A self-assembled nanodrug system for regulating mitochondrial oxidative stress and a preparation method and application thereof

Abstract

The application discloses a kind of mitochondrial oxidative stress regulation self-assembled nano-drug system and its preparation method and application, belong to the field of medicine technology.The preparation method of the self-assembled nano-drug system includes: Mdivi-1 is dissolved with natural source carrier in organic solvent, and organic phase is obtained;Second drug is dissolved in aqueous medium or ethanol, and second drug solution is obtained;Mdivi-1, second drug and carrier self-assemble to form nanoparticle precursor;Separate nanoparticle precursor, after stabilizer treatment or polysaccharide surface chemical modification, obtain the treatment drug assembly nanoparticle with targeting property.The application also discloses the self-assembled nano-drug system prepared by the method and its biomedical application as the drug for treating mitochondrial oxidative stress related diseases.The self-assembled nano-drug system of the application has good stability, can target macrophage, can target regulate mitochondrial oxidative stress and inflammatory response, and the drug efficacy is better than free drug.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a self-assembled nanomedicine system for regulating mitochondrial oxidative stress, its preparation method, and its application. Background Technology

[0002] Mitochondria, as the cell's energy factories and metabolic hubs, play a central role in maintaining cellular homeostasis. Recent studies have shown that mitochondrial dysfunction, particularly the excessive accumulation of mitochondrial reactive oxygen species (mtROS) and mitochondrial dynamic imbalance, has transcended simple energy metabolism defects and become a key pathological hub driving the development of various acute and chronic inflammatory diseases.

[0003] Although the precipitating factors and disease courses vary significantly across different diseases, mitochondrial oxidative stress and kinetic imbalance play a central driving role in both radiation enteritis and atherosclerosis, but their modes of action differ significantly. In the acute injury model of radiation enteritis, ionizing radiation directly damages mitochondrial DNA and inner membrane structure, leading to electron transport chain instability and electron leakage, instantly triggering a cascade of mtROS. The intense oxidative stress storm not only directly mediates apoptosis but also activates pathways such as NF-κB, inducing the cascade release of inflammatory factors such as tumor necrosis factor-α and interleukin-6. Simultaneously, radiation stress significantly upregulates Drp1 expression, inducing excessive mitochondrial division and fragmentation, resulting in a sharp decline in oxidative phosphorylation efficiency, plunging intestinal epithelial cells into a ATP supply crisis, ultimately damaging the intestinal mucosal barrier and exacerbating the spread of inflammation. In contrast, in chronic metabolic diseases such as atherosclerosis, mitochondrial damage manifests as a persistently amplified and difficult-to-heal positive feedback process. Under long-term stimulation from oxidized low-density lipoprotein (LDL), metabolic disorders, and abnormal blood flow shear forces, vascular endothelial cells and immune cells exhibit both increased Drp1-mediated proliferation and suppressed Mfn2-mediated fusion. Persistently damaged mitochondria continuously generate mtROS, which in turn promotes LDL oxidation, upregulates adhesion molecules such as vascular cell adhesion molecule 1 (VCM1) and monocyte chemoattractant protein 1 (MCP1), and exacerbates monocyte and macrophage infiltration. Furthermore, mtROS induces mitochondrial DNA release, activating the NLRP3 inflammasome and cGAS-STING pathways as a damage-related molecular pattern, while simultaneously driving macrophage polarization towards a pro-inflammatory phenotype and disrupting regulatory T cell homeostasis. The pathogenic positive feedback loop formed between mtROS and mitochondrial proliferation makes oxidative stress a persistent engine for maintaining and amplifying chronic vascular inflammation.

[0004] For inflammatory diseases related to mitochondrial oxidative stress, several drugs with anti-mitochondrial oxidative effects have been developed in existing technologies. For example: Mdivi-1, also known as Mitochondrial Division Inhibitor 1, has the molecular formula C1.15 H 10 Cl2N2O2S, chemical name: 3-(2,4-dichloro-5-methoxyphenyl)-2-thio-2,3-dihydroquinazoline-4(1H)-one). Mdivi-1 can inhibit mitochondrial division, affect energy metabolism, and inhibit ROS generation. However, it is highly hydrophobic, poorly soluble in water, has poor oral absorption, and low bioavailability.

[0005] SS-31 (Elamipretide): A synthetically produced mitochondrial-targeting tetrapeptide with precise mitochondrial targeting, capable of directly clearing mtROS. However, it is eliminated too quickly in vivo, and after oral administration, it is rapidly eliminated by glucuronidation.

[0006] Nicotinamide riboside (NR) and nicotinamide nucleotide (NMN): primarily by increasing NAD. + It indirectly regulates key molecules such as SIRT3 at different levels to reduce mtROS production and enhance its clearance. However, its oral formulation has significant drawbacks such as gastrointestinal instability, low bioavailability, and uncertainty in clinical efficacy.

[0007] Mitoquinone mesylate (MitoQ): A chemically modified mitochondrial-targeting antioxidant that neutralizes mtROS through a passive "quenching" mechanism. However, its oral formulation suffers from low absorption efficiency and rapid metabolism.

[0008] In summary, while existing mitochondrial-targeted drugs each have their own unique characteristics, they all suffer from insurmountable limitations: Mdivi-1 is limited by its strong hydrophobicity and low oral bioavailability; SS-31 is metabolized too rapidly in vivo; NR / NMN is gastrointestinal unstable and its clinical efficacy is uncertain; and MitoQ has low absorption efficiency and rapid metabolism. More importantly, the aforementioned strategies only target a single aspect of mitochondrial oxidative stress, lacking a comprehensive approach that simultaneously inhibits excessive mtROS production at its source and efficiently eliminates existing mtROS. This single-aspect intervention model is insufficient to effectively block the mtROS positive feedback loop in acute and chronic inflammatory diseases (especially in chronic pathological processes such as atherosclerosis), resulting in limited efficacy or failure to meet clinical needs.

[0009] Therefore, providing a drug or drug composition thereof that can achieve full-chain regulation of mitochondrial oxidative stress, in order to solve the problems that single-link intervention strategies in the prior art are difficult to block the positive feedback of oxidative stress, and that different drugs have individual defects such as poor oral absorption, excessively rapid metabolism, and insufficient targeting, has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0010] One of the objectives of this invention is to provide a method for preparing a self-assembled nanomedicine system that regulates mitochondrial oxidative stress. The self-assembled nanomedicine system prepared by this method can simultaneously inhibit the excessive production of mitochondrial reactive oxygen species (ROS) and efficiently remove existing ROS from mitochondria, and has good oral absorption characteristics and selective uptake by inflammatory cells.

[0011] A second objective of this invention is to provide a self-assembled nanomedicine system for regulating mitochondrial oxidative stress prepared using this method.

[0012] A third objective of this invention is to provide applications of this self-assembled nanomedicine system.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention discloses a method for preparing a self-assembled nanomedicine system regulated by mitochondrial oxidative stress, comprising the following steps: S1. Dissolve Mdivi-1 and the support in an organic solvent to obtain an organic phase; S2. Dissolve the second drug in an aqueous medium or ethanol to obtain a second drug solution; the second drug includes at least one of imipreptide, mitoxin mesylate, nicotinamide ribose, and nicotinamide nucleotide; S3. Use any of the following methods to enable Mdivi-1, the support, and the second drug to self-assemble into a nanoparticle precursor: Nano-coprecipitation method: The organic phase is added dropwise to an aqueous medium and mixed, and then a second drug solution is added to continue the reaction; Thin-film hydration method: The organic phase is formed into a thin film by rotary evaporation, and then hydrated with a second drug solution; Reverse solvent method: The organic phase is rapidly mixed with the second drug solution, and nanoparticles are formed by reverse solvent precipitation; Solvent evaporation method: After mixing the organic phase with the second drug solution, the organic solvent is removed by evaporation under reduced pressure; The second drug solution used in the nano-coprecipitation method, the thin film hydration method, and the reverse solvent method is prepared using an aqueous medium; the second drug solution used in the solvent evaporation method is prepared using ethanol. S4. Separate the nanoparticle precursor and mix it with a surface modifier or stabilizer for stabilization treatment to obtain drug-assembled nanoparticles; Steps S1 and S2 are not sequential.

[0014] In some embodiments of the present invention, in step S1, the carrier comprises oligoepigallocatechin gallate; The organic solvent includes at least one of dimethyl sulfoxide and anhydrous ethanol; The mass ratio of Mdivi-1 to the carrier is 1:1.5 ~ 2:1.

[0015] In some embodiments of the present invention, the aqueous medium in steps S2 and S3 is independently selected from water and phosphate buffer solution; Preferably, when the nano-coprecipitation method is used in step S3, the aqueous medium is an aqueous solution of epigallocatechin gallate with a concentration of 1~5 mg / mL, more preferably 2.6 mg / mL; More preferably, the mass ratio of Mdivi-1 to epigallocatechin gallate is 1:0.8 to 1.5, and more preferably 1:1.1.

[0016] In some embodiments of the present invention, in step S3, the mass ratio of Mdivi-1 to the second drug is 2:1 to 4:1.

[0017] In some embodiments of the present invention, the surface modifier comprises thioctic polysaccharide; the stabilizer comprises bovine serum albumin; The mass ratio of Mdivi-1 to surface modifier or stabilizer is 8~15:1, preferably 12:1; In some embodiments of the present invention, the thioctic polysaccharide includes at least one of thioctic acylated chondroitin sulfate (Chs-LA), thioctic acylated sodium hyaluronate (HA-LA), and thioctic amidated chitosan (CS-LA).

[0018] In some embodiments of the present invention, the stabilization treatment in step S4 is performed using high-energy ultrasonic treatment.

[0019] The second aspect of this invention discloses a self-assembled nanomedicine system regulated by mitochondrial oxidative stress, which is prepared by the method described above.

[0020] The second aspect of this invention discloses the application of this self-assembled nanomedicine system regulated by mitochondrial oxidative stress in the preparation of drugs for treating diseases related to mitochondrial oxidative stress.

[0021] In some embodiments of the present invention, the mitochondrial oxidative stress-related diseases include at least one of metabolic cardiovascular disease, ischemia-reperfusion injury, radiation injury, or neurodegenerative disease.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention features a scientifically designed and advanced concept. Based on the pathological axis of "mitochondrial oxidative stress-inflammation amplification," it constructs a polysaccharide-modified self-assembled nanomedicine system with active targeting, microenvironment response, and mitochondrial function regulation capabilities. This system uses a naturally derived carrier as its core. After drug molecules self-assemble, they are further surface-engineered using natural polysaccharides such as lipoic acid-acylated hyaluronic acid, lipoic acid-acylated chondroitin sulfate, or lipoic acid-amidated chitosan, thereby endowing the nanoparticles with excellent macrophage targeting and lesion enrichment capabilities. Specifically, lipoic acid-acylated hyaluronic acid and lipoic acid-acylated chondroitin sulfate, as natural ligands for the CD44 receptor, can specifically recognize activated macrophages, vascular smooth muscle cells, and damaged endothelial cells with high CD44 expression in the inflammatory microenvironment, achieving targeted enrichment of atherosclerotic plaques. In intestinal inflammation, especially radiation enteritis, CD44 expression on the surface of damaged intestinal epithelial cells and infiltrating immune cells is significantly upregulated, providing a clear active targeting biological basis for lipoic acid-acylated hyaluronic acid / lipoic acid-acylated chondroitin sulfate-modified nanoparticles. On the other hand, chitosan, due to its natural cationic charge, can electrostatically interact with the negatively charged sialic acid and sulfated glycosaminoglycans on the surface of the intestinal mucus layer and epithelial cells, thereby enhancing the adhesion and retention capacity of nanoparticles on the inflamed intestinal surface. Especially when inflammation leads to damage to the mucus barrier and exposure of the epithelium, this "physical anchoring effect" further improves local delivery efficiency and drug utilization. Simultaneously, this system uses bovine serum albumin (BSA) as a stabilizer and structural regulator in the nanoassembly process, which can effectively inhibit particle aggregation through steric hindrance, optimizing nanoparticle size uniformity and colloidal stability. Furthermore, BSA possesses excellent biocompatibility, reducing the immunogenicity of nanoformulations, prolonging the in vivo circulating half-life, and further enhancing the overall efficacy of targeted delivery.

[0023] Based on the aforementioned polysaccharide-mediated dual targeting mechanism of "receptor recognition-electrostatic anchoring," the self-assembled nanomedicine system of this invention not only possesses excellent colloidal stability and biocompatibility but also efficiently accumulates in inflammation-associated macrophages and damaged tissue regions, achieving precise regulation of mitochondrial oxidative stress and inflammatory responses. This system effectively inhibits macrophage inflammatory activation and the amplification of the inflammatory cascade by suppressing abnormal mitochondrial division, reducing intracellular reactive oxygen species accumulation, restoring mitochondrial energy metabolism homeostasis, and correcting cellular redox imbalance, thereby mitigating ionizing radiation-induced tissue damage and improving the chronic inflammatory microenvironment.

[0024] The nanomedicine system of this invention can be used for the treatment and intervention of mitochondrial oxidative stress-related diseases such as atherosclerosis and radiation enteritis. Compared with free drugs, this system significantly improves drug stability, lesion targeting, and mitochondrial protection efficiency, enhances anti-inflammatory, antioxidant, and immune microenvironment regulation effects, and overcomes the limitations of traditional free drugs, such as short in vivo circulation time, difficulty in accurately enriching lesions, and inability to effectively repair mitochondrial damage. It shows good prospects for biomedical translation and clinical application.

[0025] Physicochemical performance tests showed that the self-assembled nanomedicine system of this invention has a particle size of 120–180 nm, a PDI of less than 0.3, uniform particle size, and good dispersibility. Its high absolute value of the ζ-potential effectively prevents particle aggregation, and its stable physicochemical properties provide a reliable guarantee for in vitro and in vivo efficacy. Cellular uptake experiments confirmed that the self-assembled nanomedicine system of this invention can be efficiently taken up by M1 and M2 macrophages, exhibiting excellent targeted enrichment ability for inflammation-associated macrophages.

[0026] Both inflammatory stimuli and ionizing radiation can induce mitochondrial dysfunction and excessive accumulation of reactive oxygen species (ROS), leading to cellular oxidative stress and inflammatory damage. Compared with free drugs, the self-assembled nanomedicine system of this invention can effectively inhibit LPS+IFN-γ-induced macrophage inflammatory differentiation and alleviate the inflammatory response; simultaneously, it can scavenge excess intracellular ROS and correct mitochondrial-mediated redox imbalance. In irradiated cell models, the self-assembled nanomedicine system of this invention can significantly reduce irradiation-induced ROS accumulation, improve abnormal mitochondrial membrane potential, and repair damaged mitochondria, thereby effectively alleviating cellular oxidative stress damage caused by ionizing radiation. Its mitochondrial protective and antioxidant / anti-inflammatory effects are significantly superior to those of free drugs.

[0027] In vivo pharmacodynamic studies have shown that the pathological progression of both atherosclerosis and radiation enteritis is closely related to mitochondrial oxidative stress imbalance and inflammatory activation. Compared to the weak therapeutic effects of free drugs, the self-assembled nanomedicine system of this invention can reduce the area of ​​atherosclerotic plaques in the aorta of mice by 40%–60%, alleviate oxidative damage and inflammatory infiltration of vascular endothelial mitochondria, and effectively delay the progression of atherosclerosis. In a radiation enteritis model, prophylactic administration of the self-assembled nanomedicine system of this invention can improve leukopenia symptoms in irradiated mice, repair colorectal tissue damage, improve mouse survival rate, and effectively antagonize radiation-induced mitochondrial oxidative damage.

[0028] In summary, the self-assembled nanomedicine system of this invention has the characteristics of good physicochemical stability and strong macrophage targeting. It can target and regulate mitochondrial oxidative stress and inflammatory response, and its overall efficacy is significantly better than that of free drugs. It has extremely high application value in the prevention and treatment of inflammation, radiation damage and cardiovascular diseases related to mitochondrial oxidative stress disorder. Attached Figure Description

[0029] Appendix Figure 1 These are the NMR spectra of the chemically synthesized pharmaceutical raw materials used in this invention; wherein A is the NMR spectrum of chondroitin sulfate acylated with lipoic acid (Chs-LA), B is the NMR spectrum of sodium hyaluronate acylated with lipoic acid (HA-LA), C is the NMR spectrum of chitosan amidated with lipoic acid (CS-LA), and D is the NMR spectrum of oligoepigallocatechin gallate (OEGCG).

[0030] Appendix Figure 2 The figures show the particle size distribution and stability test results of the drug self-assembled nanoparticles in Examples 1 to 7 of Experiment 1; the left figure for each example is the particle size distribution figure, and the right figure is the stability test result figure.

[0031] Appendix Figure 3 Figure 3 shows the evaluation results of the inflammatory differentiation effect of macrophages treated with different drug self-assembled nanoparticles of the present invention; where a, b, c, and d are the evaluation results of the inflammatory differentiation effect of macrophages treated with SS-M NPs, NR-M NPs, NMN-M NPs, and MitoQ-M NPs, respectively.

[0032] Appendix Figure 4 The figure shows the results of testing the TNF-α mRNA expression level in macrophages treated with the self-assembled nanoparticles of the present invention in Experimental Example 3; where a, b, c, and d are the results of testing the TNF-α mRNA expression level in macrophages treated with SS-M NPs, NR-M NPs, NMN-M NPs, and MitoQ-M NPs, respectively.

[0033] Appendix Figure 5 The figure shows the regulation results of the redox level of macrophages after inflammation induction by the self-assembled nanoparticles of the present invention in Experiment Example 3; where a, b, and c are the regulation results of the redox level of macrophages after inflammation induction by SS-M NPs, NR-M NPs, and NMN-M NPs, respectively. Detailed Implementation

[0034] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0035] The drug source in this embodiment of the invention is: Mdivi-1: Selleck, part number S7162; Epigallocatechin gallate (EGCG): Meilun Biotechnology, Product No. N0302A; Nicotinamide ribose (NR): Source Leaf Biotechnology, catalog number S24869; Nicotinamide Nucleotide (NMN): Shanghai Aladdin Biochemical Technology Co., Ltd., Product No. N131850; Mitoquinone mesylate (MitoQ): MCE, CAS No. 845959-50-4; SS31: Qiangyao Biotechnology, custom-synthesized; Chondroitin sulfate acetylated with lipoic acid (Chs-LA): Prepared by chemical synthesis, the specific synthesis steps are as follows: Lipoic acid (LA, 50 mg, 0.242 mmol) was dissolved in 5 mL of (N,N)-dimethylformamide (DMF), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, 92.8 mg, 0.484 mmol) and 4-dimethylaminopyridine (DMAP, 35.4 mg, 0.29 mmol) were added sequentially. The mixture was stirred and activated at room temperature for 1 hour to obtain the LA activated solution. Chondroitin sulfate (0.48 g, 0.968 mmol) was separately dissolved in 15 mL of formamide, heated to 90 °C and stirred until completely dissolved, then cooled to room temperature. The above LA activated solution was slowly added dropwise to the chondroitin sulfate solution, mixed well, and reacted under light-protected conditions for 24 hours. After the reaction was completed, the reaction solution was transferred to a dialysis bag and purified by dialysis with deionized water. Finally, the target product was obtained by freeze-drying. The NMR spectrum of chondroitin sulfate acylated with lipoic acid is shown in [reference needed]. Figure 1 A.

[0036] Sodium thioctic acid acylated hyaluronic acid (HA-LA): prepared by chemical synthesis, the synthesis method of which is referred to the literature "Self-Aggregation-Induced Polymerization for Constructing Multifunctional Dynamic Zwitterionic Hydrogels" (Li X, et al. Aggregate, 2025, 6(12).DOI:10.1002 / agt2.70227.); its NMR spectrum is shown in [reference needed]. Figure 1 B.

[0037] Thioctanoic acid amide-amined chitosan (CS-LA): Prepared by chemical synthesis, the synthesis method of which refers to the literature "A simple yet effective hydrogel dressing for advanced microenvironmental management of diabetic wounds with intrinsic regulation" (Liu K, et al. Chemical Engineering Journal, 2023, 470(000):14.DOI:10.1016 / j.cej.2023.143987.), its NMR spectrum is shown in [link to NMR spectrum]. Figure 1 C.

[0038] Oligomeric epigallocatechin gallate (OEGCG): Prepared by chemical synthesis, the synthesis method of which refers to the literature "Self-assembled micellar nanocomplexes comprising green tea catechinderivatives and protein drugs for cancer therapy" (Chung JE, et al. Nature Nanotechnology, 2014, 9(11):907-912.DOI:10.1038 / nnano.2014.208.), its NMR spectrum is shown in [reference needed]. Figure 1 D.

[0039] Example 1: Preparation of SS-M NPs (nano-coprecipitation method) This embodiment provides a method for preparing drug assembly nanoparticles SS-M NPs using a nano-coprecipitation method. The specific steps are as follows: 1. Preparation of stock solution (1) Mdivi-1 stock solution: Dissolve Mdivi-1 in dimethyl sulfoxide (DMSO) to prepare a solution with a concentration of 40 mg / mL for later use; (2) OEGCG solution: Dissolve OEGCG in DMSO to prepare a solution with a concentration of 20 mg / mL for later use; (3) SS31 solution: Dissolve SS31 in sterile phosphate buffer (PBS, pH=7.4) to prepare a solution with a concentration of 10 mg / mL for later use.

[0040] (4) Chs-LA solution: Dissolve Chs-LA in water for injection to prepare a solution with a concentration of 5 mg / mL for later use.

[0041] 2. Preparation of SS-M NPs nanoparticles by nano-coprecipitation method Mdivi-1 stock solution (40 mg / mL, 40 μL) and OEGCG stock solution (20 mg / mL, 40 μL) were mixed at a mass ratio of 2:1. This mixture was then added dropwise to 585 μL of ultrapure water containing 1.5 mg EGCG, and the mixture was shaken at 1200 rpm for 2 min. Subsequently, SS31 solution (10 mg / mL, 40 μL) was added to the above reaction solution to make the final mass ratio of Mdivi-1:OEGCG:SS31 4:2:1. The mixture was further shaken and reacted at 25 °C for 1.5 h. After the reaction was completed, the precipitate was collected by centrifugation at 20000 g. The precipitate was redispersed in 500 μL of aqueous solution containing 0.5 mg / mL Chs-LA and reacted in a high-energy sonicator for 5 min (power 40%) to obtain SS-M NPs nanoparticles.

[0042] 3. Characterization of SS-M NPs nanoparticles (1) Particle size and Zeta potential: An appropriate amount of SS-M NPs was diluted 20 times with ultrapure water and then measured using a Malvern laser particle size analyzer. The results showed that the average particle size of the nanoparticles was 166.50 ± 2.10 nm, the polydispersity index (PDI) was 0.256 ± 0.05, and the Zeta potential was -28.0 ± 0.6 mV, indicating that the nanoparticle system prepared in this example had uniform particle size and good dispersion stability.

[0043] (2) Drug loading determination: The drug loading was determined using a high-performance liquid chromatograph (Agilent 1260). Mdivi-1 content detection conditions: chromatographic column: CAPCELL PAK C18 (150 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile / water (50:50 v / v); detection wavelength: 214 nm; column temperature: 30 °C; flow rate: 1 mL / min; isocratic elution. The calculated actual loading rate of Mdivi-1 was 45.23%.

[0044] SS31 content detection conditions: Chromatographic column: Kromasil 100-5C18 (4.6mm*250mm, 5μm); mobile phase: acetonitrile / water (containing 0.1% trifluoroacetic acid, TFA) = 42:58 (v / v); flow rate: 1 mL / min; isocratic elution; column temperature: 25℃; detection wavelength: 220 nm. The calculated actual loading rate of SS31 was 38.23%.

[0045] Example 2: Preparation of SS-M NPs (Thin Film Hydration Method) This embodiment provides a method for preparing drug-assembled nanoparticles SS-M NPs using a thin-film hydration method. The specific steps are as follows: 1. Preparation of stock solution (1) Prepare Mdivi-1 stock solution, OEGCG solution and SS31 solution according to the method of Example 1 for later use.

[0046] (2) 1% BSA solution: Bovine serum albumin (BSA) was dissolved in sterile phosphate buffer (PBS, pH=7.4) to prepare a solution with a concentration of 10 mg / mL for later use.

[0047] 2. Preparation of SS-M NPs nanoparticles by thin-film hydration method Mdivi-1 stock solution (40 mg / mL, 40 μL) and OEGCG stock solution (20 mg / mL, 60 μL) were mixed at a mass ratio of 1:0.75 and dissolved in 10 mL of anhydrous ethanol. The mixture was rotary evaporated at 42 °C and 100–300 rpm for 25 min to form a uniform thin film on the inner wall of the container. Then, SS31 solution (10 mg / mL, 60 μL) was added to achieve a final Mdivi-1:OEGCG:SS31 mass ratio of 8:6:3. The mixture was then hydrated at 37 °C and 200 rpm for 60 min. After the reaction was complete, the precipitate was collected by centrifugation at 20,000 g. The precipitate was redispersed in 500 μL of an aqueous solution containing 1% BSA and reacted in a high-energy sonicator for 5 min (40% power) to obtain SS-MNPs nanoparticles. 3. Characterization of SS-M NPs nanoparticles (1) Particle size and Zeta potential: The operation was the same as in Example 1. The average particle size of the nanoparticles was 175.30 ± 2.80 nm, the PDI was 0.262 ± 0.04, and the Zeta potential was -27.2 ± 0.7 mV, indicating that the nanoparticles prepared in this example had uniform particle size, good dispersibility, and stability that met the requirements of subsequent applications.

[0048] (2) Drug loading test: The test conditions for Mdivi-1 and SS31 were the same as in Example 1. Calculations showed that the actual drug loading rate of Mdivi-1 was 43.15%, and the actual drug loading rate of SS31 was 33.58%.

[0049] Example 3 Preparation of SS-M NPs (reverse solvent method) This embodiment provides a method for preparing drug-assembled nanoparticles SS-M NPs using a reverse-phase solvent method. The specific steps are as follows: 1. Preparation of stock solution Mdivi-1 stock solution, OEGCG solution, SS31 solution and Chs-LA solution were prepared according to the method in Example 1 and set aside for later use.

[0050] 2. Preparation of SS-M NPs nanoparticles by reversed-phase solvent method Mdivi-1 stock solution (40 mg / mL, 40 μL) and OEGCG stock solution (20 mg / mL, 120 μL) were mixed at a mass ratio of 1:1.5 and placed in a centrifuge tube. SS31 solution (10 mg / mL, 40 μL) was rapidly added to the mixture to achieve a final Mdivi-1:OEGCG:SS31 mass ratio of 4:6:1, and the mixture was thoroughly mixed. The reaction was then carried out in a plate shaker at 1200 rpm for 1.5 h. After the reaction was complete, the precipitate was collected by centrifugation at 20000 g. The precipitate was redispersed in 500 μL of an aqueous solution containing 0.5 mg / mL Chs-LA and reacted in a high-energy sonicator for 5 min (40% power) to obtain SS-M NPs nanoparticles.

[0051] 3. Characterization of SS-M NPs nanoparticles (1) Particle size and Zeta potential: The same operation as in Example 1 was performed. The results showed that the average particle size of the nanoparticles was 162.70 ± 2.50 nm, the PDI was 0.248 ± 0.03, and the Zeta potential was -28.8 ± 0.5 mV. This indicates that the nanoparticles prepared in this example have uniform particle size, excellent dispersion stability, and are not prone to aggregation.

[0052] (2) Drug loading determination: The detection conditions for Mdivi-1 and SS31 were the same as in Example 1. Calculations showed that the actual drug loading rate of Mdivi-1 was 47.32%, and the actual drug loading rate of SS31 was 21.15%.

[0053] Example 4: Preparation of SS-M NPs (solvent evaporation method) This embodiment provides a method for preparing drug-assembled nanoparticles SS-M NPs using a solvent evaporation method. The specific steps are as follows: 1. Preparation of stock solution Prepare Mdivi-1 stock solution, OEGCG solution and Chs-LA solution according to the method in Example 1, and set aside for later use.

[0054] SS31 ethanol solution: Dissolve SS31 in anhydrous ethanol to prepare an ethanol solution with a concentration of 10 mg / mL for later use.

[0055] 2. Preparation of SS-M NPs nanoparticles by solvent evaporation method Mix Mdivi-1 stock solution (40 mg / mL, 40 μL) and OEGCG stock solution (20 mg / mL, 80 μL) at a mass ratio of 1:1, and add SS31 ethanol solution (10 mg / mL, 80 μL) to make the final Mdivi-1:OEGCG:SS31 mass ratio 2:2:1. After thorough mixing, add 2 mL of anhydrous ethanol and stir until homogeneous to obtain the organic phase. Slowly add 25 mL of water to the organic phase and stir at 1200 rpm for 4 h at room temperature. Then transfer to a rotary evaporator and evaporate the ethanol under reduced pressure at 55 °C to a system volume of 12 mL to remove residual organic solvent, obtaining a nanoparticle suspension. The precipitate was collected by centrifugation at 18000 g for 8 min and washed twice with dispersion medium. The precipitate was redispersed in 500 μL of aqueous solution containing 0.5 mg / mL Chs-LA and reacted in a high-energy sonicator for 5 min (power 40%) to finally obtain SS-M NPs nanoparticles.

[0056] 3. Characterization of SS-M NPs nanoparticles (1) Particle size and Zeta potential: Same as in Example 1, the average particle size of the nanoparticles is 172.40 ± 3.10 nm, the PDI is 0.259 ± 0.04, and the Zeta potential is -27.9 ± 0.6 mV, indicating that the prepared nanoparticles have uniform particle size and good dispersibility, which can meet the requirements for in vitro and in vivo applications.

[0057] (2) Drug loading determination: The detection conditions for Mdivi-1 and SS31 were the same as in Example 1. Calculations showed that the actual drug loading rate of Mdivi-1 was 31.76% and that of SS31 was 20.89%, with the drug loading effect slightly lower than that of the previous methods.

[0058] Example 5: Preparation of NR-M NPs This embodiment provides a method for preparing drug-assembled nanoparticles NR-M NPs using a thin-film hydration method. The specific steps are as follows: 1. Preparation of stock solution (1) Mdivi-1 stock solution: Same as in Example 1; (2) OEGCG solution: Same as in Example 1; (3) NR solution: Dissolve NR in sterile phosphate buffer (PBS, pH=7.4) to prepare a solution with a concentration of 10 mg / mL for later use.

[0059] (4) 1% BSA solution: Dissolve BSA in sterile phosphate buffer (PBS, pH=7.4) to prepare a solution with a concentration of 10 mg / mL for later use.

[0060] 2. Preparation of NR-M NPs nanoparticles Mdivi-1 stock solution (40 mg / mL, 40 μL) and OEGCG stock solution (20 mg / mL, 40 μL) were mixed at a mass ratio of 2:1 and dissolved in 10 mL of anhydrous ethanol. The mixture was rotary evaporated at 42 °C and 100–300 rpm for 25 min to form a uniform thin film on the inner wall of the container. Subsequently, NR solution (10 mg / mL, 40 μL) was added to make the final Mdivi-1:OEGCG:NR mass ratio 4:2:1, and the mixture was hydrated at 37 °C and 200 rpm for 60 min. After the reaction was completed, the precipitate was collected by centrifugation at 20,000 g. The precipitate was redispersed in 500 μL of aqueous solution containing 1% BSA and reacted in a high-energy sonicator for 5 min (power 40%) to finally obtain NR-M NPs nanoparticles.

[0061] 3. Characterization of NR-M NPs nanoparticles (1) Particle size and Zeta potential: The operation was the same as in Example 1. The results showed that the average particle size of the nanoparticles was 126.50 ± 4.10 nm, the PDI was 0.213 ± 0.06, and the Zeta potential was -27.0 ± 0.9 mV, indicating that the nanoparticle system prepared in this example had uniform particle size and good dispersion stability.

[0062] (2) Drug loading determination: High performance liquid chromatography was used for detection.

[0063] Mdivi-1 content detection conditions: The method is the same as in Example 1, and the actual drug loading rate is 39.13%.

[0064] NR content detection conditions: chromatographic column: Zorbax SBC18 (100 mm × 4.6 mm, 5 μm); mobile phase: methanol buffer (pH 3.5, containing 0.1% sodium heptanesulfonate) = 20:80 (v / v); flow rate: 1 mL / min; isocratic elution; column temperature: 30℃; detection wavelength: 261 nm. The actual loading rate of NR was calculated to be 31.44%.

[0065] Example 6: Preparation of NMN-M NPs This embodiment provides a method for preparing drug-assembled nanoparticles NMN-M NPs using a reverse solvent method. The specific steps are as follows: 1. Preparation of stock solution (1) Mdivi-1 stock solution: Same as in Example 1; (2) OEGCG solution: Same as in Example 1; (3) NMN solution: Dissolve NMN in PBS to prepare a solution with a concentration of 10 mg / mL for later use.

[0066] (4) CS-LA solution: Dissolve CS-LA in water for injection to prepare a solution with a concentration of 5 mg / mL for later use.

[0067] 2. Preparation of NMN-NPs nanoparticles Mdivi-1 stock solution (40 mg / mL, 40 μL) and OEGCG stock solution (20 mg / mL, 40 μL) were mixed at a mass ratio of 2:1 and placed in a centrifuge tube. NMN solution (10 mg / mL, 40 μL) was quickly added to the above mixture to make the final mass ratio of Mdivi-1:OEGCG:NMN 4:2:1. The mixture was thoroughly mixed and shaken at 1200 rpm for 1.5 h in a plate shaker. After the reaction was completed, the precipitate was collected by centrifugation at 20000 g. 500 μL of CS-LA aqueous solution containing 0.5 mg / mL was added and the mixture was placed in a high-energy sonicator for 5 min (power 40%) to finally obtain NMN-MNPs nanoparticles.

[0068] 3. Characterization of NMN-M NPs nanoparticles (1) Particle size and Zeta potential: The operation was the same as in Example 1. The results showed that the average particle size of the nanoparticles was 178.23 ± 9.24 nm, the PDI was 0.167 ± 0.07, and the Zeta potential was -33.2 ± 0.13 mV, indicating that the nanoparticle system prepared in this example had uniform particle size and good dispersion stability.

[0069] (2) Drug loading determination: High performance liquid chromatography was used for detection.

[0070] Mdivi-1 content detection conditions: The method is the same as in Example 1, and the actual drug loading rate is 42.25%.

[0071] NMN content detection conditions: chromatographic column: Zorbax SBC18 (100 mm × 4.6 mm, 5 μm); mobile phase: methanol buffer (pH 3.5, containing 0.1% sodium heptanesulfonate) = 20:80 (v / v); flow rate: 1 mL / min; isocratic elution; column temperature: 30℃; detection wavelength: 261 nm. The actual loading rate of NMN was calculated to be 16.20%.

[0072] Example 7 Preparation of MitoQ-M NPs This embodiment provides a method for preparing drug-assembled nanoparticles MitoQ-M NPs using a nano-coprecipitation method. The specific steps are as follows: 1. Preparation of stock solution (1) Mdivi-1 stock solution: Same as in Example 1; (2) OEGCG solution: Same as in Example 1; (3) MitoQ solution: Dissolve MitoQ in water for injection to prepare a solution with a concentration of 10 mg / mL for later use.

[0073] (4) HA-LA solution: Dissolve HA-LA in water for injection to prepare a solution with a concentration of 5 mg / mL for later use.

[0074] 2. Preparation of MitoQ-M NPs nanoparticles Mdivi-1 stock solution (40 mg / mL, 40 μL) and OEGCG stock solution (20 mg / mL, 80 μL) were mixed at a mass ratio of 1:1, and then added dropwise to 585 μL of water for injection. The mixture was shaken at 1200 rpm for 2 min. Subsequently, MitoQ solution (10 mg / mL, 80 μL) was added to the above mixture to make the final mass ratio of Mdivi-1:OEGCG:MitoQ 2:2:1. The mixture was shaken and reacted at 25 °C for 1.5 h. After the reaction was completed, the precipitate was collected by centrifugation at 20000 g. The precipitate was redispersed in 500 μL of aqueous solution containing 0.5 mg / mL HA-LA and reacted in a high-energy sonicator for 5 min (power 40%) to obtain MitoQ-M NPs nanoparticles.

[0075] 3. Characterization of MitoQ-M NPs nanoparticles (1) Particle size and Zeta potential: The operation was the same as in Example 1. The results showed that the average particle size of the nanoparticles was 157.14±2.45 nm, the PDI was 0.235±0.09, and the Zeta potential was -28.14±0.25 mV, indicating that the nanoparticle system prepared in this example had uniform particle size and good dispersion stability.

[0076] (2) Drug loading determination: High performance liquid chromatography was used for detection. Mdivi-1 content detection conditions: The method is the same as in Example 1, and the actual loading rate of Mdivi-1 is 40.12%.

[0077] MitoQ content detection conditions: chromatographic column: Restek Allure PFP Propyl (150 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile / water (containing 0.1% TFA) = 65:35 (v / v); flow rate: 1 mL / min; isocratic elution; column temperature: 25℃; detection wavelength: 275 nm. The calculated actual MitoQ loading rate was 28.95%.

[0078] Example 8: Optimization experiment on the dosage of surface-modified chondroitin sulfate (Chs-LA) with lipoic acid. This embodiment aims to investigate the effect of the dosage of the surface modifier lipoic acid acylated chondroitin sulfate (Chs-LA) on the preparation effect of SS-MNPs nanoparticles, screen the optimal concentration of Chs-LA that can achieve nanoparticle surface modification and structural stability, and determine the best preparation process parameters.

[0079] 1. Experimental Design SS-M NPs nanoparticle precursors were prepared using the nano-coprecipitation method described in Example 1, and nanoparticle precipitates were obtained after centrifugation. The precipitates were dispersed in 500 μL of Chs-LA aqueous solutions of different concentrations for surface modification reactions. The final concentrations of Chs-LA in the final reaction system were set to 0.5 mg / mL, 1.0 mg / mL, and 2.0 mg / mL, respectively. Specific formulations are as follows: (1) 0.5 mg / mL group: Take 50 μL of Chs-LA stock solution with a concentration of 5 mg / mL, add 450 μL of ultrapure water, mix well to prepare the target concentration solution.

[0080] (2) 1.0 mg / mL group: Take 100 μL of Chs-LA stock solution with a concentration of 5 mg / mL, add 400 μL of ultrapure water, mix well to prepare the target concentration solution.

[0081] (3) 2.0 mg / mL group: Take 200 μL of Chs-LA stock solution with a concentration of 5 mg / mL, add 300 μL of ultrapure water, mix well to prepare the target concentration solution.

[0082] 2. Experimental Results and Analysis The physicochemical properties of SS-M NPs prepared under three different Chs-LA final concentration conditions were characterized, including particle size, polydispersity index (PDI), zeta potential, drug content, and system stability. The test results for each group are shown in Table 1.

[0083] Table 1

[0084] The test results show that the average particle size of SS-M NPs increases significantly with increasing final concentration of Chs-LA modifier. The 0.5 mg / mL concentration group has the lowest PDI value, indicating that the nanoparticle size distribution uniformity is optimal under this condition. The Zeta potentials of all three groups are in the negative potential range with relatively high absolute values, indicating that the nanoparticles possess basic electrostatic stability. Regarding drug loading, the 1.0 mg / mL group has a slightly higher drug content, but the overall drug content difference among the three groups is small. The stability test results indicate that excessively high Chs-LA concentrations can disrupt the equilibrium of the nanoparticle dispersion system, causing particle aggregation, system turbidity, and reduced formulation stability.

[0085] 3. Conclusion Based on the analysis of multiple indicators including particle size, particle size distribution, drug content, dispersion stability, and economic efficiency, the final concentration of the surface modifier Chs-LA was 0.5 mg / mL, resulting in SS-M NPs with the smallest particle size, the best particle size distribution uniformity, good 24-hour dispersion stability, and drug loading that met the preparation requirements. Simultaneously, it minimized the amount of excipients used, reducing preparation costs. Therefore, the optimal final concentration of the surface modifier Chs-LA in the preparation process of SS-M NPs was determined to be 0.5 mg / mL.

[0086] Example 9: Screening of the mass ratio of Mdivi-1 to OEGCG feed This embodiment aims to screen the optimal feed ratio of Mdivi-1 to OEGCG in order to optimize the stability and drug loading performance of NR-M NPs nanoparticles.

[0087] 1. Screening Method NR-M NPs nanoparticles were prepared according to the method in Example 5, with other preparation conditions kept constant, except for the mixing mass ratio of Mdivi-1 stock solution (40 mg / mL, DMSO) to OEGCG solution (20 mg / mL, DMSO). A total of four ratio groups were set up: Group 1: Mdivi-1 : OEGCG = 1 : 4 Group 2: Mdivi-1 : OEGCG = 1 : 2 Group 3: Mdivi-1 : OEGCG = 1 : 1 Group 4: Mdivi-1 : OEGCG = 4 : 1 The average particle size, polydispersity index (PDI), zeta potential, and drug loading of Mdivi-1 and NR were determined for each group of NR-M NPs nanoparticles.

[0088] 2. Screening Results Table 2 shows the key performance indicators of nanoparticles under different feeding ratios (data are expressed as mean ± standard deviation): Table 2

[0089] 3. Conclusion When the mass ratio of Mdivi-1 to OEGCG was 1:1 (Group 3), the resulting NR-M NPs had the smallest particle size (approximately 127 nm), the lowest PDI (approximately 0.21), and the most uniform particle size distribution. Simultaneously, the drug loading of Mdivi-1 and NR reached 39.13% and 36.44%, respectively, achieving a high and balanced loading of both. The overall performance of the nanoparticles at this ratio was significantly better than the other three ratios. Therefore, the optimal mass ratio of Mdivi-1 to OEGCG was determined to be 1:1.

[0090] Experimental Example 1: Stability Study of Nanoparticles 1. Experimental Grouping A temperature stability study group was set up, and nanoparticle samples prepared in Examples 1 to 7 were placed in a constant temperature environment of 4℃ and 37℃ in the dark to investigate the effect of different temperature conditions on the storage stability of nanoparticles. Three parallel samples were set up for each experimental group.

[0091] 2. Test Methods Samples were taken at regular intervals on days 1, 2, 3, 4, 5, 6, and 7 of sample storage. The particle size and polydispersity index (PDI) of the nanoparticles were determined using a Malvern laser particle size analyzer.

[0092] 3. Experimental Results The results of the storage stability test are shown in Figure 2 When the nanoparticles were stored in the dark at 4 ℃ and 37 ℃ for 7 days, the particle size and PDI value of each sample fluctuated very little, and there were no significant changes in any of the indicators, indicating that the series of nanoparticles have good short-term storage stability under the above temperature conditions.

[0093] Nanoparticles prepared in Examples 1, 5, 6, and 7 were selected for comprehensive characterization of particle size, PDI, and ζ-potential. The physicochemical parameters of each sample are shown in Table 3.

[0094] Table 3. Particle size potential characterization of nanoparticles prepared in Examples 1, 5, 6, and 7.

[0095] As shown in Table 3, the particle sizes of the four types of nanoparticles are all in the range of 120–180 nm, and the overall particle size is uniform. The PDI of all samples is less than 0.3, indicating that the nanoparticles are well dispersed and there is no obvious agglomeration. At the same time, the absolute value of the ζ-potential of the nanoparticles is relatively high, indicating that the surface charge of the nanoparticles is sufficient and can effectively inhibit particle aggregation, further confirming that this series of nanoparticles has excellent physicochemical stability.

[0096] Experimental Example 2: Investigation of Cellular Uptake Efficiency This experiment used three types of macrophages, M0, M1, and M2, differentiated from mouse bone marrow-derived macrophages (BMDM), as cell models to conduct in vitro cell uptake experiments on the nanoparticles prepared in Examples 1, 4, 5, and 6. The aim was to explore the uptake capacity of macrophages in different polarization states for each nanoparticle and to verify that the prepared nanoparticles can be effectively uptaken by macrophages.

[0097] 1. Raw materials and reagents The reagents, specifications, product numbers, and manufacturer information used in this experiment are detailed in Table 4.

[0098] Table 4 Reagent List

[0099] 2. Test Methods This experiment used mouse bone marrow-derived macrophages (BMDM) to induce differentiation into M1 pro-inflammatory phenotype and M2 anti-inflammatory phenotype macrophages, respectively. Using unpolarized M0 macrophages as the basic model, the co-incubation time of nanoparticles with cells was uniformly set at 6 h to investigate the uptake efficiency of each nanoparticle by macrophages in different polarization states.

[0100] 2.1 Core Materials and Cell Induction Parameters Working concentration of fluorescently labeled nanoparticles: 1 mg / mL; Cell model: Mouse bone marrow-derived macrophages (BMDM).

[0101] Cell polarization induction conditions: M1 pro-inflammatory macrophages were co-stimulated with lipopolysaccharide (LPS, final concentration 100 ng / mL) and interferon-γ (IFN-γ, final concentration 4 ng / mL) for 24 h; M2 anti-inflammatory macrophages were stimulated with interleukin-4 (IL-4, final concentration 20 ng / mL) for 24 h; BMDM without induction agents was used as a blank model of M0 macrophages.

[0102] 2.2 Preparation of fluorescently labeled nanoparticles Following the preparation processes described in Examples 1, 5, 6, and 7, SS-M NPs, NR-M NPs, NMN-M NPs, and MitoQ-M NPs nanoparticles were prepared, respectively. Subsequently, each group of nanoparticles was fluorescently labeled with DiO fluorescent dye to obtain DiO-labeled fluorescent nanoparticles, which were then purified for later use.

[0103] 2.3 Cell uptake experiment procedure After macrophage polarization induction was completed, the corresponding DiO-labeled nanoparticle solutions were added to each cell group. Cells were incubated at 37°C in a 5% CO2 incubator for 6 hours, and the culture was terminated after the preset time. Flow cytometry was used to detect the fluorescence signal of each group of cells, and the mean fluorescence intensity (MFI) was used to characterize the uptake of nanoparticles by macrophages, quantitatively analyzing the cellular uptake efficiency of nanoparticles in each group.

[0104] 3. Test Results All data in this experiment are expressed as mean ± standard deviation. One-way ANOVA was used to test for statistical differences, with P < 0.05 considered statistically significant. *P < 0.05 and **P < 0.01 indicated a significant difference compared to the blank control group. The uptake results of different nanoparticles by macrophages in each group are shown in Table 5.

[0105] Table 5. Average fluorescence intensity of nanoparticle uptake by macrophages in each group

[0106] As shown in Table 5, compared with the blank control group, the four DiO-labeled nanoparticles—SS-M NPs(+Dio), NR-M NPs(+Dio), NMN-M NPs(+Dio), and MitoQ-M NPs(+Dio)—all exhibited significantly increased uptake in macrophages under different polarization states. Specifically, in M1 pro-inflammatory macrophages, the uptake of each nanoparticle was approximately three times that of the control group; and in M2 anti-inflammatory macrophages, the uptake was approximately twice that of the control group. These results confirm that the series of nanoparticles prepared in this invention can be effectively uptaken by M1 and M2 polarized macrophages, demonstrating excellent macrophage-targeted uptake capability.

[0107] Experimental Example 3: Nanoparticle pretreatment inhibits LPS+IFN-γ-induced inflammatory differentiation of macrophages. This experiment aims to evaluate the inhibitory effect of pretreatment with SS-M NPs, NR-M NPs, NMN-M NPs, and Mito-M NPs prepared in Examples 1, 5, 6, and 7 on the pro-inflammatory phenotype polarization of RAW 264.7 macrophages induced by lipopolysaccharide (LPS) combined with interferon-gamma (IFN-γ), and to verify the in vitro anti-inflammatory activity of each drug-loaded nanoparticle.

[0108] I. Experimental Methods (I) Core Material Parameters Drug concentration: Free Mdivi-1 and each nanoparticle group were set at an equivalent concentration of 15 μM for Mdivi-1.

[0109] (II) Cell drug delivery and modeling protocols This study included four independent control groups to evaluate the regulatory effects of four nanoparticles—SS-M NPs, NR-M NPs, NMN-M NPs, and MitoQ-M NPs—on macrophage polarization. Each group included: a blank control group (Control), a pro-inflammatory model group (LPS), a free Mdivi-1 group (Free Mdivi-1), an IL-4 positive control group (IL4), and the corresponding nanoparticle administration group.

[0110] (1) Blank control group: cultured in 1640 complete medium without any treatment.

[0111] (2) Pro-inflammatory model group (Lps): A macrophage pro-inflammatory polarization model was constructed by combined stimulation with LPS (100ng / mL) and IFN-γ (4ng / mL).

[0112] (3) Free Mdivi-1 group: Add medium containing free Mdivi-1 (final concentration 15 μM); pre-treat for 24 h, then add LPS+IFN-γ for 24 h of combined stimulation.

[0113] (4) IL-4 positive control group (IL4): Only IL-4 (20ng / mL) was used to construct a macrophage anti-inflammatory polarization model.

[0114] (5) SS-M NPs group (SS-M): Pretreatment with culture medium containing SS-M NPs nanoparticles (Mdivi-1 equivalent concentration 15μM) for 24h, followed by LPS+IFN-γ combined stimulation for 24h.

[0115] (6) NR-M NPs group (NR-M): Pretreated with culture medium containing NR-M nanoparticles (NR equivalent concentration 5 μM) for 24 h, followed by LPS+IFN-γ co-stimulation for 24 h. (7) NMN-M NPs group (NMN-M): Pretreated with culture medium containing NMN-M NPs nanoparticles (NMM equivalent concentration 5 μM) for 24 h, followed by LPS+IFN-γ co-stimulation for 24 h. (8) MitoQ-M NPs group (MitoQ-M): Pretreatment with culture medium containing MitoQ-M nanoparticles (MitoQ-equivalent concentration 5μM) for 24h, followed by LPS+IFN-γ combined stimulation for 24h.

[0116] All cells were pretreated in a 37℃, 5% CO2 incubator for 6 h. Except for the blank control group, all other groups were incubated with LPS (final concentration 100 ng / mL) and IFN-γ (final concentration 4 ng / mL) for another 24 h to complete the induction of macrophage pro-inflammatory phenotype.

[0117] (III) Flow cytometry detection of CD86, a marker of M1 macrophages Macrophages from each group were collected after treatment and stained with macrophage-specific antibody Anti-F4 / 80 and pro-inflammatory marker antibody Anti-CD86 (working concentration 0.2 μg / test). After incubation at 4°C in the dark, the cells were analyzed by flow cytometry, and the F4 / 80 ratio was statistically analyzed. + CD86 + The percentage of double-positive cells was used to evaluate the level of pro-inflammatory polarization of macrophage M1 cells.

[0118] (iv) Detection of pro-inflammatory gene expression levels by qPCR Total RNA was extracted from cells in each group and reverse transcribed. Real-time quantitative PCR was used to detect the relative expression levels of mRNA of pro-inflammatory factors in cells, and the inhibitory effect of the samples on macrophage inflammatory activation was verified at the gene level.

[0119] II. Test Results The flow cytometry results are attached. Figure 3 As shown: Compared with the positive control group, all nanoparticle pretreatment groups significantly reduced CD86 levels. + The proportion of positive macrophages inhibits M1 type macrophage polarization. Among them, CD86 of the SS-M nanoparticle pretreatment group + The proportion of cells decreased by 63.52% compared to the positive control group, which was significantly better than the free Mdivi-1 group (15.65% reduction). CD86 of the NR-M nanoparticle pretreatment group + The proportion of cells decreased by 61.13% compared to the positive control group, which was better than the free Mdivi-1 group (10.25% reduction). CD86 of the NMN-M nanoparticle pretreated group+ The proportion of cells decreased by 33.18% compared to the positive control group, which was significantly better than the free Mdivi-1 group (18.23% reduction). CD86 of the MitoQ-M nanoparticle pretreatment group + The proportion of cells decreased by 53.12% compared to the positive control group, which was better than the free Mdivi-1 group (reduced by 9.613%).

[0120] The qPCR results are attached. Figure 4 As shown, LPS+IFN-γ can significantly upregulate macrophage mRNA expression, while each nanoparticle pretreatment group can effectively downregulate the relative expression of pro-inflammatory gene mRNA, and the anti-inflammatory effect is significantly better than that of the free drug Mdivi-1 under the same conditions.

[0121] The above results demonstrate that the nanoparticle drug delivery system of this invention, especially SS-M NPs, can more effectively inhibit the activation of macrophages to a pro-inflammatory phenotype under inflammatory stimulation than free Mdivi-1. The nanoparticles of this invention possess excellent inflammation-regulating capabilities, highlighting the efficacy advantages of the nanoparticle drug delivery system.

[0122] Experiment 4: The regulatory effects of SS-M NPs, NR-M NPs, and NMN-M NPs (Examples 1, 5, and 6) on LPS+IFN-γ-induced redox imbalance in macrophages. This experiment, based on an LPS+IFN-γ-induced RAW 264.7 macrophage inflammation model, evaluated the regulatory effects of the self-assembled nanosystems of the present invention prepared in Examples 1, 5, and 6 on the accumulation of intracellular reactive oxygen species (ROS) and redox imbalance in inflammatory macrophages.

[0123] I. Experimental Methods (I) Core Material Parameters Beyotime reactive oxygen species detection kit (S0033S): working concentration 8.3μM.

[0124] (II) Drug Treatment Plan (1) Blank control group: Pretreatment was the same as in Experiment 3 (2) Pro-inflammatory model group (Lps): Pretreatment was the same as in case 3. (3) Free Mdivi-1 group: Pretreatment was the same as in Experiment 3 (4) SS-M NPs group (SS-M NPs): Pretreatment was the same as in Experiment 3 (5) NR-M NPs group (NR-M NPs): Pretreatment same as in Experiment 3 (6) NMN-M NPs group (NMN-M NPs): Pretreatment same as in Experiment 3 Cells from all groups were pretreated at 37°C and 5% CO2 for 6 h. Except for the blank control group, the other groups were continuously stimulated with LPS+IFN-γ for 24 h to construct an inflammatory oxidative stress model.

[0125] (III) Flow cytometry detection of intracellular ROS levels After cell modeling, the cells were washed twice with serum-free culture medium, and then incubated with ROS fluorescent probes in the dark for staining. The average fluorescence intensity of intracellular ROS in each group of cells was detected by flow cytometry to quantify the level of cellular oxidative stress.

[0126] II. Test Results The flow cytometry results are attached. Figure 5 As shown, the average fluorescence intensity of the ROS fluorescent probe in the nanoparticle pretreated group was significantly lower than that in the positive control group, and the effect was significantly better than that in the free Mdivi-1 group.

[0127] Intracellular ROS detection results are attached. Figure 5 As shown, LPS+IFN-γ inflammatory stimulation can significantly induce a large accumulation of ROS in macrophages, causing cellular oxidative stress damage. Compared with the positive control group, the average fluorescence intensity of ROS in cells pretreated with each nanoparticle was significantly reduced, and the effects of ROS scavenging and oxidative balance regulation were significantly better than those of the free drug Mdivi-1 group, confirming that the nanoparticles of this invention can effectively improve the redox imbalance of inflammatory macrophages.

[0128] Example 5: The role of SS-M NPs and MitoQ-M NPs in regulating cellular redox balance in an irradiation damage model. This experiment used ionizing radiation (IR)-induced damage to rat small intestinal crypt epithelial cells (IEC-6) as a model to evaluate the regulatory effects of SS-M NPs and MitoQ-M NPs pretreatment prepared in Examples 1 and 7 on redox imbalance and mitochondrial function protection in irradiated cells.

[0129] I. Experimental Methods (I) Core Material Parameters Dosing concentration: All drug formulations were administered at the equivalent concentration of Mdivi-1, 15 μM; Reactive oxygen species (ROS) detection: Beyotime DCFH-DA ROS detection kit (S0033S), probe working concentration 8.3 μM; Mitochondrial membrane potential detection: Thermo Fisher MitoProbe™ JC-1 Assay Kit; Irradiation equipment: Small animal X-ray irradiator, irradiation dose rate 6.5 Gy / min.

[0130] (II) Cell pretreatment IEC-6 cells in the logarithmic growth phase were harvested and cultured at a concentration of 2.5 × 10⁻⁶. 6 Cells were seeded at a density of cells / mL in 12-well plates for later use.

[0131] (III) Drug pretreatment and irradiation modeling Blank control group: Cells were cultured normally and placed statically next to an irradiator, without ionizing radiation or drug intervention.

[0132] Irradiation treatment: No drug pretreatment was performed; a single X-ray ionizing radiation of 15 Gy was administered to create the model.

[0133] Free Mdivi-1 group: Pretreated with culture medium containing free Mdivi-1 (final concentration 15 μM) for 12 h, and then subjected to a single ionizing radiation of 15 Gy; SS-M NPs group: Pretreated with culture medium containing SS-M NPs nanoparticles (Mdivi-1 equivalent concentration 15 μM) for 12 h, and then subjected to a single ionizing radiation of 15 Gy; MitoQ-M NPs group: Pretreated with culture medium containing MitoQ-M NPs nanoparticles (Mdivi-1 equivalent concentration 15 μM) for 12 h, and then subjected to a single ionizing radiation of 15 Gy; Post-irradiation culture: After irradiation, the cells were returned to the incubator and cultured for another 12 hours to complete the model construction.

[0134] (iv) Indicator Testing Methods 1. Intracellular ROS detection: After cell culture, the cells were washed twice with serum-free medium, and then incubated with the DCFH-DA probe in the dark. The intracellular ROS fluorescence intensity was detected by flow cytometry.

[0135] 2. Mitochondrial membrane potential detection: JC-1 probe staining was used, and the red-green fluorescence ratio of cells was detected by flow cytometry to quantify the mitochondrial membrane potential (ΔΨm) level and evaluate the degree of mitochondrial damage.

[0136] II. Test Results The experimental results are shown in Table 6. All data are expressed as mean ± standard deviation. Parallel one-way ANOVA was performed, with P < 0.05 indicating statistical significance. *P < 0.05 and **P < 0.01 vs. control group. Experimental results are as follows: Table 6 Test Results

[0137] As shown in Table 6, compared with the blank control group, the intracellular ROS level in the irradiation-damaged group was significantly increased, indicating that ionizing radiation can successfully induce severe oxidative stress damage in IEC-6 cells, and the model construction is effective. Compared with the irradiation-damaged group, free Mdivi-1, SS-M NPs, and MitoQ-M NPs can all reduce cellular ROS levels. Among them, the two nanoparticle formulations have significantly better ROS scavenging effects than the free drug Mdivi-1, and can effectively improve the irradiation-induced cellular redox imbalance.

[0138] Mitochondrial membrane potential detection results showed that ionizing radiation causes abnormal mitochondrial membrane potential and damage to mitochondrial function. Drug pretreatment can effectively alleviate mitochondrial damage. Compared with the free drug Mdivi-1 group, the red-green fluorescence ratio of mitochondria in the SS-M NPs and MitoQ-M NPs groups was significantly optimized, and the mitochondrial membrane potential recovery effect was better, which confirms that the nanoparticles of this invention have a good protective effect on mitochondria after irradiation.

[0139] Experimental Example 6: Evaluation of the efficacy of nanoparticles in the treatment of atherosclerosis This study involved feeding ApoE cells with a high-fat diet. - / - Mice were used as an animal model of atherosclerosis. The in vivo efficacy of the nanoparticles of this invention in inhibiting the progression of atherosclerosis was verified through in vivo drug intervention and aortic pathological staining analysis.

[0140] I. Experimental Materials Experimental animals: Male ApoE- / - mice, aged 6-8 weeks, were fed a high-fat diet (HCD) to induce an atherosclerosis model.

[0141] Animal grouping: 6-10 mice per group, all drug interventions were administered twice a week.

[0142] II. Experimental Grouping and Dosing Regimen (1) Control group: Only saline was injected into the tail vein, and the patient was fed a high-fat diet without any drug intervention.

[0143] (2) Free Mdivi-1 group: Mdivi-1 solution was injected intraperitoneally; the dosage was equivalent to that of the nanoparticle group.

[0144] (3) SS-M NPs group: SS-M NPs (prepared according to the method of Example 1) were injected via the tail vein; the dose was 5 mg / kg based on Mdivi-1.

[0145] (4) NR-M NPs group: NR-M NPs (prepared according to the method of Example 5) were injected via the tail vein; the dose was 1.8 mg / kg based on Mdivi-1.

[0146] (5) NMN-M NPs group: NMN-M NPs (prepared according to the method of Example 6) were injected via tail vein; the dose was 1.8 mg / kg based on Mdivi-1.

[0147] After four weeks of drug treatment, the mice were euthanized, and the aorta and aortic root were separated to prepare paraffin sections. The morphology of vascular plaques was observed by hematoxylin-eosin (H&E) staining and Oil Red O staining. The plaque area and lipid deposition area were counted to evaluate the anti-atherosclerotic effect of the drug.

[0148] III. Test Results All data are expressed as mean ± standard deviation. Parallel one-way ANOVA was performed, with P < 0.05 indicating statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. control group. Specific results are shown in Table 7.

[0149] Table 7. Experimental results of each nanoparticle group inhibiting the progression of atherosclerosis.

[0150] Pathological staining results showed that the aortic root of the high-lipid-induced model mice had a large amount of lipid deposition and atherosclerotic plaques. Free Mdivi-1 intervention only slightly reduced the plaque area, with limited therapeutic effect; while the three nano-formulations SS-M NPs, NR-M NPs, and NMN-M NPs could significantly reduce atherosclerotic plaques, with plaque area reduction reaching 40%–60%, effectively inhibiting vascular lipid deposition and delaying the progression of atherosclerotic lesions. Their in vivo therapeutic effect was significantly superior to that of free drugs.

[0151] Experimental Example 7: Pharmacodynamic Evaluation of the Prevention of Radiation Enteritis This experiment uses an ionizing radiation-induced mouse model of radiation enteritis to evaluate the preventive and radiation protection effects of the nanoparticles of this invention on radiation enteritis.

[0152] I. Experimental Materials Experimental animals: C57BL / 6J mice, male, 8-10 weeks old.

[0153] SS-M NPs nanoparticles: prepared according to the method of Example 1.

[0154] Mito-M NPs nanoparticles: prepared according to the method in Example 7.

[0155] II. Experimental Grouping C57BL / 6J mice were randomly divided into four groups of 15 mice each: a normal control group, a model group, an SS-M NPs prevention group, and a Mito-M NPs prevention group. Drug intervention was administered from 5 days before irradiation to 10 days after irradiation, with administration via gavage every other day, for a total of 8 administrations. The model group and the normal control group were administered the same volume of physiological saline via gavage. On day 10 after irradiation, all mice underwent the same sampling procedure, collecting whole blood and colorectal tissue for relevant evaluation indicators.

[0156] III. Test Results All experimental data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA. A p-value < 0.05 was considered statistically significant, and *p < 0.05, **p < 0.01, and ***p < 0.001 indicated significant differences compared to the model group. Detailed experimental results are shown in Table 8.

[0157] Table 8. Trial results of NR-M NPs and Mito-M NPs in preventing radiation enteritis.

[0158] Statistical results on animal survival and physiological indicators showed that ionizing radiation caused severe radiation damage in mice. The survival rate of mice in the model group was only 13.33%, accompanied by a significant decrease in peripheral blood leukocyte (WBC) count and a significant shortening of the colon and rectum, consistent with the characteristics of a radiation enteritis model. Compared with the model group, prophylactic administration of SS-M NPs and Mito-M NPs significantly improved the survival rate of irradiated mice, alleviated radiation-induced leukopenia symptoms, and effectively relieved colorectal tissue atrophy and damage.

[0159] The results in summary confirm that the nanoparticles of this invention have excellent radiation protection properties and can effectively prevent radiation-induced enteritis caused by ionizing radiation, and the overall preventive effect of Mito-M NPs is even better.

[0160] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A method for preparing a self-assembled nanomedicine system regulated by mitochondrial oxidative stress, characterized in that, Includes the following steps: S1. Dissolve Mdivi-1 and the support in an organic solvent to obtain an organic phase; S2. Dissolve the second drug in an aqueous medium or ethanol to obtain a second drug solution; the second drug includes at least one of imipreptide, mitoxin mesylate, nicotinamide ribose, and nicotinamide nucleotide; S3. Use any of the following methods to self-assemble Mdivi-1, the support, and the second drug to form a nanoparticle precursor: Nano-coprecipitation method: The organic phase is added dropwise to an aqueous medium and mixed, and then a second drug solution is added to continue the reaction; Thin-film hydration method: The organic phase is formed into a thin film by rotary evaporation, and then hydrated with a second drug solution; Reverse solvent method: The organic phase is rapidly mixed with the second drug solution, and nanoparticles are formed by reverse solvent precipitation; Solvent evaporation method: After mixing the organic phase with the second drug solution, the organic solvent is removed by evaporation under reduced pressure; The second drug solution used in the nano-coprecipitation method, the thin film hydration method, and the reverse solvent method is prepared using an aqueous medium; the second drug solution used in the solvent evaporation method is prepared using ethanol. S4. Separate the nanoparticle precursor and mix it with a surface modifier or stabilizer for stabilization treatment to obtain drug-assembled nanoparticles; Steps S1 and S2 are not sequential.

2. The preparation method according to claim 1, characterized in that, In step S1, the carrier comprises oligoepigallocatechin gallate; The organic solvent includes at least one of dimethyl sulfoxide and anhydrous ethanol; The mass ratio of Mdivi-1 to the carrier is 1:1.5 ~ 2:

1.

3. The preparation method according to claim 1, characterized in that, The aqueous medium in steps S2 and S3 is independently selected from water and phosphate buffer. Preferably, when the nano-coprecipitation method is used in step S3, the aqueous medium is an aqueous solution of epigallocatechin gallate with a concentration of 1~5 mg / mL, more preferably 2.6 mg / mL; More preferably, the mass ratio of Mdivi-1 to epigallocatechin gallate is 1:0.8 to 1.5, and more preferably 1:1.

1.

4. The preparation method according to claim 1, characterized in that, In step S3, the mass ratio of Mdivi-1 to the second drug is 2:1 to 4:

1.

5. The preparation method according to claim 1, characterized in that, The surface modifier includes thioctic polysaccharide; the stabilizer includes bovine serum albumin. The mass ratio of Mdivi-1 to surface modifier or stabilizer is 8~15:1, preferably 12:

1.

6. The preparation method according to claim 5, characterized in that, The thioctic polysaccharide includes at least one of thioctic acid-acylated chondroitin sulfate, thioctic acid-acylated sodium hyaluronate, and thioctic acid-amidated chitosan.

7. The preparation method according to claim 1, characterized in that, The stabilization process described in step S4 employs high-energy ultrasonic treatment.

8. A self-assembled nanomedicine system regulated by mitochondrial oxidative stress, characterized in that, It is prepared by the method described in any one of claims 1 to 7.

9. The application of the self-assembled nanomedicine system regulated by mitochondrial oxidative stress according to claim 8, characterized in that, Application in the preparation of drugs for treating diseases related to mitochondrial oxidative stress.

10. The application of the self-assembled nanomedicine system regulated by mitochondrial oxidative stress according to claim 9, characterized in that, The mitochondrial oxidative stress-related diseases include at least one of metabolic cardiovascular disease, ischemia-reperfusion injury, radiation injury, or neurodegenerative disease.

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