Preparation method and application of a fasudil multi-morphology targeted nanomedicine system for diabetes osteoarthritis

Abstract

The application discloses a preparation method and application of a multi-morphology fasudil targeted nanomedicine system for diabetes osteoarthritis, and belongs to the technical field of medicines, and comprises the following steps: 1) weighing free fasudil, dissolving the free fasudil in an organic phase solvent, fully stirring and dissolving, and preparing fasudil organic drug stock solution, which is marked as Fas; 2) slowly adding an acid solution to the Fas; the application takes free fasudil as an active drug, constructs Fas@PB-Sr 2+ The composite nano intermediate is subjected to CAP polypeptide targeted modification to obtain multi-morphology targeted nanoparticles. Through a low-temperature hydrothermal process, drug thermal degradation is avoided, square, rod, spherical and porous mesoporous composite morphologies are generated by regulating raw material ratio and reaction conditions, and cartilage specificity enrichment is realized in combination with the CAP polypeptide, multiple pathological damages such as cartilage wear, synovial inflammation and abnormal vascular hyperplasia of diabetes osteoarthritis are improved, joint local drug residence time is prolonged, and systemic drug toxic side effects are reduced.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to a method for preparing and applying a multimorphological method for preparing a nanomedicine system targeting diabetic osteoarthritis. Background Technology

[0002] Diabetic osteoarthritis (D-OA) is the most common musculoskeletal complication of diabetes. Unlike ordinary osteoarthritis, the high-glucose microenvironment continuously overactivates the intra-articular Rho / ROCK signaling pathway, inducing multiple pathological damages: on the one hand, it upregulates matrix-degrading enzymes such as MMP-13 and ADAMTS5, accelerating the loss of type II collagen and proteoglycans, causing continuous cartilage wear; on the other hand, it promotes the massive release of TNF-α, IL-1β, and IL-6, forming chronic low-grade inflammation of the synovium; simultaneously, high glucose induces abnormal proliferation of endothelial cells, and a large amount of pannus erodes the subchondral bone tissue. Coupled with diabetic microcirculatory disorders and oxidative stress damage, the disease progresses much faster than in ordinary OA. Conventional clinical interventions have significant limitations: nonsteroidal anti-inflammatory drugs (NSAIDs) can only provide short-term analgesia and cannot block the ROCK pathway or reverse cartilage matrix degradation; long-term use of intra-articular glucocorticoids can inhibit chondrocyte activity and aggravate bone loss; hypoglycemic drugs only regulate blood sugar and cannot act on local pathological targets in the joint, making it difficult to simultaneously meet the dual needs of blood sugar control and joint repair. Therefore, there is an urgent need for targeted therapeutic agents that can specifically block the ROCK pathway and adapt to the joint microenvironment of diabetes.

[0003] Fasudil is a highly selective ROCK1 / ROCK2 inhibitor that can simultaneously achieve anti-inflammatory, cartilage protection, inhibition of abnormal angiogenesis, and improvement of local microcirculation. Theoretically, it is an ideal active drug for the treatment of D-OA. However, free fasudil alone has several formulation shortcomings: free fasudil has extremely poor water solubility and cannot be directly formulated into an injectable form. Fasudil hydrochloride, after salt formation, is too hydrophilic and is rapidly absorbed into the bloodstream within minutes after intra-articular injection, with a half-life of only 16 minutes in vivo. The local effective duration is extremely short, requiring frequent repeated injections, resulting in poor patient compliance. Small molecule drugs do not have cartilage-targeting ability and are widely distributed throughout the body. To achieve effective joint concentrations, the dosage needs to be increased, which can easily cause systemic adverse reactions such as hypotension, elevated transaminase levels, and increased bleeding risk, which is not conducive to long-term use in diabetes. A single free molecule only has a burst-release effect and cannot simultaneously meet the needs of stratified treatment, such as rapid penetration into the cartilage surface, long-term retention in the joint cavity, and sustained-release repair of deep cartilage.

[0004] Traditional hydrothermal synthesis of nanoparticle drug delivery systems often employs high-temperature conditions above 80°C. These high temperatures can disrupt the structure of free fasudil sulfonamide bonds, leading to drug degradation and inactivation. Conventional processes can only produce single spherical nanoparticles with uniform morphology, making it difficult to simultaneously achieve multiple functions such as rapid penetration, long-term retention, and sustained release. The carriers lack cartilage-targeting modification, making it difficult to specifically accumulate in damaged cartilage tissue after intravenous or intra-articular injection, resulting in low drug utilization efficiency. Most systems are not structurally designed for the high-glucose microenvironment of diabetes, failing to achieve controlled-release of drugs from lesions and failing to effectively avoid systemic toxicity.

[0005] Currently, there is a lack of a nanosystem that can be prepared at low temperatures, has multiple morphologies, can be targeted to cartilage, and can stably load free fasudil. Existing formulations cannot simultaneously solve a series of problems with free fasudil, such as poor water solubility, short half-life, lack of targeting, lack of microenvironment response, and single morphology with limited effect, which restricts the clinical translation of fasudil in the long-acting targeted treatment of diabetic osteoarthritis. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing and applying a multimorphological method for sudil-targeted diabetic osteoarthritis nanomedicine system in order to solve the above-mentioned problems.

[0007] On the one hand, in order to achieve the above objectives, the present invention employs the following method: a method for preparing a multimorphological method of sudil-targeted diabetic osteoarthritis nanomedicine system, comprising the following steps: 1) Weigh out free fasudil, dissolve it in an organic solvent, stir thoroughly to dissolve, and prepare a fasudil organic drug stock solution, labeled as Fas; 2) Slowly add acidic solution to Fas, then add K3[Fe(CN)6]·3H2O and SrCl2 in sequence. After mixing evenly, transfer to a hydrothermal reactor for low-temperature hydrothermal reaction. After the reaction is completed, centrifuge and wash to obtain the drug-loaded Prussian blue strontium composite nano-intermediate Fas@PB-Sr 2+ ; 3) The reaction of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with N-hydroxysuccinimide with Fas@PB-Sr 2+ The surface carboxyl groups were activated; after activation, chondroitin-affinity CAP peptides were added, and the peptide coupling modification was completed by continuous stirring; the reaction solution was purified by dialysis to remove unreacted small molecules and free peptides, and then lyophilized to obtain Fas@PB-Sr. 2+ @CAP multimorphic targeted nanoparticles are the multimorphic fasudil-targeted diabetic osteoarthritis nanomedicine system.

[0008] As a further description of the above technical solution: In step 1), the organic phase is one or more mixed solvents selected from dimethyl sulfoxide, anhydrous methanol, and anhydrous ethanol.

[0009] As a further description of the above technical solution: In step 2), the acidic solution is one or more of the following: citric acid aqueous solution, glacial acetic acid aqueous solution, and dilute hydrochloric acid with a concentration not exceeding 1 mol / L.

[0010] As a further description of the above technical solution: In step 2), the hydrothermal reaction conditions are: reaction temperature 45-55℃, and reaction time 24-72h.

[0011] As a further description of the above technical solution: In step 3), the activation conditions are: activation temperature from room temperature to 40°C, and activation time from 0.2 to 2 hours.

[0012] As a further description of the above technical solution: Raw material mass ratio: The mass ratio of fasudil: K3[Fe(CN)6]·3H2O: CAP is 1:0.4~20:0.02~1.5.

[0013] On the other hand, in order to achieve the above objectives, the present invention adopts the following technical solution: a multimorphological method for sudil-targeted diabetic osteoarthritis nanomedicine system, which is prepared by the above preparation method.

[0014] On the other hand, in order to achieve the above objectives, the present invention adopts the following technical solution: the application of a multimorphological method of sudil-targeted diabetic osteoarthritis nanomedicine system, wherein the drug system prepared by the above method is used in intra-articular injection drugs for the treatment of diabetic osteoarthritis.

[0015] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1. In this invention, the constructed multi-morphological composite nanocarrier balances high drug loading capacity and excellent tissue penetration. Compared with traditional single spherical or rod-shaped nanocarriers, the multi-morphological synergistic structure can significantly improve the drug encapsulation efficiency and loading stability of fasudil, effectively solving the problems of poor water solubility, rapid in vivo clearance, and short half-life of free fasudil. This enables long-term sustained release of the drug at joint lesions, significantly reduces the frequency of administration, and improves treatment compliance.

[0016] 2. In this invention, through targeted modification, the nanomedicine system can specifically recognize the target receptors highly expressed at the lesion site of diabetic osteoarthritis, accurately accumulate in the damaged cartilage tissue and inflamed synovial cells, and effectively penetrate the thickened joint synovium and dense cartilage matrix under the pathological state of diabetes. This overcomes the technical bottleneck of traditional nanomedicines, which have weak targeting, low lesion accumulation, and difficulty in deeply acting on the damaged site, and achieves precise targeted drug delivery to the lesion.

[0017] 3. In this invention, without damaging the activity of fasudil, the drug is loaded and embedded simultaneously with the in-situ growth of the drug-loaded nanocrystals, achieving uniform doping of drug molecules and anchoring them inside and on the surface of the multi-morphology nanocarrier lattice. This avoids the defects of uneven drug loading, easy burst release, and poor drug loading stability of traditional post-adsorption drug loading methods, significantly improving drug loading capacity and structural stability, and ensuring long-lasting and stable sustained-release drug effect.

[0018] 4. In this invention, a local targeted accumulation and local response drug release mode is adopted, which greatly reduces the distribution of drugs in normal tissues and systemic blood circulation, significantly reduces systemic toxic side effects such as hypotension and headache caused by high doses of free drugs, and improves the safety and bioavailability of drug use.

[0019] 5. In this invention, the preparation process is simple, controllable, and reproducible. The multimorphic nanostructure regulation method is mild, and the hydrothermal temperature is below 55°C, which does not destroy the drug activity of fasudil. It is suitable for large-scale preparation and production and has good prospects for clinical translation and practical application. Attached Figure Description

[0020] Figure 1 This is a flowchart of a multimorphological method for preparing a sudil-targeted diabetic osteoarthritis nanomedicine system.

[0021] Figure 2 SEM image of a multimorphic sudil nanomedicine system prepared by a method for preparing a multimorphic sudil-targeted diabetic osteoarthritis nanomedicine system.

[0022] Figure 3 Ultraviolet images of a fasudil nanomedicine system prepared by a method for preparing a multimorphic fasudil-targeted diabetic osteoarthritis nanomedicine system and free fasudil.

[0023] Figure 4 Image showing the loading rate of a multimorphic sudil nanomedicine system prepared by a method for preparing a multimorphic sudil-targeted diabetic osteoarthritis nanomedicine system. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1 S01: Weigh 10mg of free fasudil raw material, dissolve it in 1ml of anhydrous dimethyl sulfoxide (DMSO) solvent, stir thoroughly at room temperature until completely dissolved, and prepare a homogeneous and stable fasudil organic drug stock solution, labeled as Fas stock solution, and store it in a light-proof and sealed container for later use. S02: Slowly add citric acid (3 mg / mL, 10 mL) dropwise to the Fas stock solution, and adjust the pH to 2.5-3.0 with 0.1 M hydrochloric acid (HCl); weigh 116.13 mg of K3[Fe(CN)]6 sample and 3 g of PVP (MWCO: 58000) and dissolve them in 40 mL of CH3COOH (0.01 M, 0.1 M, 1.011 M) solution and stir magnetically for 2 h; then mix with the Fas stock solution above and transfer to a reaction vessel, react at 50 °C for 24 h; after the reaction is complete, add 10.68 mg of SrCl2·6H2O and stir at 40 °C for 4 h; then centrifuge the precipitate at 12000 rpm, discard the supernatant, and wash three times with deionized water; freeze-dry the sample to obtain square Fas@PB-Sr 2+ Nanoparticles.

[0026] Example 2 S01: Weigh 10mg of free fasudil raw material, dissolve it in 1ml of anhydrous dimethyl sulfoxide (DMSO) solvent, stir thoroughly at room temperature until completely dissolved, and prepare a homogeneous and stable fasudil organic drug stock solution, labeled as Fas stock solution, and store it in a light-proof and sealed container for later use. S02: Slowly add the Fas stock solution dropwise to 40 ml of 0.1 M hydrochloric acid (HCl); weigh 116.13 mg of K3[Fe(CN)]6 sample and 10.68 mg of SrCl2·6H2O, dissolve them separately in 3 g (MWCO: 10000) PVP and 15 ml of deionized water, and then mix them together; then mix with the Fas stock solution mentioned above, and transfer to a reaction vessel, react at 50 °C for 48 h; after the reaction, centrifuge the precipitate at 12000 rpm, discard the supernatant, and wash three times with deionized water; freeze-dry the sample to obtain rod-shaped Fas@PB-Sr 2+ Nanoparticles.

[0027] Example 3 S01: Weigh 20 mg of free fasudil raw material and place it in a light-protected reaction bottle. Add 4 mL of an equal volume of methanol / ethanol (1:1) mixed solvent and stir at room temperature for 30 min until the solid is completely dissolved. Prepare a 5 mg / mL fasudil organic stock solution, seal and store in the dark for later use. Stir thoroughly at room temperature until completely dissolved to prepare a homogeneous and stable fasudil organic drug stock solution, which is labeled as Fas stock solution and stored in a light-protected and sealed container for later use. S02: Slowly add 30 ml of 0.1 M hydrochloric acid (HCl) dropwise to the Fas stock solution; weigh 87.10 mg of K3[Fe(CN)]6 sample and 12.0 mg of SrCl2·6H2O2 and dissolve them separately in 2.25 g (MWCO: 58000) PVP; then mix with the Fas stock solution above and transfer to a reaction vessel, react at 55 °C for 16 h; after the reaction is complete, centrifuge the precipitate at 12000 rpm, discard the supernatant, and wash three times with deionized water; freeze-dry the sample to obtain square Fas@PB-Sr 2+ Nanoparticles.

[0028] Example 4 S01: Weigh 20 mg of free fasudil raw material and place it in a light-protected reaction bottle. Add 4 mL of an equal volume of methanol / ethanol (1:1) mixed solvent and stir at room temperature for 30 min until the solid is completely dissolved. Prepare a 5 mg / mL fasudil organic stock solution, seal and store in the dark for later use. Stir thoroughly at room temperature until completely dissolved to prepare a homogeneous and stable fasudil organic drug stock solution, which is labeled as Fas stock solution and stored in a light-protected and sealed container for later use. S02: Slowly add 30 ml of 0.1 M hydrochloric acid (HCl) dropwise to the Fas stock solution; weigh 87.10 mg of K3[Fe(CN)]6 sample and 70.67 mg of SrCl2·6H2O, and dissolve them separately in 2.25 g (MWCO: 58000) PVP; then mix with the Fas stock solution mentioned above, transfer to a reaction vessel, react at 55 °C for 16 h, and keep warm at 45 °C for 24 h; after the reaction, centrifuge the precipitate at 12000 rpm, discard the supernatant, and wash three times with deionized water; freeze-dry the sample to obtain square Fas@PB-Sr 2+ Nanoparticles.

[0029] Current treatments and nanodelivery carriers for diabetes mellitus complicated with osteoarthritis generally suffer from the following technological shortcomings: The conventional fasudil has poor water solubility and a short half-life in vivo. Systemic administration can easily cause vasodilation, leading to systemic side effects such as hypotension and headache. It also lacks the ability to target and enrich joint lesions, requiring repeated administration of large doses. Conventional single-morphology nanocarriers have a single structure, making it difficult to achieve both drug encapsulation efficiency and controllable sustained release. They cannot adapt to the lesion penetration and responsive drug release requirements under the diabetic microenvironment, such as high glucose, high oxidative stress, and high inflammatory factors. In diabetic bone and joint areas, matrix metalloproteinases and inflammatory chemokines are highly expressed. However, traditional nanocarriers lack specific targeting modification and are difficult to penetrate the thickened synovial membrane and cartilage matrix. Drugs are also difficult to accumulate in damaged chondrocytes and synovial macrophages. In diabetic patients, chondrocyte apoptosis, microvascular complications, and subchondral bone remodeling disorders occur simultaneously. Single-morphology drug delivery systems can only achieve simple anti-inflammatory effects or single-pathway regulation, and cannot synergistically improve multiple pathological problems such as high glucose damage, inflammatory infiltration, and bone matrix degradation.

[0030] This invention uses free fasudil as the active drug. The drug is loaded and embedded simultaneously with the in-situ growth of drug-loaded nanocrystals. By controlling the nanomaterial growth process, a multi-morphological carrier framework with square, rod-shaped, spherical, and porous mesoporous structures is prepared and coupled with cartilage-targeting functional peptides for surface modification. The multi-morphological synergistic structure can simultaneously achieve: high drug loading, high synovial membrane penetration efficiency, and drug release in a specific response to the high glucose inflammatory microenvironment. The targeting peptide mediates the specific enrichment of nanoparticles in diabetic articular cartilage and synovial inflammatory cells, and locally and continuously releases fasudil, which improves high glucose-induced oxidative stress apoptosis of chondrocytes, inhibits synovial inflammatory infiltration, regulates abnormal subchondral bone remodeling, and reduces the toxic side effects of systemic administration, thus achieving precise and long-term synergistic treatment of diabetes mellitus complicated with osteoarthritis.

[0031] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a multimorphological sulfachlor-targeted diabetic osteoarthritis nanomedicine system, characterized in that: Includes the following steps: 1) Weigh out free fasudil, dissolve it in an organic solvent, stir thoroughly to dissolve, and prepare a fasudil organic drug stock solution, labeled as Fas; 2) Slowly add acidic solution to Fas, then add K3[Fe(CN)6]·3H2O and SrCl2 in sequence. After mixing evenly, transfer to a hydrothermal reactor for low-temperature hydrothermal reaction. After the reaction is completed, centrifuge and wash to obtain the drug-loaded Prussian blue strontium composite nano-intermediate Fas@PB-Sr 2+ ; 3) The reaction of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with N-hydroxysuccinimide with Fas@PB-Sr 2+ The surface carboxyl groups were activated; after activation, chondroitin-affinity CAP peptides were added, and the peptide coupling modification was completed by continuous stirring; the reaction solution was purified by dialysis to remove unreacted small molecules and free peptides, and then lyophilized to obtain Fas@PB-Sr. 2+ @CAP multimorphic targeted nanoparticles are the multimorphic fasudil-targeted diabetic osteoarthritis nanomedicine system.

2. The method for preparing a multimorphological sudil-targeted diabetic osteoarthritis nanomedicine system according to claim 1, characterized in that, In step 1), the organic phase is one or more mixed solvents selected from dimethyl sulfoxide, anhydrous methanol, and anhydrous ethanol.

3. The method for preparing a multimorphological sulfamethoxazole-targeted diabetic osteoarthritis nanomedicine system according to claim 1, characterized in that, In step 2), the acidic solution is one or more of the following: citric acid aqueous solution, glacial acetic acid aqueous solution, and dilute hydrochloric acid with a concentration not exceeding 1 mol / L.

4. The method for preparing a multimorphological method for a sudil-targeted diabetic osteoarthritis nanomedicine system according to claim 1, characterized in that, In step 2), the hydrothermal reaction conditions are: reaction temperature 45-55℃, and reaction time 24-72h.

5. The method for preparing a multimorphological method for a sudil-targeted diabetic osteoarthritis nanomedicine system according to claim 1, characterized in that, In step 3), the activation conditions are: activation temperature from room temperature to 40°C, and activation time from 0.2 to 2 hours.

6. The method for preparing a multimorphological sudil-targeted diabetic osteoarthritis nanomedicine system according to claim 1, characterized in that, Raw material mass ratio: The mass ratio of fasudil: K3[Fe(CN)6]·3H2O: CAP is 1:0.4~20:0.02~1.

5.

7. A multimorphological sulfachlor-targeted diabetic osteoarthritis nanomedicine system, which is prepared by the preparation method described in any one of claims 1-6.

8. The application of a multimorphological method for sudil-targeted diabetic osteoarthritis nanomedicine system, wherein the drug system prepared according to claim 7 is used in intra-articular injection for the treatment of diabetic osteoarthritis.

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