Rifapentine-loaded bone-targeted nano-micelle as well as preparation method and application thereof

Abstract

The invention relates to the technical field of nano-micelles, in particular to rifapentine-loaded bone targeting nano-micelles as well as a preparation method and application thereof.The rifapentine-loaded bone targeting nano-micelles are prepared by accurately controlling a synthesis process (shading, catalyst ratio, temperature and feed ratio) of a bone targeting nano-micelle carrier (ALN-PLGA-mPEG) modified by alendronate sodium (ALN). The bone targeting ability of alendronate sodium (ALN) is combined with the drug loading and long circulation advantages of polymer mPEG-PLGA micelles to construct a bone targeting nano-micelle carrier (ALN-PLGA-mPEG), so that the obtained rifapentine-loaded bone targeting nano-micelle (ALN-PLGA-mPEG (at) RPT) can significantly improve the bone targeting and slow release performance, and provides a new strategy with high efficiency and low toxicity for precise treatment of bone tuberculosis.

Description

Technical Field

[0001] This invention relates to the field of nanomicelle technology, specifically to bone-targeting nanomicelles loaded with rifapentine, their preparation method, and their applications. Background Technology

[0002] Tuberculosis (TB) is a global infectious disease caused by Mycobacterium tuberculosis. According to the World Health Organization's "Global Tuberculosis Report 2024," there are 10.8 million new cases worldwide each year, making it the leading cause of death from a single infectious disease globally. Bone tuberculosis accounts for 35% of extrapulmonary tuberculosis cases and is characterized by high disability rates, slow healing, and complex treatment. Currently, isoniazid and rifampin are the first-line drugs for anti-tuberculosis treatment. However, isoniazid is a water-soluble drug, and after bone grafting during surgery, it can spread rapidly with body fluids, reducing its local residence time and affecting its optimal choice for local administration. Furthermore, approximately 500,000 new cases of rifampin-resistant tuberculosis are diagnosed each year, which may limit its clinical application to some extent.

[0003] Rifapentine (RTP) is a semi-synthetic cyclopentylrifamycin antibiotic with an antibacterial spectrum similar to rifampin, but its anti-tuberculosis activity is 2 to 10 times higher, with a minimum inhibitory concentration of only 0.02 μg / mL to 0.06 μg / mL. It also has fewer adverse reactions and a longer half-life. Unlike other water-soluble anti-tuberculosis drugs, rifapentine has good stability and is less prone to leakage in vitro and in vivo after being formulated into targeted drugs. However, currently only rifapentine capsules are available for oral administration in clinical practice, which have the following key problems: (1) The drug itself is limited. Oral RPT is affected by the first-pass effect and food interference, resulting in low bioavailability. Traditional administration has high systemic exposure and significant toxic side effects; (2) The drug delivery efficiency to bone tissue is low. Bone tissue has poor blood supply, and the lesion area is often accompanied by necrosis and calcification, forming a physical barrier, which makes it difficult for traditional oral or injectable anti-tuberculosis drugs to penetrate effectively and reach a sufficient therapeutic concentration at the lesion site; (3) There is a lack of efficient targeting strategies. Developing a new drug delivery system with non-surgical routes and both efficient bone targeting and sustained-release characteristics is the key to breaking through the existing treatment dilemma.

[0004] Polymer micelles, particularly amphiphilic block copolymers (such as mPEG-PLGA) based on polylactic-co-glycolic acid (PLGA) and polyethylene glycol (PEG) (e.g., mPEG), are highly promising drug delivery carriers due to their hydrophobic core which can solubilize poorly soluble drugs (such as RPT), their hydrophilic shell (PEG) which can prolong in vivo circulation time, their small particle size (10 nm to 200 nm) which facilitates passive targeting (EPR effect), and their good biocompatibility and degradability. However, traditional PLGA nanoparticles have a highly hydrophobic surface and are easily cleared by the mononuclear phagocytic system (MPS). While using mPEG-PLGA micelles alone can prolong circulation time, it lacks the ability to actively target bone tissue.

[0005] Bone-targeting agents can utilize hydroxyapatite (HA), which is abundant in bone tissue (60% to 70%), as a target. Alendronate sodium (ALN), as a bisphosphonate drug, has bisphosphonate groups in its molecule that can react with calcium ions (Ca) in HA. 2+ It exhibits specific and strong coordination binding, possesses excellent bone-targeting ability, and is often used as a bone-targeting ligand modified nanocarrier.

[0006] Therefore, in order to address the issues of first-pass effect and low targeted accumulation of rifapentine in the treatment of bone tuberculosis, there is an urgent need to develop a new type of highly efficient bone-targeting nanomicelles. Summary of the Invention

[0007] This invention provides a bone-targeting nanomicelle loaded with rifapentine, its preparation method, and its application, overcoming the shortcomings of the prior art. It can effectively solve the problems of first-pass effect and low targeted accumulation of rifapentine in the existing treatment of bone tuberculosis.

[0008] One of the technical solutions of the present invention is achieved through the following measures: a bone-targeting nanomicelle loaded with rifapentine is obtained by the following method: The first step involves dissolving the required amounts of methoxy polyethylene glycol (mPEG), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and 4-dimethylaminopyridine (DMAP) in an organic solvent under light-protected conditions, followed by stirring and activation to obtain a mixture. The second step involves adding the required amount of polylactic acid-glycolic acid copolymer (PLGA) to the mixture to continue the reaction and obtain the initial product. The third step involves dynamic dialysis and freeze-drying of the primary product to obtain the polymer (mPEG-PLGA). The fourth step involves dissolving the polymer (mPEG-PLGA) in water, ultrasonically dispersing it into micelles, adding the required amounts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the micelles for activation, and then adding sodium alendronate (ALN) and stirring the reaction to obtain the final product. The fifth step involves subjecting the final product to ultrafiltration, centrifugation, and freeze-drying to obtain bone-targeting nanomicelle carriers (ALN-PLGA-mPEG). Step 6: Dissolve the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) in water, ultrasonically disperse it into micelles, add an ethanol solution of rifapentine (RPT) to the micelles, stir, and then obtain bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT) after ultrafiltration, centrifugation and freeze drying.

[0009] The following are further optimizations and / or improvements to one of the above-mentioned technical solutions: In the first step above, the molar ratio of methoxy polyethylene glycol (mPEG), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP) is 1:(10 to 20):(10 to 20).

[0010] In the second step above, the molar ratio of methoxy polyethylene glycol (mPEG) to polylactic acid-glycolic acid copolymer (PLGA) is (1 to 5):1.

[0011] In the fourth step above, the amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) added is 10 to 20 times the molar amount of polymer (mPEG-PLGA), and the molar ratio of sodium alendronate (ALN) to polymer (mPEG-PLGA) is (5 to 10):1.

[0012] In step six above, the mass ratio of bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) to rifapentine (RPT) is 10:(2 to 5).

[0013] In the first step above, the organic solvent is one of dimethyl sulfoxide, acetone, and chloroform.

[0014] In the first step above, the temperature during stirring is 40℃ to 80℃, and the activation time is 20min to 40min.

[0015] In the second step mentioned above, the reaction time continues for 24 to 48 hours.

[0016] In the third step above, during dynamic dialysis, the molecular weight cutoff is 3.5 kDa, and the dialysis time is 24 to 36 hours.

[0017] In the fourth step above, the activation time is 3 to 6 hours, and the stirring time is 24 to 36 hours.

[0018] In step 5 above, the molecular weight cutoff during ultrafiltration centrifugation is 3 kDa.

[0019] In step six above, the stirring time is 2 to 4 hours, and the molecular weight cutoff during ultrafiltration centrifugation is 3 kDa.

[0020] The second technical solution of the present invention is achieved through the following measures: a method for preparing bone-targeting nanomicelles loaded with rifapentine, which is carried out according to the following method: The first step involves dissolving the required amounts of methoxy polyethylene glycol (mPEG), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and 4-dimethylaminopyridine (DMAP) in an organic solvent under light-protected conditions, followed by stirring and activation to obtain a mixture. The second step involves adding the required amount of polylactic acid-glycolic acid copolymer (PLGA) to the mixture to continue the reaction and obtain the initial product. The third step involves dynamic dialysis and freeze-drying of the primary product to obtain the polymer (mPEG-PLGA). The fourth step involves dissolving the polymer (mPEG-PLGA) in water, ultrasonically dispersing it into micelles, adding the required amounts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the micelles for activation, and then adding sodium alendronate (ALN) and stirring the reaction to obtain the final product. The fifth step involves subjecting the final product to ultrafiltration, centrifugation, and freeze-drying to obtain bone-targeting nanomicelle carriers (ALN-PLGA-mPEG). Step 6: Dissolve the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) in water, ultrasonically disperse it into micelles, add an ethanol solution of rifapentine (RPT) to the micelles, stir, and then obtain bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT) after ultrafiltration, centrifugation and freeze drying.

[0021] The third technical solution of the present invention is achieved through the following measures: the application of bone-targeting nanomicelles loaded with rifapentine in the preparation of drugs for treating bone tuberculosis.

[0022] This invention combines the bone-targeting ability of alendronate sodium (ALN) with the drug-loading and long-cycle advantages of polymer mPEG-PLGA micelles by precisely controlling the synthesis process (light shielding, catalyst ratio, temperature, and feed ratio) of alendronate sodium (ALN) modified bone-targeting nanomicelle carrier (ALN-PLGA-mPEG). The resulting rifapentine-loaded bone-targeting nanomicelles (ALN-PLGA-mPEG@RPT) significantly improve bone targeting and sustained-release performance, providing a new, efficient, and low-toxicity strategy for the precise treatment of bone tuberculosis. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the synthesis of bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT).

[0024] Figure 2 The graph shows the value of I1 / I3 as a function of c (polymer (mPEG-PLGA) nanomicelles) when the feed ratio n (mPEG:PLGA) is 0.5:1.

[0025] Figure 3 The graph shows the value of I1 / I3 as a function of c (polymer (mPEG-PLGA) nanomicelles) when the feed ratio n (mPEG:PLGA) is 1:1.

[0026] Figure 4 The graph shows the value of I1 / I3 as a function of c (polymer (mPEG-PLGA) nanomicelles) when the feed ratio n (mPEG:PLGA) is 2:1.

[0027] Figure 5 The graph shows the value of I1 / I3 as a function of c (polymer (mPEG-PLGA) nanomicelles) when the feed ratio n (mPEG:PLGA) is 5:1.

[0028] Figure 6 This is the 1H NMR spectrum of methoxy polyethylene glycol (mPEG).

[0029] Figure 7 This is the 1H NMR spectrum of polylactic acid-glycolic acid copolymer (PLGA).

[0030] Figure 8 This is the 1H NMR spectrum of the polymer (mPEG-PLGA).

[0031] Figure 9 This is the infrared spectrum of methoxy polyethylene glycol (mPEG).

[0032] Figure 10This is the infrared spectrum of polylactic acid-glycolic acid copolymer (PLGA).

[0033] Figure 11 This is the infrared spectrum of the polymer (mPEG-PLGA).

[0034] Figure 12 This is the colorimetric reaction of sodium alendronate (ALN) with ninhydrin solution.

[0035] Figure 13 This is a standard curve showing the relationship between alendronate sodium (ALN) concentration and absorbance value.

[0036] Figure 14 The UV absorption spectra of sodium alendronate (ALN) reference standard, test solution, and blank solution are shown.

[0037] Figure 15 The standard curve for rifapentine (RPT) was established based on HPLC.

[0038] Figure 16 The in vitro cumulative release curve of rifapentine (RPT) is shown.

[0039] Figure 17 Results of in vitro bone affinity of bone-targeting nanomicelle carrier (ALN-PLGA-mPEG).

[0040] Figure 18 These are the results of fluorescence scanning of small animals using an in vivo imaging system.

[0041] Figure 19 The results are fluorescence scans of isolated mouse bone tissue.

[0042] Figure 20 A standard curve for the determination of rifapentine (RPT) in biological samples by HPLC.

[0043] Figure 21 HE staining results of rat organs loaded with rifapentine-targeted nanomicelles (ALN-PLGA-mPEG@RPT). Detailed Implementation

[0044] This invention is not limited to the following embodiments, and specific implementation methods can be determined according to the technical solutions and actual conditions of this invention. Unless otherwise specified, all chemical reagents and chemicals mentioned in this invention are well-known and commonly used chemical reagents and chemicals in the prior art; unless otherwise specified, all percentages in this invention are mass percentages; unless otherwise specified, all solutions in this invention are aqueous solutions with water as the solvent, for example, hydrochloric acid solution is an aqueous solution of hydrochloric acid; room temperature in this invention generally refers to a temperature between 15°C and 25°C, generally defined as 25°C.

[0045] The present invention will be further described below with reference to embodiments: Example 1: The bone-targeting nanomicelles loaded with rifapentine were obtained by the following method: The first step involves dissolving the required amounts of methoxy polyethylene glycol (mPEG), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and 4-dimethylaminopyridine (DMAP) in an organic solvent under light-protected conditions, followed by stirring and activation to obtain a mixture. The second step involves adding the required amount of polylactic acid-glycolic acid copolymer (PLGA) to the mixture to continue the reaction and obtain the initial product. The third step involves dynamic dialysis and freeze-drying of the primary product to obtain the polymer (mPEG-PLGA). The fourth step involves dissolving the polymer (mPEG-PLGA) in water, ultrasonically dispersing it into micelles, adding the required amounts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the micelles for activation, and then adding sodium alendronate (ALN) and stirring the reaction to obtain the final product. The fifth step involves subjecting the final product to ultrafiltration, centrifugation, and freeze-drying to obtain bone-targeting nanomicelle carriers (ALN-PLGA-mPEG). Step 6: Dissolve the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) in water, ultrasonically disperse it into micelles, add an ethanol solution of rifapentine (RPT) to the micelles, stir, and then obtain bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT) after ultrafiltration, centrifugation and freeze drying.

[0046] Example 2: As an optimization of the above example, in the first step, the molar ratio of methoxy polyethylene glycol (mPEG), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP) is 1:(10 to 20):(10 to 20).

[0047] Example 3: As an optimization of the above example, in the second step, the molar ratio of methoxy polyethylene glycol (mPEG) to polylactic acid-glycolic acid copolymer (PLGA) is (1 to 5):1, preferably, the molar ratio of methoxy polyethylene glycol (mPEG) to polylactic acid-glycolic acid copolymer (PLGA) is 2:1.

[0048] Example 4: As an optimization of the above example, in the fourth step, the amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) added is 10 to 20 times the molar amount of polymer (mPEG-PLGA), and the molar ratio of sodium alendronate (ALN) to polymer (mPEG-PLGA) is (5 to 10):1. Preferably, the molar ratio of sodium alendronate (ALN) to polymer (mPEG-PLGA) is 10:1.

[0049] Example 5: As an optimization of the above example, in the sixth step, the mass ratio of bone-targeted nanomicelle carrier (ALN-PLGA-mPEG) to rifapentine (RPT) is 10:(2 to 5), preferably, the mass ratio of bone-targeted nanomicelle carrier (ALN-PLGA-mPEG) to rifapentine (RPT) is 10:4.

[0050] Example 6: As an optimization of the above example, in the first step, the organic solvent is one of dimethyl sulfoxide, acetone and chloroform.

[0051] Example 7: As an optimization of the above example, in the first step, the temperature during stirring is 40°C to 80°C, and the activation time is 20 min to 40 min.

[0052] Example 8: As an optimization of the above example, in the second step, the reaction time is continued for 24 hours to 48 hours.

[0053] Example 9: As an optimization of the above example, in the third step, during dynamic dialysis, the molecular weight cutoff is 3.5 kDa, and the dialysis time is 24 h to 36 h.

[0054] Example 10: As an optimization of the above example, in the fourth step, the activation time is 3h to 6h during activation and the stirring time is 24h to 36h during stirring.

[0055] Example 11: As an optimization of the above example, in the fifth step, the molecular weight cutoff is 3kDa during ultrafiltration centrifugation.

[0056] Example 12: As an optimization of the above example, in step 6, the stirring time is 2h to 4h, and the molecular weight cutoff is 3kDa during ultrafiltration centrifugation.

[0057] Compared with the prior art, the beneficial effects of the present invention are as follows: First, the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) prepared in the fifth step of this invention exhibits several advantages. Firstly, its particle size is uniform and stable, with an average particle size of 89.34 nm ± 1.91 nm and a polydispersity index (PDI) of 0.259 ± 0.055. A PDI < 0.3 indicates excellent monodispersity. Secondly, the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) demonstrates high stability, specifically a Zeta potential of -21.17 ± 1.46 mV. An absolute Zeta potential > 20 mV indicates strong colloid stability. Furthermore, the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) exhibits excellent self-assembly ability, specifically a critical micelle concentration (CMC) as low as 0.0050 mg / mL, indicating strong surface tension and solubilization capabilities. Secondly, the bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT) of this invention not only have a high drug loading capacity of 16.74% and an encapsulation efficiency of 50.27% (superior to traditional liposomes, whose encapsulation efficiency is <10%), but also have good long-term sustained-release ability. The drug release is only 25% after 12 hours, which is significantly lower than that of the original drug group (90%) and the physical mixture (65%), and 65% after 48 hours, demonstrating good sustained-release ability. Third, the bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT) of this invention exhibit outstanding bone targeting properties. Not only does it have strong in vitro binding ability, as evidenced by the significantly higher HA fluorescence intensity in the ALN-modified group compared to the unmodified group, but it also demonstrates high in vivo targeting efficiency, as evidenced by the significantly higher fluorescence signal intensity in bone tissue as shown in in vivo imaging compared to the unmodified group. Furthermore, its in vivo distribution characteristics show a bone / blood drug concentration ratio of 1.93, which is superior to the non-targeted group (only 0.33). Fourth, the bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT) of this invention have good safety and no obvious toxicity. HE staining of the major organs (heart, liver, spleen, lung and kidney) of rats showed no inflammation or necrosis, which confirms good biocompatibility. In summary, this invention first optimizes the synthesis process of alendronate sodium (ALN) modified bone-targeting nanomicelle carriers (ALN-PLGA-mPEG). The optimized synthesis process includes reacting under light-shielding conditions, a molar ratio of mPEG, EDC, and DMAP of 1:10:10, an activation temperature of 60°C, an activation time of 30 min, and a molar ratio of mPEG:PLGA = 2:1 and ALN:mPEG-PLGA = 10:1. Then, based on the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG), the anti-tuberculosis drug rifapentine (RPT) is loaded, and a bone-targeting delivery system is constructed with a mass ratio of ALN-PLGA-mPEG:RPT = 10:4, namely, rifapentine-loaded bone-targeting nanomicelles (ALN-PLGA-mPEG@RPT). This results in the alendronate sodium (ALN) modified bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) of this invention having uniform and stable particle size, strong colloidal stability, and excellent self-assembly ability. This results in the bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT) of the present invention having high drug loading capacity and long-lasting sustained release capability, as well as outstanding bone targeting, good safety, and good biocompatibility, providing a new strategy for the precise treatment of bone tuberculosis that is highly efficient and low in toxicity.

[0058] Example 13: The bone-targeting nanomicelles loaded with rifapentine were obtained by the following method: The first step involves dissolving methoxy polyethylene glycol (mPEG), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and 4-dimethylaminopyridine (DMAP) in an organic solvent (dimethyl sulfoxide) at a molar ratio of 1:10:10 under light-protected conditions. After stirring and activating at a constant temperature of 40°C for 30 minutes, a mixture is obtained. The second step involves adding polylactic acid-glycolic acid copolymer (PLGA) to the mixture and continuing the reaction for 24 hours to obtain the initial product, wherein the molar ratio of methoxy polyethylene glycol (mPEG) to polylactic acid-glycolic acid copolymer (PLGA) is 1:1. The third step involves dynamically dialyzing the initial product through a dialysis bag (MWCO, 3.5 kDa) for 24 hours, followed by freeze-drying to obtain the polymer (mPEG-PLGA). In the fourth step, the polymer (mPEG-PLGA) was dissolved in water and ultrasonically dispersed (30% amplitude, 3s on / 2s off) to form nanomicelles. The required amounts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to the nanomicelles for activation for 3 hours. Then, sodium alendronate (ALN) was added and stirred for 24 hours to obtain the final product. The amounts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) added were 10 times the molar amount of the polymer (mPEG-PLGA), and the molar ratio of sodium alendronate (ALN) to polymer (mPEG-PLGA) was 10:1. The fifth step involves subjecting the final product to ultrafiltration centrifugation (MWCO, 3 kDa) and freeze-drying to obtain bone-targeting nanomicelle carriers (ALN-PLGA-mPEG). Step 6: Dissolve the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) in water, ultrasonically disperse it into micelles, add an ethanol solution of rifapentine (RPT) to the micelles, stir for 2 hours, and then perform ultrafiltration centrifugation (MWCO, 3kDa) and freeze-drying to obtain bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT). The mass ratio of the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) to rifapentine (RPT) is 10:2.

[0059] Example 14: The bone-targeting nanomicelles loaded with rifapentine were obtained by the following method: The first step involves dissolving methoxy polyethylene glycol (mPEG), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and 4-dimethylaminopyridine (DMAP) in an organic solvent (acetone) at a molar ratio of 1:20:20 under light-protected conditions. After stirring and activating at 80°C for 20 minutes, a mixture is obtained. The second step involves adding polylactic acid-glycolic acid copolymer (PLGA) to the mixture and continuing the reaction for 48 hours to obtain the initial product, wherein the molar ratio of methoxy polyethylene glycol (mPEG) to polylactic acid-glycolic acid copolymer (PLGA) is 3:1. The third step involved dynamically dialyzing the initial product through a dialysis bag (MWCO, 3.5 kDa) for 36 hours, followed by freeze-drying to obtain the polymer (mPEG-PLGA). In the fourth step, the polymer (mPEG-PLGA) was dissolved in water and ultrasonically dispersed (30% amplitude, 3s on / 2s off) to form nanomicelles. The required amounts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to the nanomicelles for activation for 6 hours. Then, sodium alendronate (ALN) was added and stirred for 36 hours to obtain the final product. The amounts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) added were 20 times the molar amount of the polymer (mPEG-PLGA), and the molar ratio of sodium alendronate (ALN) to polymer (mPEG-PLGA) was 15:1. The fifth step involves subjecting the final product to ultrafiltration centrifugation (MWCO, 3 kDa) and freeze-drying to obtain bone-targeting nanomicelle carriers (ALN-PLGA-mPEG). Step 6: Dissolve the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) in water, ultrasonically disperse it into micelles, add an ethanol solution of rifapentine (RPT) to the micelles, stir for 4 hours, and then perform ultrafiltration centrifugation (MWCO, 3kDa) and freeze drying to obtain bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT). The mass ratio of the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) to rifapentine (RPT) is 10:6.

[0060] Example 15: The bone-targeting nanomicelles loaded with rifapentine were obtained by the following method: The first step involves dissolving methoxy polyethylene glycol (mPEG), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and 4-dimethylaminopyridine (DMAP) in an organic solvent (chloroform) at a molar ratio of 1:15:15 under light-protected conditions. After stirring and activating at 60°C for 25 minutes, a mixture is obtained. The second step involves adding polylactic acid-glycolic acid copolymer (PLGA) to the mixture and continuing the reaction for 36 hours to obtain the initial product, wherein the molar ratio of methoxy polyethylene glycol (mPEG) to polylactic acid-glycolic acid copolymer (PLGA) is 2:1. The third step involved dynamically dialyzing the initial product through a dialysis bag (MWCO, 3.5 kDa) for 30 hours, followed by freeze-drying to obtain the polymer (mPEG-PLGA). In the fourth step, the polymer (mPEG-PLGA) was dissolved in water and ultrasonically dispersed (30% amplitude, 3s on / 2s off) to form nanomicelles. The required amounts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to the nanomicelles for activation for 4 hours. Then, sodium alendronate (ALN) was added and stirred for 30 hours to obtain the final product. The amounts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) added were 15 times the molar amount of the polymer (mPEG-PLGA), and the molar ratio of sodium alendronate (ALN) to polymer (mPEG-PLGA) was 12:1. The fifth step involves subjecting the final product to ultrafiltration centrifugation (MWCO, 3 kDa) and freeze-drying to obtain bone-targeting nanomicelle carriers (ALN-PLGA-mPEG). Step 6: Dissolve the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) in water, ultrasonically disperse it into micelles, add an ethanol solution of rifapentine (RPT) to the micelles, stir for 3 hours, and then perform ultrafiltration centrifugation (MWCO, 3 kDa) and freeze drying to obtain bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT). The mass ratio of the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) to rifapentine (RPT) is 10:4.

[0061] Example 16: The bone-targeting nanomicelles loaded with rifapentine were obtained by the following method: Step 1, activate methoxylated polyethylene glycol (mPEG): Weigh out 400 mg of methoxy polyethylene glycol (mPEG, Mn=1000), 766.8 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and 488.7 mg of 4-dimethylaminopyridine (DMAP), i.e., the molar ratio of mPEG:EDC:DMAP = 1:10:10. Place the mixture in a 50 mL light-proof, dry round-bottom flask, add 30 mL of dimethyl sulfoxide (DMSO), and stir magnetically in a 60 °C water bath (200 rpm) for 30 min to activate the carboxyl groups of mPEG, thus obtaining a mixture. The second step is the coupling of polylactic-co-glycolic acid copolymer (PLGA): Add 2g of polylactic acid-glycolic acid copolymer (PLGA, with a molar ratio of lactic acid to glycolic acid of 75:25 and Mw=10,000) to the mixture (the molar ratio of mPEG to PLGA is 2:1), and continue the reaction in the dark for 24h to obtain the initial product; Step 3, purification: The initial product was transferred to a dialysis bag (MWCO 3.5 kDa) for dynamic dialysis (ultrapure water, 2000 mL) for 48 h, with the water changed every 6 h. The dialysate was collected and freeze-dried to obtain the polymer (mPEG-PLGA) with a yield of 76.7%. Step 4, modification of alendronate sodium (ALN): 550 mg of polymer (mPEG-PLGA) was dissolved in ultrapure water and ultrasonically dispersed (30% amplitude, 3s on / 2s off) to form nanomicelles. 95.9 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 57.5 mg of N-hydroxysuccinimide (NHS) were added to the nanomicelles (the amounts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were each 10 times the molar amount of polymer (mPEG-PLGA)). The mixture was stirred at room temperature in the dark for 4 h to activate the carboxyl groups. Then, 135.5 mg of sodium alendronate (ALN) (molar ratio ALN:mPEG-PLGA:=10:1) was added and stirred in the dark for 24 h to obtain the final product. Step 5, further purification: The final product was purified by ultrafiltration and centrifugation (MWCO = 3 kDa, 4000 × g), and the supernatant was collected and freeze-dried to obtain the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG); the synthetic route is as follows. Figure 1 As shown; Step 6, loading the drug: 100 mg of bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) was dissolved in 100 mL of ultrapure water and sonicated with a probe (30% amplitude, 3 s on / 2 s off) for 15 min to disperse it into micelles. An ethanol solution of rifapentine (RPT) (40 mg RPT dissolved in 10 mL of ethanol, i.e., the mass ratio of bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) to rifapentine (RPT) was added dropwise to the micelles and stirred for 3 h. Then, the mixture was subjected to ultrafiltration and centrifugation (MWCO 3 kDa, 4000 × g) to remove free drug, and the retained layer was collected. After freeze-drying, a light red powder was obtained, which is the bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT).

[0062] Experimental Example 1: The effect of different feed ratios n(mPEG:PLGA) of the raw materials methoxy polyethylene glycol (mPEG) and polylactic acid-glycolic acid copolymer (PLGA) on the properties of polymer (mPEG-PLGA) nanomicelles was investigated.

[0063] Experimental Methods: Following the preparation method in Example 16, the first step involved weighing methoxy polyethylene glycol (mPEG), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and 4-dimethylaminopyridine (DMAP), placing them in a 50 mL dry round-bottom flask, and then adding dimethyl sulfoxide (DMSO). Under light-shielding conditions, the mixture was magnetically stirred in a 60°C water bath (200 rpm) for 30 min to activate the terminal carboxyl groups of the mPEG, yielding a mixture. The second step involved adding polylactic acid-glycolic acid copolymer (PLGA) to the round-bottom flask at different molar ratios to mPEG (0.5:1, 1:1, 1:2, and 2:1), and continuing the reaction for 24 h. After the reaction was complete, the initial product was obtained. The third step involved placing the initial product into a dialysis bag (MWCO 3.5). In a dialysis bag, the dialysis bag was completely immersed in 2000 mL of ultrapure water for dynamic dialysis for 48 h, during which the external dialysis medium was replaced periodically. After freeze-drying, the polymer (mPEG-PLGA) was obtained. In the fourth step, an appropriate amount of polymer (mPEG-PLGA) was weighed into ultrapure water and dissolved by sonication (amplitude 30, sonication for 3 min, sonication for 3 s, pause for 2 s) to obtain polymer (mPEG-PLGA) nanomicelles. The particle size, PDI, and zeta potential of the nanomicelles were determined using a nanoparticle size and zeta potential analyzer. The critical micelle concentration was determined using the pyrene fluorescent probe method.

[0064] Experimental results: The effect of the feed ratio n(mPEG:PLGA) on the properties of polymer (mPEG-PLGA) nanomicelles is shown in Table 1. Figures 2 to 5 As shown, Figure 2 The curve shows the change of the I1 / I3 value with c(polymer (mPEG-PLGA) nanomicelles) when the feed ratio n(mPEG:PLGA) is 0.5:1. Figure 3 The curve shows the value of I1 / I3 as a function of c(polymer (mPEG-PLGA) nanomicelles) when n(mPEG:PLGA) is 1:1. Figure 4 The curve shows the value of I1 / I3 as a function of c(polymer (mPEG-PLGA) nanomicelles) when n(mPEG:PLGA) is 2:1. Figure 5 The curves showing the I1 / I3 values ​​as a function of c(polymer (mPEG-PLGA) nanomicelles) when n(mPEG:PLGA) is 5:1 are shown; Table 1 shows the effect of the feed ratio n(mPEG:PLGA) on the properties of polymer (mPEG-PLGA) nanomicelles. ±s, n =3).

[0065] from Figure 2It can be seen that when the feed ratio n(mPEG:PLGA) is 0.5:1, the critical micelle concentration of the polymer (mPEG-PLGA) nanomicelles is 0.0025 mg / mL. From... Figure 3 It can be seen that when the feed ratio n(mPEG:PLGA) is 1:1, the critical micelle concentration of the polymer (mPEG-PLGA) nanomicelles is 0.0030 mg / mL. From... Figure 4 It can be seen that when the feed ratio n(mPEG:PLGA) is 1:2, the critical micelle concentration of the polymer (mPEG-PLGA) nanomicelles is 0.0050 mg / mL. From... Figure 5 It can be seen that when the feed ratio n(mPEG:PLGA) is 2:1, the critical micelle concentration of polymer (mPEG-PLGA) nanomicelles is 0.0075 mg / mL.

[0066] As shown in Table 1, the particle size of the polymer (mPEG-PLGA) nanomicelles obtained by the four feed ratios is within the range of nanomicelles. Among them, the polymer (mPEG-PLGA) nanomicelles obtained by the feed ratio of mPEG to PLGA of 2:1 have the lowest molecular weight distribution (PDI), more uniform particle size distribution, and higher yield (96.47% ± 3.69%), with a critical micelle concentration (CMC) of 0.0050 mg / mL. Therefore, the optimal feed ratio of the raw materials methoxy polyethylene glycol (mPEG) and polylactic acid-glycolic acid copolymer (PLGA) is 2:1, i.e., n(mPEG:PLGA) is 2:1.

[0067] Experimental Example 2: Structural characterization of the polymer (mPEG-PLGA) nanomicelles prepared in this invention.

[0068] Experimental Methods: Structural analysis of the polymer (mPEG-PLGA) obtained in step 3 of Example 16 was performed using a 600 MHz nuclear magnetic resonance (NMR) spectrometer. mPEG, PLGA, and their copolymers were accurately weighed and dissolved in 0.6 mL of deuterated chloroform (CDCl3) to prepare test solutions. After thorough dissolution, the sample solutions were transferred to a 5 mm NMR tube, and proton NMR data for each component were acquired. By comparing the characteristic proton signal shifts and integral area changes of the monomers and copolymers, and combining the assignment of characteristic resonance peaks of ether bonds and ester groups in the polymer molecular structure, the chemical structural characteristics of the copolymer were systematically analyzed, providing spectroscopic evidence for successful molecular chain bonding. Infrared spectra were recorded using an infrared spectrometer, and the structure of mPEG-PLGA was confirmed by comparing the spectra of PEG, PLGA, and mPEG-PLGA.

[0069] Experimental results: The 1H NMR spectrum (600 MHz, CDCl3) of methoxy polyethylene glycol (mPEG) is as follows: Figure 6 As shown, the 1H NMR spectrum (600 MHz, CDCl3) of polylactic acid-glycolic acid copolymer (PLGA) is as follows. Figure 7 As shown, the proton NMR spectrum (600 MHz, CDCl3) of the polymer (mPEG-PLGA) is as follows. Figure 8 As shown, from Figures 6 to 8 As can be seen from the 1H NMR spectrum analysis, in terms of PLGA structural characterization, the characteristic signals of the lactic acid unit in the PLGA block are as follows: the peak of methyl hydrogen (-CH3) at 1.59 ppm and the peak of methylene hydrogen (-CH-) at 5.22 ppm; while the characteristic peak of methylene hydrogen (-CH2-) in the glycolic acid unit appears at 4.84 ppm. In terms of mPEG structural characterization, the peak at 3.65 ppm corresponds to the proton signal of the oxyethane repeating unit (-OCH2CH2-), while the peak at 3.38 ppm belongs to the terminal methoxy group (-OCH3). Notably, the residual solvent peak at 7.27 ppm originates from deuterated chloroform (CDCl3). The chemical shifts of each characteristic peak are highly consistent with the expected molecular structure, clearly confirming the successful synthesis of the polymer (mPEG-PLGA).

[0070] The infrared spectrum of methoxy polyethylene glycol (mPEG) is as follows: Figure 9 As shown, the infrared spectrum of polylactic-co-glycolic acid copolymer (PLGA) is as follows. Figure 10 As shown, the infrared spectrum of the polymer (mPEG-PLGA) is as follows: Figure 11 As shown, from Figures 9 to 11 As can be seen, infrared spectroscopy analysis shows that the synthesized polymer (mPEG-PLGA) has the following characteristic absorption peaks: (1) High frequency region: 2500~3300 cm⁻¹ -1 A broad absorption peak appears at -2870~2950 cm⁻¹, which is attributed to the stretching vibration of OH in free carboxylic acids; -1 The characteristic peaks within this range correspond to the CH stretching vibration of the methylene (-CH2-) group in the PEG chain segment; -2830cm -1 The weak absorption peak can be attributed to the CH stretching vibration of the methoxy group (-OCH3). (2) Carbonyl vibration region: -1750cm -1 The strong absorption peak at -1700 cm⁻¹ corresponds to the characteristic C=O stretching vibration of the ester bond (lactic acid and glycolic acid units) in the PLGA chain segment; -1 The nearby absorption peak corresponds to the C=O stretching vibration of the carboxylic acid group, and this peak may partially overlap with the C=O vibration peak of the PLGA ester group. (3) Vibration region of ether bond and ester bond: -1180~1260cm -1 The absorption peaks within the range originate from the COC asymmetric stretching vibration of the PLGA ester bond; -1100cm-1 The strong absorption peak is a characteristic stretching vibration of the ether bond (COC) in the PEG chain.

[0071] Key synthetic evidence shows that three characteristic absorption peaks are present simultaneously in the spectrum: (1) the characteristic peak of the ether bond of the PEG segment (1100 cm⁻¹). -1 (2) C=O vibration peak of PLGA ester group (1750 cm⁻¹) -1 ); and (3) the C=O vibration peak of the carboxylic acid group (1700 cm⁻¹). -1 The synergistic presence of these characteristic peaks effectively confirmed the successful synthesis of the target product, indicating that the polymer (mPEG-PLGA) simultaneously contains PEG segments, PLGA ester bonds, and terminal carboxylic acid groups. This further confirms the successful synthesis of the polymer (mPEG-PLGA).

[0072] Experimental Example 3: To investigate the method for determining the ultraviolet content of alendronate sodium (ALN).

[0073] Experimental Method: 0 mL, 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL, 3.0 mL, 3.5 mL, 4.0 mL, and 4.5 mL of 200 µg / mL alendronate sodium solution were respectively placed into 10 mL volumetric flasks. 0.8 mL of 0.01 mol / L sodium bicarbonate solution was added sequentially. All volumetric flasks were heated in a 90°C water bath for 10 min. Then, 3.5 mL of 0.2 wt% water and ninhydrin ethanol solution were added sequentially. The solutions were then diluted to the mark with ultrapure water and thoroughly mixed to obtain ALN ​​standard solutions of different concentrations. All volumetric flasks were heated in a 90°C water bath for 30 min, cooled to room temperature, and then diluted to the mark with ultrapure water. The sodium alendronate standard solution was scanned at 300 nm to 800 nm using an ultraviolet-visible spectrophotometer (UV-Vis), and the chromatogram was recorded. The wavelength of maximum absorption was used as the detection wavelength. The absorbance (OD) of ALN standard solutions of different concentrations was measured sequentially. A standard curve of sodium alendronate was plotted with concentration on the x-axis and absorbance on the y-axis, and the equation of the standard curve was determined. For specificity experiments, blank solvent, colorimetric reference solution, and test solution were scanned at the full wavelength range of 300–800 nm. By comparing the UV absorption spectra of the three solutions, it was confirmed that the blank solvent had no interfering peaks at the target wavelength, and that the reference and test solutions were consistent at their characteristic absorption peaks.

[0074] Experimental results: The colorimetric reaction of sodium alendronate (ALN) with ninhydrin solution is as follows: Figure 12 As shown, the standard curve of alendronate sodium (ALN) concentration versus absorbance value is as follows: Figure 13As shown, the UV absorption spectra of alendronate sodium (ALN) reference standard, test solution, and blank solution are as follows: Figure 14 As shown.

[0075] Figure 12 The colorimetric reaction of alendronate sodium (ALN) with ninhydrin hydrate is demonstrated. Figure 12 It can be seen that no characteristic color development was observed in the blank control group (sample 0), while in the gradient concentration experimental groups (samples 1 to 5), the system showed a change from light to dark blue-purple as the concentration of sodium alendronate (ALN) increased, indicating that the color intensity was significantly correlated with the concentration of sodium alendronate (ALN).

[0076] The absorbance values ​​(OD568) of each concentration gradient were measured at a wavelength of 568 nm using ultraviolet spectrophotometry, and a standard curve was established as follows: Figure 13 As shown. From Figure 14 Quantitative analysis results show that, within the selected concentration range, absorbance exhibits a good linear relationship with alendronate sodium (ALN) concentration, with the regression equation being: y = 16.108x - 0.1733, R0 2 =0.9964. The establishment of this standard curve confirms that the colorimetric reaction has a stable quantitative detection function in the concentration range of 0.01 mg / mL to 0.10 mg / mL.

[0077] from Figure 14 As can be seen, the alendronate sodium (ALN) reference solution, test solution, and blank solution were analyzed across the entire wavelength range of 300 nm to 800 nm. Experimental data show that the UV spectral characteristics of the test and reference solutions are highly consistent, both exhibiting symmetrical single-peak absorption characteristics at 568 nm, with the absorption intensity reaching its maximum at this peak. Notably, no interfering absorption signal was detected in the blank matrix solution at this characteristic wavelength, proving that this method can effectively eliminate background interference. Based on the above spectral characteristic analysis, 568 nm was determined to be the optimal detection wavelength. The research results fully verify that the raw material alendronate sodium (ALN) selected in this invention possesses ideal specificity in the UV detection system.

[0078] Experiment Example 4: The effect of alendronate sodium (ALN) dosage on the synthesis of bone-targeting nanomicelle carriers (ALN-PLGA-mPEG) was investigated.

[0079] Experimental Methods: First, 20 mg of the synthesized polymer (mPEG-PLGA) was accurately weighed and dissolved in ultrapure water by slow dropwise addition. Then, the mixture was treated with an ultrasonic disruptor (30% amplitude, 3 seconds of sonication followed by a 2-second pause) for 3 minutes to form a uniform nanomicelle dispersion. This nanomicelle dispersion was transferred to a round-bottom flask, and carboxyl activation was performed by adding chemical crosslinking agents sequentially in a light-protected environment: first, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was added and pre-activated for 30 minutes using a magnetic stirrer; then, N-hydroxysuccinimide (NHS) was introduced and the reaction continued for 4 hours. The molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to N-hydroxysuccinimide (NHS) was 10 times that of the polymer (mPEG-PLGA). Finally, sodium alendronate (ALN) was added, and a covalent coupling reaction was carried out for 24 hours under constant temperature and stirring conditions. Sodium alendronate (ALN) and polymer (mPEG-PLGA) were reacted at molar ratios of 1:1, 5:1, and 10:1, respectively. After complete reaction, the mixture was transferred to an ultrafiltration tube (MWCO, 3 kDa) and centrifuged in a high-capacity, high-speed benchtop refrigerated centrifuge. The supernatant was collected and freeze-dried to obtain the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG). The dosage of sodium alendronate (ALN) was determined based on the particle size, PDI, and zeta potential of the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG).

[0080] Experimental results: The effect of the amount of alendronate sodium (ALN) added on the synthesis of bone-targeted nanomicelle carrier (ALN-PLGA-mPEG) is shown in Table 2.

[0081] Table 2 shows the effect of the feed ratio of sodium alendronate (ALN) to polymer (mPEG-PLGA) on the synthesis of bone-targeted nanomicelle carriers (ALN-PLGA-mPEG). As can be seen from Table 2, the particle size of the bone-targeted nanomicelle carriers (ALN-PLGA-mPEG) meets the requirements. When the feed ratio n(ALN:mPEG-PLGA) is 10:1, the resulting bone-targeted nanomicelle carriers (ALN-PLGA-mPEG) have a smaller molecular weight distribution (PDI), a more uniform particle size distribution, and a larger absolute potential value, indicating that the bone-targeted nanomicelle carriers (ALN-PLGA-mPEG) prepared in this invention have good stability. Therefore, the optimal feed ratio of sodium alendronate (ALN) to polymer (mPEG-PLGA) for synthesizing bone-targeted nanomicelle carriers (ALN-PLGA-mPEG) is 10:1.

[0082] Experimental Example 5: Determination of grafting rate of alendronate sodium (ALN).

[0083] Experimental Method: The grafting rate of ALN was determined using the ninhydrin hydrate reaction method. First, a 0.01 mol / L sodium bicarbonate solution and a 0.2 wt% ninhydrin hydrate ethanol solution were prepared. Following step 5 of Example 16, an appropriate amount of the lower layer solution after ultrafiltration and centrifugation was added to a 10 mL volumetric flask. Then, 0.8 mL of the 0.01 mol / L sodium bicarbonate solution was added sequentially. The volumetric flask was heated in a 90°C water bath for 10 min. Next, 3.5 mL of the 0.2 wt% ninhydrin hydrate ethanol solution was added sequentially. The solution was then diluted to volume with ultrapure water and thoroughly mixed. The volumetric flask was heated in a 90°C water bath for 30 min, cooled to room temperature, and then diluted to volume with ultrapure water again. The absorbance was measured at a wavelength of 568.0 nm. The absorbance value was substituted into the standard curve equation to calculate the concentration, and the grafting rate of alendronate sodium (ALN) was further calculated. ALN grafting rate = (ALN input amount - measured ALN amount) / ALN input amount × 100%.

[0084] Experimental Results: A bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) synthesized with an alendronate sodium (ALN) to polymer (mPEG-PLGA) ratio of 10:1 was used. Sample solutions were prepared according to the above method, and absorbance was measured. The results were then substituted into the standard curve of alendronate sodium (ALN) concentration versus absorbance (i.e., in Experimental Example 3). Figure 13 From the obtained standard curve, the concentration of unreacted sodium alendronate (ALN) was calculated, and the final calculated grafting rate of sodium alendronate (ALN) was 51.16%, which also indicates that the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) was successfully synthesized.

[0085] Experimental Example 6: Establish an HPLC standard curve for rifapentine (RPT).

[0086] Experimental Methods: Accurately weigh 5.00 mg of rifapentine (RPT), dissolve it in methanol, transfer it to a 50 mL amber volumetric flask, and dilute to the mark to prepare a standard stock solution with a concentration of 100 µg / mL. Prepare a series of standard working solutions (5 µg / mL, 10 µg / mL, 20 µg / mL, 30 µg / mL, 40 µg / mL, 50 µg / mL) using a serial dilution method. Analyze the solutions using high-performance liquid chromatography (HPLC), and record the integrated peak area values ​​for each concentration. Plot the peak area (A) on the ordinate and the concentration (C) on the abscissa to obtain the standard curve regression equation.

[0087] Preparation of mobile phase in chromatographic conditions: Isocratic elution was used in this experiment. Mobile phase A was chromatographically pure methanol. The preparation method of mobile phase B was as follows: 5.00 g of potassium dihydrogen phosphate (KH2PO4, AR grade) was accurately weighed, dissolved in ultrapure water with a resistivity of 18.25 MΩ·cm, degassed by sonication, and then diluted to volume in a 1 L volumetric flask to obtain a buffer solution with a concentration of 0.0367 mol / L.

[0088] Chromatographic parameters used: An Agilent C18 column (4.6 × 250 mm, 5 μm) was used, and the column oven temperature was maintained at 25 ± 0.5 °C. A UV-Vis detector was used, with the detection wavelength set to 254 nm. The elution program was in isocratic mode, with an organic-to-aqueous phase volume ratio of 7:3 (methanol-phosphate buffer), a flow rate of 1.00 mL / min, and an autosampler injection volume of 10.0 μL. All analytical procedures were performed before filtration through a 0.22 μm microporous membrane and ultrasonic degassing.

[0089] Experimental Results: The rifapentine (RPT) standard curve established based on HPLC is shown in Figure 15. In the figure, the drug concentration of the series of standard solutions is taken as the independent variable (C), and the corresponding chromatographic response value is taken as the dependent variable (A). The regression equation obtained by linear fitting is A = 281.75C - 125.37, R0 2 =0.9995. This indicates that the HPLC detection system exhibits good linearity in the concentration range of 5 µg / mL to 50 µg / mL, meeting the quantitative analysis requirements for rifapentine (RPT).

[0090] Experiment Example 7: The encapsulation efficiency and drug loading of bone-targeting nanomicelles (ALN-PLGA-mPEG@RPT) loaded with rifapentine were determined in this invention.

[0091] Experimental Methods: Rifapentine (RPT) in bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT) was quantitatively analyzed by HPLC. Drug loading (DLC) was characterized as the percentage of rifapentine (RPT) loaded in the nanomicelles to the total mass of the drug-loaded nanomicelles, while encapsulation efficiency (DLE) reflected the percentage of the actual rifapentine (RPT) loaded to the initial drug dosage. In the formulation process, the unretained liquid after ultrafiltration was collected, and the free drug concentration was determined based on the rifapentine (RPT) standard curve to calculate the unloaded drug mass.

[0092] Experimental results: The encapsulation efficiency and drug loading of rifapentine (RPT) are shown in Table 3.

[0093] As shown in Table 3, when the mass ratio of bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) to rifapentine (RPT) is 10:4, the prepared rifapentine-loaded bone-targeting nanomicelles (ALN-PLGA-mPEG@RPT) exhibits the best drug loading efficiency, with a drug loading of (16.74±0.51)% and a corresponding encapsulation efficiency of (50.27±1.91)%. This ratio is significantly better than other experimental groups in terms of drug loading. Therefore, the optimal mass ratio of bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) to rifapentine (RPT) is 10:4.

[0094] Experimental Example 8: This invention presents an in vitro drug release kinetics study of bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT).

[0095] Experimental Methods: The in vitro release characteristics of the rifapentine-loaded bone-targeting nanomicelles of this invention were investigated using dynamic dialysis technology. Rifapentine-loaded bone-targeting nanomicelles (ALN-PLGA-mPEG@RPT), rifapentine (RPT), and a physical mixture of rifapentine-loaded bone-targeting nanomicelles (ALN-PLGA-mPEG@RPT) and rifapentine (RPT) (mixed in equal volumes at a 1:1 ratio, labeled ALN-PLGA-mPEG+RPT) were placed in a dialysis bag (MWCO, 3.5 kDa) and 100 mL of phosphate buffer (pH 7.4) was used as the release medium. The dialysis was performed under isothermal shaking conditions at 37 ± 0.5 °C (100 rpm). 1 mL samples were taken at time points of 1 h, 2 h, 5 h, 8 h, 12 h, 24 h, 48 h, 72 h, and 120 h, and an equal volume of pre-warmed phosphate buffer was immediately added. All samples were filtered through a 0.22 μm microporous membrane, and the rifapentine (RPT) content was quantitatively analyzed by HPLC. Three parallel samples were set up for each experimental group, and data are expressed as mean ± standard deviation. The cumulative release of rifapentine (RPT) was calculated using the following formula: Where Cn is the drug concentration in the buffer solution taken out for the nth time (μg / mL); V is the volume of the buffer solution in the release container (mL); Cx is the drug concentration in the buffer solution taken out for the xth time (μg / mL); V' is the volume of the buffer solution taken out each time (mL); and M is the amount of drug loaded in each dialysis bag (μg).

[0096] Experimental results: The in vitro cumulative release curve of rifapentine (RPT) is as follows: Figure 16 As shown, from Figure 16It can be seen that the free RPT group achieved a cumulative release rate of 90% within 12 hours, and the physical mixture group (ALN-PLGA-mPEG+RPT) achieved a release rate of 90% within 48 hours. In contrast, the bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT) of this invention only released 25% within 12 hours and 65% within 48 hours. These kinetic data indicate that the bone-targeting nanomicelles loaded with rifapentine of this invention (ALN-PLGA-mPEG@RPT) exhibits a significantly lower burst release effect compared to the free drug (rifapentine (RPT)) and the physical mixture (ALN-PLGA-mPEG+RPT), confirming that the bone-targeting nanomicelles loaded with rifapentine of this invention (ALN-PLGA-mPEG@RPT) possesses significant sustained-release properties.

[0097] Experiment Example 9: In vitro bone targeting evaluation of the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) prepared in the fifth step of this invention.

[0098] Experimental Methods: The affinity of three groups of micelles for hydroxyapatite in vitro was investigated using DIR-labeled mPEG-PLGA, ALN and mPEG-PLGA physical mixtures, and ALN-PLGA-mPEG groups. The fluorescently labeled mPEG-PLGA, ALN and mPEG-PLGA micelle mixtures, and ALN-PLGA-mPEG groups were incubated with hydroxyapatite (HA) in a light-protected, temperature-controlled shaker (200 rpm, 37℃) for 3 h, 6 h, and 18 h, respectively, followed by centrifugation to separate the unbound nanomicelles from HA. The nanomicelles adsorbed on HA at different incubation times were observed using a fluorescence inverted microscope.

[0099] Experimental Results: The in vitro bone affinity of the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) prepared in step five of this invention is as follows: Figure 17 As shown, Figure 17 The fluorescence intensity images of the mPEG-PLGA group, the ALN and mPEG-PLGA physical mixture group, and the ALN-PLGA-mPEG group are shown after 3h, 6h, and 18h ​​of incubation. Figure 17 As shown, the fluorescence intensity of HA increased with increasing incubation time. The fluorescence intensity differences between the mPEG-PLGA group, the ALN group, and the mPEG-PLGA physical mixture group were not significant. HA hatched in the ALN-PLGA-mPEG group exhibited stronger fluorescence than the mPEG-PLGA group. This is because sodium alendronate (ALN) reacts with the Ca in HA. 2+ The specific binding of hydroxyapatite to hydroxyapatite indicates that the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) prepared in step five of this invention has good affinity with hydroxyapatite in vitro.

[0100] Experimental Example 10: In vivo bone targeting evaluation of the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) prepared in the fifth step of this invention.

[0101] Experimental methods: ICR mice were anesthetized before drug administration and at 1h, 3h, 6h, 10h, 24h, 48h, and 72h after drug administration, and then fluorescence imaging was performed in a small animal in vivo imaging system. In some experimental animals, after a tail vein injection of fluorescently labeled micelles for 24 hours, the spine was dislocated, and the long bones of the limbs, vertebrae, and associated ribs were completely separated. Residual soft tissue was removed using ophthalmic surgical scissors, and the samples were transferred to 10mL sterile culture dishes for in vitro fluorescence imaging.

[0102] The instrument parameters were set using the LivingImage 4 program. The cooling temperature was adjusted to -90℃ to maintain detector sensitivity. The exposure parameters were set to automatic mode. The optical system used a lens with an f-number of 2. The imaging range was set to D-23cm, and the objective lens focusing height was fixed at 1.5cm to suit the mouse's body size. The excitation wavelength was set to 745nm, and the emission wavelength was set to 800nm.

[0103] Experimental results: The fluorescence scanning results of the mouse in vivo imaging system are as follows: Figure 18 As shown, A represents the ALN-PLGA-mPEG group; B represents the PLGA-mPEG group; and C represents the free DIR group. Figure 18 The results show that small animal in vivo imaging technology can effectively trace the in vivo distribution characteristics of DIR-labeled nanomicelles. From Figure 18 As shown in A, B, and C, after intravenous administration, the fluorescence signals of the mPEG-PLGA group and the free DIR group mainly accumulated in the liver, while the fluorescence signal of the ALN-PLGA-mPEG group showed significant accumulation in both the limbs and liver tissue.

[0104] Limb bones, spine, and thoracic rim of mice in the ALN-PLGA-mPEG group, mPEG-PLGA group, and free DIR group were removed and simultaneously exposed to the same mode. The fluorescence scanning results of the isolated mouse bone tissue are shown below. Figure 19 As shown.

[0105] from Figure 19As can be seen, the ALN-PLGA-mPEG group exhibited higher fluorescence intensity in bone, while no significant fluorescence signal enrichment was detected in the mPEG-PLGA group and the free DIR group, indicating that the bone tissue targeting efficiency of DIR molecules in the mPEG-PLGA group and the free DIR group was low. These data confirm that the introduction of alendronate sodium (ALN) ligand in the ALN-PLGA-mPEG group can significantly enhance the active targeting ability of the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) in bone tissue.

[0106] Experimental Example 11: This invention investigates the in vivo distribution characteristics of bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT).

[0107] Experimental methods: Thirty-six healthy male SD rats were randomly divided into two groups: the RPT administration group and the ALN-PLGA-mPEG@RPT administration group. Each group was given an equal dose of RPT.

[0108] Blood samples were collected from the orbital venous plexus of rats at 1 h, 24 h, and 48 h after drug administration, followed by liver and bone tissue collection. Tissue samples were precisely weighed and prepared into 20% (w / v) saline homogenates. The RPT content in each sample was quantitatively determined using HPLC. The in vivo accumulation characteristics of the bone-targeting nanomicelle carrier (ALN-PLGA-mPEG) were systematically evaluated by calculating the ratio of tissue drug concentration (μg / g) to concurrent plasma drug concentration (μg / mL) (Ctissue / Cplasma). Data are presented as mean ± standard deviation.

[0109] Experimental Results: This experiment successfully established a quantitative analysis method for rifapentine (RPT). The standard curve for rifapentine (RPT) in biological samples by HPLC is shown below. Figure 20 As shown, with concentration (C) as the horizontal axis and chromatographic peak area (A) as the vertical axis, the equation obtained by linear regression analysis is: A=256.29C+187.59 (R²=0.9993). This model shows excellent linearity in the detection range of 0.25g / mL to 25μg / mL, meets the requirements for biological sample detection, and can be accurately applied to the quantitative analysis of rifapentine (RPT) in rat plasma.

[0110] The distribution of RPT in rats at 1 h, 24 h, and 48 h was determined by HPLC. The distribution ratios of RPT in different tissues of rats are shown in Table 4.

[0111] As shown in Table 4, 1 hour after administration of the non-targeted rifapentine (RPT) group, the highest drug concentration was observed in the liver, with the distribution order in vivo being: liver > blood > bone. At this point, the drug content in bone was only 23% of the plasma concentration. During continuous observation from 24 to 48 hours, the formulation maintained its original distribution characteristics, with liver tissue consistently being the main accumulation site, and the RPT concentration in bone tissue being significantly lower than that in the liver and blood. In contrast, after 1 hour of administration of the bone-targeted nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT) of this invention, although the overall distribution gradient still followed liver > blood > bone, the drug concentration in bone tissue was close to the plasma level, and the rifapentine (RPT) concentration in the liver was lower than that in the non-targeted group (P < 0.05), suggesting that nanocarriers can effectively alter the in vivo distribution pattern of drugs. Furthermore, after 24 h and 48 h of administration, the distribution of rifapentine-loaded bone-targeting nanomicelles (ALN-PLGA-mPEG@RPT) showed a change from liver > bone > blood. Specifically, at 48 h after administration, the drug concentration in bone tissue was 1.93 times that in blood, while it was only 33% in the non-targeted group. Moreover, the rifapentine (RPT) concentration in bone tissue at each sampling point was higher than that in the non-targeted group (P<0.05), indicating that the rifapentine-loaded bone-targeting nanomicelles (ALN-PLGA-mPEG@RPT) of the present invention has good bone targeting properties.

[0112] Experimental Example 12: Safety evaluation of bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT) of the present invention.

[0113] Experimental methods: HE staining was used to assess the HE staining of rat organs by the ALN-PLGA-mPEG@RPT group, and then the safety of ALN-PLGA-mPEG@RPT was evaluated.

[0114] Experimental results: HE staining results of rat organs are as follows Figure 21 As shown, from Figure 21 As can be seen, 48 hours after drug administration, compared with the saline control group, there were no significant morphological differences in the heart, spleen, lungs, kidneys, and liver of rats in the ALN-PLGA-mPEG@RPT experimental group after HE staining, and no significant signs of necrosis or inflammation were observed. This indicates that the bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT) of this invention have good safety.

[0115] The experimental data above demonstrate that the bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT) prepared by the optimal synthesis process of this invention have a particle size of 89.34±1.91 nm, a drug loading of 16.74%, an encapsulation efficiency of 50.27%, and a critical micelle concentration of 0.0050 mg / mL, exhibiting excellent stability. Alendronate sodium (ALN) achieves active targeting by specifically binding to the bone matrix hydroxyapatite through bisphosphonate groups; the polymer (PLGA-mPEG) core provides sustained-release properties, with a 12-hour drug release rate of only 25%, which is superior to the original drug group (90%). In vitro / in vivo experiments confirmed that the bone-targeting group has a stronger affinity for hydroxyapatite, with the drug concentration in bone tissue being 1.93 times that in blood (0.33 times that in the non-targeting group). In vivo imaging showed significantly enhanced fluorescence signals in the bone area, and no pathological damage to major organs. Therefore, the bone-targeting nanomicelles loaded with rifapentine (ALN-PLGA-mPEG@RPT) of this invention can significantly improve the bone-targeting efficiency of drugs and reduce systemic toxicity, providing a new strategy for the precision treatment of bone tuberculosis.

[0116] In summary, this invention combines the bone-targeting ability of alendronate sodium (ALN) with the drug-loading and long-cycle advantages of polymer mPEG-PLGA micelles by precisely controlling the synthesis process (light shielding, catalyst ratio, temperature, and feed ratio) of alendronate sodium (ALN) modified bone-targeting nanomicelle carrier (ALN-PLGA-mPEG). This results in rifapentine-loaded bone-targeting nanomicelles (ALN-PLGA-mPEG@RPT) that significantly improves bone targeting and sustained-release performance, providing a highly efficient and low-toxicity new strategy for the precise treatment of bone tuberculosis.

[0117] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Unnecessary technical features can be added or removed according to actual needs to meet the needs of different situations.

Claims

1. A bone-targeting nanomicelle loaded with rifapentine, characterized in that... Obtained using the following method: The first step involves dissolving the required amounts of methoxy polyethylene glycol, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 4-dimethylaminopyridine in an organic solvent under light-protected conditions, followed by stirring and activation to obtain a mixture. The second step involves adding the required amount of polylactic acid-glycolic acid copolymer to the mixture and continuing the reaction to obtain the initial product. The third step involves dynamic dialysis and freeze-drying of the initial product to obtain the polymer. The fourth step involves dissolving the polymer in water, ultrasonically dispersing it into micelles, adding the required amounts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the micelles for activation, and then adding sodium alendronate and stirring the reaction to obtain the final product. The fifth step involves subjecting the final product to ultrafiltration, centrifugation, and freeze-drying to obtain a bone-targeting nanomicelle carrier. Step 6: Dissolve the bone-targeting nanomicelle carrier in water, ultrasonically disperse it into micelles, add an ethanol solution of rifapentine to the micelles, stir, and then obtain bone-targeting nanomicelles loaded with rifapentine after ultrafiltration, centrifugation and freeze-drying.

2. The bone-targeting nanomicelles loaded with rifapentine according to claim 1, characterized in that... In the first step, the molar ratio of methoxy polyethylene glycol, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine is 1:10 to 20:10 to 20.

3. The bone-targeting nanomicelles loaded with rifapentine according to claim 1 or 2, characterized in that... In the second step, the molar ratio of methoxy polyethylene glycol to polylactic acid-hydroxyacetic acid copolymer is 1 to 5:

1.

4. The bone-targeting nanomicelles loaded with rifapentine according to claim 1, 2, or 3, characterized in that... In the fourth step, the amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide added is 10 to 20 times the molar amount of the polymer, and the molar ratio of sodium alendronate to the polymer is 5 to 10:

1.

5. Bone-targeting nanomicelles loaded with rifapentine according to any one of claims 1 to 4, characterized in that... In step six, the mass ratio of bone-targeting nanomicelle carrier to rifapentine is 10:2 to 5.

6. Bone-targeting nanomicelles loaded with rifapentine according to any one of claims 1 to 5, characterized in that... In the first step, the organic solvent is one of dimethyl sulfoxide, acetone, and chloroform.

7. Bone-targeting nanomicelles loaded with rifapentine according to any one of claims 1 to 6, characterized in that... In the first step, the temperature is 40℃ to 80℃ during stirring, and the activation time is 20min to 40min; or / and in the second step, the reaction time is 24h to 48h; or / and in the third step, the molecular weight cutoff is 3.5kDa during dynamic dialysis, and the dialysis time is 24h to 36h.

8. Bone-targeting nanomicelles loaded with rifapentine according to any one of claims 1 to 7, characterized in that... In step four, the activation time is 3 to 6 hours, and the stirring time is 24 to 36 hours; or / and in step five, the molecular weight cutoff is 3 kDa during ultrafiltration centrifugation; or / and in step six, the stirring time is 2 to 4 hours, and the molecular weight cutoff is 3 kDa during ultrafiltration centrifugation.

9. A method for preparing bone-targeting nanomicelles loaded with rifapentine according to any one of claims 2 to 8, characterized in that... Perform the following steps: The first step involves dissolving the required amounts of methoxy polyethylene glycol, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 4-dimethylaminopyridine in an organic solvent under light-protected conditions, followed by stirring and activation to obtain a mixture. The second step involves adding the required amount of polylactic acid-glycolic acid copolymer to the mixture and continuing the reaction to obtain the initial product. The third step involves dynamic dialysis and freeze-drying of the initial product to obtain the polymer. The fourth step involves dissolving the polymer in water, ultrasonically dispersing it into micelles, adding the required amounts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the micelles for activation, and then adding sodium alendronate and stirring the reaction to obtain the final product. The fifth step involves subjecting the final product to ultrafiltration, centrifugation, and freeze-drying to obtain a bone-targeting nanomicelle carrier. Step 6: Dissolve the bone-targeting nanomicelle carrier in water, ultrasonically disperse it into micelles, add an ethanol solution of rifapentine to the micelles, stir, and then obtain bone-targeting nanomicelles loaded with rifapentine after ultrafiltration, centrifugation and freeze-drying.

10. The use of bone-targeting nanomicelles loaded with rifapentine according to any one of claims 1 to 8 in the preparation of a drug for treating bone tuberculosis.

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