-PEG nanoparticles, and metal-organic frameworks, have achieved the loading and targeted release of metformin, enhancing therapeutic safety and ultimately significantly improving treatment outcomes, indicating that metformin nanomaterials hold great promise for the treatment of osteoporosis.

[0004] A summary of existing technologies reveals that metformin nanomaterials currently employ diverse therapeutic strategies: utilizing various carriers such as tetrahedral framework nucleic acids, cartilage-targeted self-assembled structures, phosphine nanosheets, PLGA microspheres, sodium alginate / chitosan, or SO2-derived materials, they achieve transdermal, oral, intra-articular, or macrophage-directed delivery, and leverage mechanisms such as AMPK / NF-κB, ROS scavenging, and pyroptosis inhibition to treat various diseases including diabetic complications, osteoarthritis, and tumors. However, metformin nanomaterials still face many challenges in clinical application: the therapeutic effect of metformin on osteoporosis needs improvement, drug delivery efficiency is low, bioavailability is low, in vivo targeting efficiency is low, long-term safety data is insufficient, and clinical scale-up processes are unstable, limiting their translational application. Therefore, there is an urgent need to design a more effective metformin nanomaterial to address these existing problems. Summary of the Invention

[0005] The problem the invention aims to solve

[0006] This invention aims to provide a novel metformin nanomaterial. By preparing metformin into a nanomaterial, systemic delivery can be achieved. Furthermore, the nanomaterial regulates both bone resorption and bone formation within the bone microenvironment, thereby enhancing the therapeutic effect of metformin on osteoporosis and improving its delivery efficiency, bioavailability, and in vivo bone targeting efficiency. Additionally, this invention also aims to provide a method for preparing the aforementioned nanomaterial and its application in the field of osteoporosis.

[0007] Solution for solving the problem

[0008] In a first aspect, the present invention provides a metformin nanomaterial, wherein the metformin nanomaterial has an average hydrodynamic diameter of less than 500 nm, and the metformin nanomaterial comprises a polymeric compound represented by formula (I):

[0009]

[0010] (I)

[0011] Where n represents any integer from 30 to 60;

[0012] The metformin nanomaterial was prepared by a method including the following steps:

[0013] Step S1: Metformin and polyphenolic compounds self-assemble to obtain metformin-polyphenolic nanoparticles;

[0014] Step S2: Bisphosphonate and polyethylene glycol react to obtain bisphosphonate-polyethylene glycol organic segments;

[0015] Step S3: The metformin-polyphenol nanoparticles and the bisphosphonate-polyethylene glycol organic segments react to obtain the metformin nanomaterials;

[0016] The structural formula of the polyethylene glycol is as follows: n represents any integer between 30 and 60;

[0017] In step S1, the mass ratio of metformin to the polyphenolic compound is 1:1 to 1:5;

[0018] In step S2, the mass ratio of the bisphosphonate to the polyethylene glycol is 1:1 to 1:5;

[0019] In step S3, the mass ratio of the metformin-polyphenol nanoparticles to the bisphosphonate-polyethylene glycol organic segments is 1:1 to 1:5.

[0020] Preferably, the polyphenolic compound is tannic acid.

[0021] Preferably, the bisphosphonate is sodium alendronate.

[0022] Preferably, in step S1, the mass ratio of metformin to the polyphenol compound is 1:1 to 1:2.

[0023] Preferably, in step S2, the mass ratio of the bisphosphonate to the polyethylene glycol is 1:1 to 1:3.

[0024] Preferably, in step S3, the mass ratio of the metformin-polyphenol nanoparticles to the bisphosphonate-polyethylene glycol organic segments is 1:1 to 1:2.

[0025] Preferably, in step S1, the self-assembly is carried out in the presence of water.

[0026] Preferably, in step S2, the polyethylene glycol is activated by NHS and EDC and then reacted with the bisphosphonate.

[0027] Preferably, in step S2, the solvent for the reaction is water.

[0028] Preferably, in step S2, the reaction is carried out under alkaline conditions.

[0029] Preferably, in step S3, the solvent for the reaction is a mixture of PBS and DMSO.

[0030] Preferably, the average hydrodynamic diameter of the metformin nanomaterial is 30-100 nm.

[0031] Secondly, the present invention provides a method for preparing metformin nanomaterials, comprising the following steps:

[0032] Step S1: Metformin and polyphenolic compounds self-assemble to obtain metformin-polyphenolic nanoparticles;

[0033] Step S2: Bisphosphonate and polyethylene glycol react to obtain bisphosphonate-polyethylene glycol organic segments;

[0034] Step S3: The metformin-polyphenol nanoparticles and the bisphosphonate-polyethylene glycol organic segments react to obtain the metformin nanomaterials;

[0035] The structural formula of the polyethylene glycol is as follows: n represents any integer between 30 and 60;

[0036] In step S1, the mass ratio of metformin to the polyphenolic compound is 1:1 to 1:5;

[0037] In step S2, the mass ratio of the bisphosphonate to the polyethylene glycol is 1:1 to 1:5;

[0038] In step S3, the mass ratio of the metformin-polyphenol nanoparticles to the bisphosphonate-polyethylene glycol organic segments is 1:1 to 1:5.

[0039] Preferably, in step S1, the mass ratio of metformin to the polyphenol compound is 1:1 to 1:2.

[0040] Preferably, in step S2, the mass ratio of the bisphosphonate to the polyethylene glycol is 1:1 to 1:3.

[0041] Preferably, in step S3, the mass ratio of the metformin-polyphenol nanoparticles to the bisphosphonate-polyethylene glycol organic segments is 1:1 to 1:2.

[0042] Thirdly, the present invention provides a pharmaceutical formulation comprising the metformin nanomaterials described in the first aspect.

[0043] Preferably, the pharmaceutical preparation is an intravenous injection preparation.

[0044] Fourthly, the present invention provides the use of the metformin nanomaterials according to the first aspect or the pharmaceutical formulations according to the third aspect in the preparation of medicaments for the prevention and / or treatment of osteoporosis.

[0045] The effects of the invention

[0046] The present invention has the following advantages and effects compared with the prior art:

[0047] 1. Overcome the limitations of existing metformin administration methods and improve drug delivery efficiency.

[0048] Current metformin administration relies on oral administration, which is affected by the acidic environment of the stomach and digestive enzyme degradation, resulting in problems such as drug inactivation, low bioavailability, and gastrointestinal irritation. This invention constructs a novel nanomaterial by self-assembling metformin and polyphenols (e.g., tannic acid) and modifying them with polyethylene glycol and bisphosphonates (e.g., alendronate sodium). This nanomaterial enables systemic delivery without oral administration, avoiding the destruction of the drug by the gastrointestinal environment, significantly improving drug bioavailability, and reducing adverse reactions such as gastrointestinal ulcers. It overcomes the limitation of "requiring frequent medication" and reduces the burden of medication for patients.

[0049] 2. It bidirectionally regulates bone metabolism and enhances the anti-osteoporosis effect.

[0050] In existing technologies, while metformin can exert osteoprotective effects through the AMPK pathway, it lacks direct targeted regulation of bone resorption and bone formation; some nanocarriers only achieve drug delivery without enhancing bone metabolism regulation. This invention innovatively introduces bisphosphonates (e.g., alendronate sodium) and polyphenols (e.g., tannic acid). The synergistic effect of bisphosphonates, polyphenols, and metformin not only regulates osteoblast activity but also inhibits excessive osteoclast proliferation, achieving bidirectional regulation of "bone resorption inhibition + bone formation promotion." This precisely targets the pathological basis of osteoporosis—"bone metabolism imbalance (bone resorption > bone formation)"—and synergistically maintains bone balance, improving the efficacy of treatment for osteoporosis. Furthermore, its efficacy is superior to metformin and existing nano-formulations that simply deliver metformin.

[0051] In addition, most existing technologies only study the osteogenic differentiation effect of metformin. This invention focuses on the osteogenic mineralization effect of metformin and found that when metformin and bisphosphonates without osteogenic differentiation and mineralization effects are prepared into nanomaterials, the effect on osteoblast differentiation and mineralization is better than that of metformin or metformin-polyphenol products.

[0052] 3. Bisphosphonate-polyethylene glycol organic segment grafting modification to improve bone targeting efficiency.

[0053] The metformin nanomaterials of the present invention, after being grafted with bisphosphonate-polyethylene glycol organic segments, exhibit significantly enhanced bone-targeting capabilities both in vitro and in vivo. By improving the bone-targeting efficiency of metformin, the concentration of metformin in bone tissue is significantly increased, thereby further enhancing the therapeutic effect.

[0054] 4. The preparation method is simple.

[0055] The preparation method of metformin nanomaterials provided by this invention has simple steps, mild reaction conditions, and stable process, making it suitable for industrial-scale production. Attached Figure Description

[0056] Figure 1This is a schematic diagram of the preparation of Met NPs.

[0057] Figure 2 This is a particle size distribution diagram of Met NPs dissolved in deionized water.

[0058] Figure 3 Transmission electron microscopy (TEM) images of Met NPs.

[0059] Figure 4 High-angle annular dark-field transmission image (HAADF-TEM) and elemental mapping image of Met NPs.

[0060] Figure 5 The Fourier transform infrared (FTIR) spectrum of the Met NPs.

[0061] Figure 6 This is a schematic diagram illustrating the establishment and treatment experiments of an osteoporosis mouse model.

[0062] Figure 7 Representative Micro-CT reconstructed images of the femur of ovariectomized osteoporotic female mice treated with Met, Met-TA, and Met NPs.

[0063] Figure 8 Quantitative analysis of Micro-CT-derived BMD, Tb.Th, and Tb.Sp in the femur after different treatments.

[0064] Figure 9 H&E staining images of femoral tissue from OVX mice after various treatments at different magnifications.

[0065] Figure 10 TRAP staining images of femoral tissue from OVX mice after various treatments at different magnifications.

[0066] Figure 11 A schematic diagram of in vivo and in vitro bone targeting experiments to test Met NPs.

[0067] Figure 12 A comparison of the HAP adsorption affinity of Met-TA and Met NPs.

[0068] Figure 13 Fluorescence distribution images of Cy5-labeled Met-TA and Cy5-labeled Met NPs in the heart, liver, spleen, lungs, kidneys, femur, and tibia at 24 h after intravenous injection.

[0069] Figure 14 ALP staining image of MC3T3-E1 cells induced by Met, Met-TA and Met NPs in vitro for osteogenic differentiation.

[0070] Figure 15 ARS staining image of osteogenic mineralization in MC3T3-E1 cells induced by Met, Met-TA, and Met NPs in vitro.

[0071] Figure 16 F-actin / nuclear staining image showing the inhibition of osteoclast formation in RAW264.7 cells by Met, Met-TA, and Met NPs.

[0072] Figure 17 TRAP staining image of RAW264.7 cells inhibited in vitro osteoclast differentiation using Met, Met-TA, and Met NPs. Detailed Implementation

[0073] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0074] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0075] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0076] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0077] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0078] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0079] In this instruction manual, when "room temperature" or "room temperature" is used, the temperature can be 15-30℃, or more specifically 20-30℃, such as 25℃.

[0080] The English abbreviations and their full Chinese names used in this instruction manual are shown in the table below:

[0081]

[0082] Through dedicated research, the inventors have developed a novel metformin nanomaterial. Specifically, metformin is reacted with polyphenols, polyethylene glycol, and bisphosphonates through a specific reaction to prepare metformin-loaded nanomaterials. This enables systemic delivery of metformin, improving its efficacy and bioavailability in treating osteoporosis, while also enhancing its bone-targeting properties. Furthermore, this invention provides a simple and stable method for preparing the metformin nanomaterials, suitable for industrial-scale production.

[0083] Preparation method of metformin nanomaterials

[0084] First, this invention provides a method for preparing metformin nanomaterials, which includes the following steps:

[0085] Step S1: Metformin and polyphenolic compounds self-assemble to obtain metformin-polyphenolic nanoparticles;

[0086] Step S2: Bisphosphonate and polyethylene glycol react to obtain bisphosphonate-polyethylene glycol organic segments;

[0087] Step S3: The metformin-polyphenol nanoparticles and the bisphosphonate-polyethylene glycol organic segments react to obtain the metformin nanomaterials;

[0088] The structural formula of the polyethylene glycol is as follows: , where n represents any integer between 30 and 60.

[0089] In some implementations, n represents any integer from 35 to 50, preferably any integer from 38 to 47, such as 40, 42, 45, etc.

[0090] In some embodiments, the polyphenolic compound is one or more of tannic acid, epigallocatechin, catechin, epicatechin, gallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, epigallocatechin gallate, and kaempferol.

[0091] In some specific embodiments, the polyphenolic compound comprises tannic acid, preferably tannic acid.

[0092] In this invention, the tannic acid used comprises the following compounds:

[0093] .

[0094] In some embodiments, the bisphosphonate is a bisphosphonate having an amino group (-NH2), such as sodium alendronate or sodium pamidronate.

[0095] In some specific embodiments, the bisphosphonate comprises sodium alendronate, preferably sodium alendronate.

[0096] In some embodiments, the structure of the bisphosphonate-polyethylene glycol organic segment is as follows:

[0097]

[0098] Where n represents any integer between 30 and 60.

[0099] In some implementations, n represents any integer from 35 to 50, preferably any integer from 38 to 47, such as 40, 42, 45, etc.

[0100] In some embodiments, in step S1, the mass ratio of metformin to the polyphenolic compound is 1:1 to 1:5, preferably 1:1 to 1:2, such as 1:1.1, 1:1.2, 1:1.5, etc.

[0101] In some embodiments, in step S2, the mass ratio of the bisphosphonate to the polyethylene glycol is 1:1 to 1:5, preferably 1:1 to 1:3, such as 1:1.5, 1:2, 1:2.2, 1:2.5, 1:2.8, etc.

[0102] In some embodiments, in step S3, the mass ratio of the metformin-polyphenol nanoparticles to the bisphosphonate-polyethylene glycol organic segments is 1:1-1:5, preferably 1:1-1:2, such as 1:1, 1:1.2, 1:1.3, 1:1.5, 1:1.8, etc.

[0103] In some implementations, in step S1, the self-assembly is carried out in the presence of water.

[0104] In some implementations, the solvent for the reaction in step S2 is water.

[0105] In some embodiments, in step S2, the polyethylene glycol is activated by NHS and EDC and then reacted with the bisphosphonate.

[0106] In some embodiments, in step S2, the mass ratio of polyethylene glycol, NHS and EDC is 1:(0.1-1):(0.1-1), preferably 1:(0.1-0.5):(0.1-0.5), and more preferably 1:(0.15-0.35):(0.1-0.3).

[0107] In some embodiments, in step S2, the reaction is carried out under alkaline conditions, for example, pH 7-10, preferably pH 7-8. In some specific embodiments, an alkali may be added to adjust the pH to alkaline, for example, sodium hydroxide or an aqueous solution thereof may be added to adjust the pH to alkaline.

[0108] In some implementations, in step S2, the reaction is carried out in an inert gas environment, such as in a nitrogen or argon environment.

[0109] In some embodiments, in step S3, the solvent for the reaction is a mixture of PBS and DMSO, for example, a mixture of PBS and DMSO in a volume ratio of 1:1 to 1:5.

[0110] In some implementations, in step S1, the self-assembly temperature is 20-50°C, preferably 30-45°C, for example 37°C.

[0111] In some embodiments, in step S2, the reaction temperature is 20-50°C, preferably 20-35°C, for example 25°C.

[0112] In some embodiments, in step S3, the reaction temperature is 20-50°C, preferably 20-30°C, for example 25°C.

[0113] In some implementations, the self-assembly time in step S1 is 12-48 h, preferably 20-36 h, for example 24 h.

[0114] In some implementations, the reaction time in step S2 is 12-48 h, preferably 20-36 h, for example 24 h.

[0115] In some implementations, the reaction time in step S3 is 5-15 h, preferably 6-10 h, for example 7 h.

[0116] In some embodiments, step S1 further includes a post-processing step: drying after self-assembly. In some specific embodiments, the drying is freeze-drying.

[0117] In some embodiments, step S2 further includes a post-processing step: after the reaction is complete, dialysis and drying. In some specific embodiments, the molecular weight cutoff for dialysis is 800-1500 Da, preferably 800-1200 Da, for example 1000 Da. In some specific embodiments, the drying is freeze-drying.

[0118] In some embodiments, step S3 further includes a post-processing step: after the reaction is complete, dialysis and drying. In some specific embodiments, the molecular weight cutoff for dialysis is 800-1500 Da, preferably 800-1200 Da, for example 1000 Da. In some specific embodiments, the drying is freeze-drying.

[0119] Metformin nanomaterials

[0120] The present invention also provides a metformin nanomaterial, wherein the average hydrodynamic diameter of the metformin nanomaterial is less than 500 nm, and it is prepared by the above preparation method.

[0121] In some embodiments, the metformin nanomaterial comprises a polymeric compound of formula (I):

[0122]

[0123] (I)

[0124] Where n represents any integer between 30 and 60.

[0125] In some implementations, n represents any integer from 35 to 50, preferably any integer from 38 to 47, such as 40, 42, 45, etc.

[0126] In some embodiments, the metformin nanomaterial is a nanoparticle.

[0127] In some embodiments, the average hydrodynamic diameter of the metformin nanomaterial is 30-100 nm, preferably 40-70 nm, such as 45 nm, 50 nm, 55 nm, 60 nm, etc.

[0128] In some embodiments, the metformin and the polyphenolic compound self-assemble through at least one of hydrogen bonding and ionic bonding. Specifically, in the self-assembly of the metformin and the polyphenolic compound, the binding site of the metformin is an imine group, and the binding site of the polyphenolic compound is a phenolic hydroxyl group, and the two are bonded through at least one of hydrogen bonding and ionic bonding.

[0129] In some embodiments, the bisphosphonate and the polyethylene glycol are covalently bonded, for example, via an amide bond. Specifically, in the reaction between the bisphosphonate and the polyethylene glycol, the binding site of the bisphosphonate is an amino group, and the binding site of the polyethylene glycol is a carboxyl group; the two bind through a condensation reaction.

[0130] In some embodiments, the metformin-polyphenol nanoparticles and the bisphosphonate-polyethylene glycol organic segments are covalently bonded. Specifically, in the reaction between the metformin-polyphenol nanoparticles and the bisphosphonate-polyethylene glycol organic segments, the binding site of the metformin-polyphenol nanoparticles is the hydrogen atom on the benzene ring of the polyphenol, and the binding site of the bisphosphonate-polyethylene glycol organic segments is the thiol group on the polyethylene glycol; the two bind through a nucleophilic substitution reaction.

[0131] Pharmaceutical Composition

[0132] The present invention also provides a pharmaceutical composition comprising metformin nanomaterials prepared according to the above preparation method or the above metformin nanomaterials.

[0133] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

[0134] pharmaceutical preparations

[0135] The present invention also provides a pharmaceutical formulation comprising metformin nanomaterials prepared according to the above preparation method, or the above metformin nanomaterials or the above pharmaceutical composition.

[0136] In some implementations, the pharmaceutical preparation is an intravenous injection preparation, which can achieve systemic delivery.

[0137] application

[0138] The present invention also provides the use of metformin nanomaterials prepared according to the above preparation method, or the above metformin nanomaterials, or the above pharmaceutical compositions, or the above pharmaceutical preparations in the preparation of medicaments for the prevention and / or treatment of osteoporosis.

[0139] In some embodiments, the osteoporosis is systemic osteoporosis or regional osteoporosis, preferably systemic osteoporosis.

[0140] In some implementations, the osteoporosis is osteoporosis caused by diabetes.

[0141] Example

[0142] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0143] Example 1: Preparation of Met NPs (metformin nanoparticles)

[0144] A schematic diagram of the synthesis of Met NPs can be found in [link to diagram]. Figure 1 Where PEG represents COOH-PEG-SH, the structural formula is The average molecular weight was 2000, purchased from Shanghai Feline Biotechnology Co., Ltd., brand name J&K; TA was purchased from Shanghai Feline Biotechnology Co., Ltd., specification 100g, brand name Maclean. The specific preparation steps of Met NPs are as follows:

[0145] Step 1: Preparation of Met-TA (Metformin-Tanonic Acid Nanoparticles)

[0146] Met (20.64 mg) and TA (22 mg) were weighed sequentially and dissolved in 20 mL and 1 mL of deionized water, respectively, to prepare Met solution and TA solution. The TA solution was rapidly added dropwise to the Met solution with stirring and stirred for 10 minutes. The solution showed an obvious yellow color change. The reaction was then carried out at 37 °C and 800 rpm for 24 h with shaking and then freeze-dried to obtain the product Met-TA.

[0147] Step 2: Preparation of Aln-PEG (sodium alendronate-polyethylene glycol organic segments)

[0148] Activation of COOH-PEG-SH: NHS (23 mg), EDC (19.1 mg), and deionized water (25 mL) were added to COOH-PEG-SH (100 mg), and the mixture was shaken at 37 °C and 800 rpm. After activation of COOH-PEG-SH, Aln (40.65 mg) was added, and the pH was adjusted to 7-8 with NaOH aqueous solution. The mixture was stirred at 300 rpm for 24 h under an inert gas environment at room temperature. After 24 h, the mixture was dialyzed (dialysis molecular weight cutoff value was 1000 Da) for 48 h and then freeze-dried to obtain the product Aln-PEG.

[0149] Step 3: Preparation of Met NPs

[0150] Met-TA (10 mg) and Aln-PEG (10 mg) were dissolved in a mixture of PBS and dimethyl sulfoxide (DMSO) (20 mL, PBS and DMSO volume ratio 1:1); the pH was adjusted to 8-9 with NaOH aqueous solution, and then the reaction was carried out at 25 °C and 800 rpm for 7 h with shaking; after 7 h, hydrochloric acid (4 wt% of the reaction system) was added to terminate the reaction; the resulting solution was dialyzed (dialysis molecular weight cutoff value was 1000 Da) and freeze-dried to obtain the product Met NPs.

[0151] Experimental Example 1: Characterization of Met NPs

[0152] 1. Surface morphology and dimensions of Met NPs:

[0153] The hydrodynamic diameters of Met NPs in aqueous solution were determined by dynamic light scattering (DLS), and the results are shown in [Figure number missing]. Figure 2 The results showed that the average hydrodynamic diameter of the Met NPs was 50.3 nm, indicating that the nanoparticles have a small hydrodynamic size, which is beneficial for their circulation and targeted delivery in vivo.

[0154] Meanwhile, the surface morphology of Met NPs was observed by transmission electron microscopy (TEM), and the results are shown in [Figure number missing]. Figure 3 The images show that the nanoparticles exhibit a well-dispersed granular structure with uniform morphology and no obvious aggregation, further verifying their stable dispersion characteristics at the microscale and providing a structural basis for subsequent biological applications.

[0155] 2. Element distribution of Met NPs:

[0156] The elemental composition of Met NPs was analyzed using high-angle annular dark-field imaging (HAADF) and elemental mapping (TEM mapping). The results are shown in [Figure number missing]. Figure 4 The HAADF images clearly show the microscopic distribution of the nanoparticles, and the corresponding elemental spectrum analysis ( Figure 4 Results (including C, N, and O elements) showed that Met NPs contain C, N, and O elements, and each element exhibits a uniform distribution within the nanoparticle region. This result demonstrates that Met, TA, Aln, and PEG achieve stable binding and uniform dispersion within the nanoparticles, further validating the successful synthesis of Met NPs.

[0157] 3. Chemical structure of Me NPs

[0158] Fourier transform infrared spectroscopy (FTIR) Figure 5The chemical structures of Met NPs were analyzed. By comparing the characteristic peaks of Met, Met-TA, and Met NPs using FTIR, the infrared spectrum of Met-TA showed characteristic absorption peaks different from those of Met, such as a decreased intensity of the broad phenolic peak and the C=O shoulder peak of the ester, indicating the possibility of hydrogen bonding between the phenolic group of TA and the imino group of Met. The infrared spectra of Met NPs also showed characteristic absorption peaks different from those of Met-TA, such as those in the 1400-1600 cm⁻¹ range. -1 The peaks in this band changed, presumably due to a reaction between the thiol group and the benzene ring, such as the formation of thiophenolic compounds or the substitution of a hydrogen atom on the benzene ring by the thiol group. This change could include a slight shift in peak position or a change in peak intensity. Simultaneously, the original characteristic peaks showed a characteristic shift, indicating chemical bonding between the components (Met and TA are bonded via hydrogen and ionic bonds; Aln and PEG are bonded via amide bonds, i.e., Aln and PEG are bonded via covalent bonds; the reaction site for Met-TA is the hydrogen atom on the benzene ring of TA, and the reaction site for Aln-PEG is the thiol group of PEG; the reaction is a nucleophilic addition reaction, meaning Met-TA and Aln-PEG are bonded via covalent bonds). This characterization result verifies that the chemical structure of Met NPs has been successfully synthesized, and the functional components have been effectively combined.

[0159] Experimental Example 2: In vivo osteoporosis treatment effect of Met NPs

[0160] To investigate the in vivo therapeutic effect of Met NPs on osteoporosis, female C57BL / 6 mice were randomly divided into 5 groups: (1) Sham group (sham-operated mice); (2) OVX group (ovariectomized mice); (3) Met group (ovariectomized mice treated with Met, which was purchased from Tianjin Puxitang Biomedical Technology Co., Ltd., specification: 1g); (4) Met-TA group (ovariectomized mice treated with Met-TA, which is the product of step 1 of Example 1); (5) Met NPs group (ovariectomized mice treated with Met NPs).

[0161] See the experimental design and flowchart. Figure 6 First, mice were used to establish a model (0-4 weeks). In the Sham group, only the abdominal cavity was opened without removing the ovaries, while the other groups underwent bilateral ovariectomy. After the model was established (4-8 weeks), mice in each group were treated with drugs via tail vein injection. After the treatment was completed (12 weeks), the mice were sacrificed, and their femurs and other bone tissues were dissected for detection and analysis such as micro-computed tomography (Micro-CT).

[0162] Micro-CT images (see) Figure 7The femoral trabecular structure in the Sham group was dense and continuous; the trabecular structure in the OVX group was significantly sparse and fractured, showing typical osteoporosis; the trabecular damage in the Met group was improved but still relatively sparse; the Met-TA group showed improvement compared to the Met group; while the trabecular structure in the Met NPs group was closer to that of the Sham group, with significant recovery of density and continuity, and the improvement effect was better than that of the Met group and the Met-TA group.

[0163] Quantitative bone parameters (see) Figure 8 Compared with the OVX group, the Met NPs group showed significantly increased bone mineral density (BMD), significantly increased trabecular bone thickness (Tb.Th), and significantly decreased trabecular bone separation (Tb.Sp); at the same time, the improvement effect of the Met NPs group was better than that of the Met group and the Met-TA group.

[0164] Tissue section staining: Compared with the OVX group, the Met NPs group showed significantly improved trabecular density, significantly reduced fractures, and bone morphology closer to normal physiological state (HE staining results are shown in...). Figure 9 Simultaneously, the number of osteoclasts decreased significantly, their activity was markedly reduced, and bone resorption was effectively inhibited (TRAP staining results are shown in [reference needed]). Figure 10 Furthermore, the bone tissue morphology repair effect and osteoclast inhibition effect of the Met NPs group were superior to those of the Met group and the Met-TA group.

[0165] Experimental Example 3: In vivo and in vitro bone targeting capabilities of Met NPs

[0166] To verify the in vitro bone-targeting ability of Met NPs, the experimental schematic diagram is shown below. Figure 11 .

[0167] Hydroxyapatite (HAP), a core inorganic component of bone tissue, was selected as a target to simulate the interaction between nanoparticles and the bone matrix. 200 µg / mL solutions of Me-TA and Met NPs were prepared, and the initial fluorescence intensity was measured using a microplate reader. Subsequently, 10 mg / mL HAP was added to each solution and incubated in the dark for 2 h. After centrifugation, the supernatant was collected, and the fluorescence intensity was measured and calculated using the following formula:

[0168]

[0169] The results showed that Met NPs achieved a binding rate of 30.81% for HAP, while Met-TA only achieved 7.08% (see...). Figure 12 This indicates that Met NPs can significantly enhance the binding efficiency of nanoparticles to the bone matrix.

[0170] To further verify the in vivo bone-targeting ability of the nanoparticles, OVX-induced model mice were randomly selected, and Met-TA and MetNPs were intravenously injected into the mice. Twenty-four hours later, major organs such as the heart, liver, spleen, lungs, and kidneys, as well as bone tissue, were dissected and collected. The in vivo distribution of the nanoparticles was analyzed using fluorescence imaging technology (see [link to article]). Figure 13 The fluorescence distribution images clearly show that the bone tissue regions in the MetNPs group exhibit bright and concentrated fluorescence signals, while the fluorescence intensity in the bone tissue of the Met-TA group is significantly weaker. These results directly corroborate that Met NPs have superior in vivo bone targeting properties.

[0171] Experimental Example 4: Study on the biological mechanism of Met NPs on osteoblast MC3T3-E1

[0172] To investigate the in vitro therapeutic effect of Met NPs on osteoporosis, osteoblasts MC3T3-E1 were cultured in the following conditioned media: blank osteogenic induction group (control group), Met+ osteogenic induction group, Met-TA+ osteogenic induction group, and Met NPs+ osteogenic induction group. After incubation in conditioned media for 14 and 18 days, in vitro osteogenic differentiation and mineralization experiments were performed, respectively.

[0173] ALP staining images (see) Figure 14 The expression levels of ALP activity in MC3T3-E1 cells were significantly increased in the Met, Met-TA, and Met NPs groups compared to the control group. This indicates that Met-TA and Met NPs have a better promoting effect on ALP activity in MC3T3-E1 cells than Met, and Met NPs have the best effect on promoting osteogenic differentiation.

[0174] ARS staining images (see) Figure 15 Materials from the Met, Met-TA, and Met NPs groups can promote osteogenic mineralization in MC3T3-E1 cells, with significant formation of mineralized nodules. Furthermore, Met-TA and Met NPs have a better promoting effect on ARS activity in MC3T3-E1 cells than Met, with Met NPs showing the best effect in promoting osteogenic mineralization.

[0175] Experimental Example 5: Study on the biological mechanism of Met NPs on osteoclast RAW264.7

[0176] To investigate the in vitro therapeutic effect of Met NPs on osteoporosis, RAW264.7 osteoclasts were cultured in the following conditioned media: blank osteoclast induction group (control group), Met+ osteoclast induction group, Met-TA+ osteoclast induction group, and Met NPs+ osteoclast induction group. After incubation in conditioned media for 3 and 7 days, in vitro osteoclast formation and differentiation experiments were performed, respectively.

[0177] F-actin (green) / cell nucleus (blue) staining image (see Figure 16 In the control group, mononuclear RAW264.7 cells had largely fused to form multinucleated mature osteoclasts, and a more complete and distinct osteoclast cytoskeleton, namely the F-actin ring, was observed. Compared with the control group, the number and size of F-actin rings in RAW264.7 cells in the Met, Met-TA, and Met NPs groups were significantly reduced. Most mononuclear RAW264.7 cells maintained aggregated growth without showing a fusion trend, indicating that the above three drugs can inhibit the formation of mature multinucleated osteoclasts in the later stage of RAW264.7 cells, and the inhibitory effect of Met NPs is more significant.

[0178] TRAP-stained images (see) Figure 17 The large area of ​​TRAP positivity in the control group indicates that RAW264.7 cells successfully differentiated into osteoclasts under RANKL induction. Compared with the control group, the TRAP positivity area in the Met, Met-TA, and Met NPs groups was reduced, indicating that Met, Met-TA, and Met NPs can all inhibit the differentiation of RAW264.7 cells into osteoclasts under RANKL induction, and Met NPs have a better inhibitory effect on osteoclast differentiation.

[0179] The above results indicate that Met NPs treatment can significantly alleviate bone loss caused by ovariectomy, effectively improve bone microstructure and bone parameters in osteoporotic mice, and demonstrate a good in vivo anti-osteoporosis therapeutic effect.

[0180] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0181] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A metformin nanomaterial, characterized in that, The metformin nanomaterial has an average hydrodynamic diameter of less than 500 nm, and the metformin nanomaterial comprises the polymer compound shown in formula (I): （I） Where n represents any integer from 30 to 60; The metformin nanomaterial was prepared by a method including the following steps: Step S1: Metformin and polyphenolic compounds self-assemble to obtain metformin-polyphenolic nanoparticles; Step S2: Bisphosphonate and polyethylene glycol react to obtain bisphosphonate-polyethylene glycol organic segments; Step S3: The metformin-polyphenol nanoparticles and the bisphosphonate-polyethylene glycol organic segments react to obtain the metformin nanomaterials; The structural formula of the polyethylene glycol is as follows: n represents any integer between 30 and 60; In step S1, the mass ratio of metformin to the polyphenolic compound is 1:1 to 1:5; In step S2, the mass ratio of the bisphosphonate to the polyethylene glycol is 1:1 to 1:5; In step S3, the mass ratio of the metformin-polyphenol nanoparticles to the bisphosphonate-polyethylene glycol organic segments is 1:1 to 1:5; The polyphenolic compound is tannic acid, and the bisphosphonate is sodium alendronate.

2. The metformin nanomaterial according to claim 1, characterized in that, In step S1, the mass ratio of metformin to the polyphenolic compound is 1:1 to 1:2; In step S2, the mass ratio of the bisphosphonate to the polyethylene glycol is 1:1 to 1:3; In step S3, the mass ratio of the metformin-polyphenol nanoparticles to the bisphosphonate-polyethylene glycol organic segments is 1:1 to 1:

2.

3. The metformin nanomaterial according to claim 1 or 2, characterized in that, In step S1, the self-assembly is carried out in the presence of water; In step S2, the polyethylene glycol is activated by NHS and EDC and then reacts with the bisphosphonate. The solvent for the reaction is water, and the reaction is carried out under alkaline conditions. In step S3, the solvent for the reaction is a mixture of PBS and DMSO.

4. The metformin nanomaterial according to claim 1 or 2, characterized in that, The average hydrodynamic diameter of the metformin nanomaterial is 30-100 nm.

5. A method for preparing metformin nanomaterials, characterized in that, The preparation method includes the following steps: Step S1: Metformin and polyphenolic compounds self-assemble to obtain metformin-polyphenolic nanoparticles; Step S2: Bisphosphonate and polyethylene glycol react to obtain bisphosphonate-polyethylene glycol organic segments; Step S3: The metformin-polyphenol nanoparticles and the bisphosphonate-polyethylene glycol organic segments react to obtain the metformin nanomaterials; The structural formula of the polyethylene glycol is as follows: n represents any integer between 30 and 60; In step S1, the mass ratio of metformin to the polyphenolic compound is 1:1 to 1:5; In step S2, the mass ratio of the bisphosphonate to the polyethylene glycol is 1:1 to 1:5; In step S3, the mass ratio of the metformin-polyphenol nanoparticles to the bisphosphonate-polyethylene glycol organic segments is 1:1 to 1:5; The polyphenolic compound is tannic acid, and the bisphosphonate is sodium alendronate.

6. The preparation method according to claim 5, characterized in that, In step S1, the mass ratio of metformin to the polyphenolic compound is 1:1 to 1:2; In step S2, the mass ratio of the bisphosphonate to the polyethylene glycol is 1:1 to 1:3; In step S3, the mass ratio of the metformin-polyphenol nanoparticles to the bisphosphonate-polyethylene glycol organic segments is 1:1 to 1:

2.

7. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation comprises metformin nanomaterials according to any one of claims 1-4.

8. The pharmaceutical preparation according to claim 7, characterized in that, The drug preparation is an intravenous injection preparation.

9. The use of the metformin nanomaterial according to any one of claims 1-4 or the pharmaceutical formulation according to claim 7 or 8 in the preparation of a medicament for the prevention and / or treatment of osteoporosis.

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