An amphiphilic nicotinamide adenine dinucleotide conjugate, a nanomicelle and application thereof

Abstract

This invention relates to the field of biomedical technology and discloses an amphiphilic nicotinamide adenine dinucleotide (NAD) + Couplings, nanomicelles and their applications. Amphiphilic NAD+ + Couplings have the following general structural formula: ; where NAD + R is a hydrophilic group, R is a hydrophobic antioxidant group, and phenylboronic acid derivative is the linking bond; R is selected from free radical scavengers, small molecule organoselenic compounds that mimic the function of glutathione peroxidase 4, iron chelating agents, 7-dehydrocholesterol, royal jelly acid, α-lipoic acid, oleic acid, or derivatives of the above molecules. In this invention, NAD... + Conjugates and their self-assembled nanomicelles possess functions such as promoting DNA repair, maintaining cellular homeostasis, and delaying aging. Simultaneously, nanomicelles can inhibit inflammation and oxidative stress, and can physically encapsulate hydrophobic active drugs, showing broad application prospects in inflammatory diseases, age-related diseases, organ damage, and the prevention and treatment of toxic side effects from chemotherapy drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an amphiphilic nicotinamide adenine dinucleotide conjugate, nanomicelles, and their applications. Background Technology

[0002] Inflammation is a key characteristic of many diseases, especially age-related diseases such as arthritis. Clinically used anti-inflammatory drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids, often face numerous side effects, including gastrointestinal damage, cardiovascular risk, liver and kidney damage, metabolic disorders, and immunosuppression. Biologics targeting specific inflammatory factors and immune cells, as well as small-molecule targeted drugs, are expensive and require monitoring for infection risks. Therefore, developing novel, highly effective anti-inflammatory drugs with fewer side effects is of great significance.

[0003] NAD + NAD is a key coenzyme and signaling molecule widely distributed within cells. As an electron carrier, it participates in the breakdown and metabolism of substances such as sugars, fats, and alcohol, and is a core component of cellular energy production. As an essential substrate for many key enzymes, it participates in regulating gene expression, metabolism and aging, DNA damage repair, immune regulation, and many other cellular functions. + As a core node connecting cellular metabolism and functional regulation, NAD is an important target for the prevention and treatment of aging and various chronic diseases. + It plays a crucial role in regulating the body's redox balance, resisting oxidative stress under inflammatory conditions, and reducing the levels of reactive oxygen species and free radicals. In uncontrolled inflammation, abnormal metabolism exacerbates the inflammatory response, and NAD+... + It can maintain normal metabolism, ensure cellular energy supply, and reduce the activation of inflammatory signals caused by metabolic disorders. NAD + It is an important coenzyme of the deacetylase SIRT1. The combination of the two activates SIRT1, triggering the deacetylation of various transcription factors and inflammation-related proteins, thereby inhibiting the expression of inflammation-related genes and alleviating the inflammatory response. In addition, NAD... + It can also inhibit the assembly and activation of the NLRP3 inflammasome and reduce the maturation and release of inflammatory factors such as IL-1β by regulating intracellular metabolic state and redox balance. Finally, NAD + It can regulate metabolic reprogramming within macrophages, promoting their polarization towards the M2 type and enhancing their anti-inflammatory capabilities, while simultaneously inhibiting the pro-inflammatory function of M1 macrophages and reducing the release of inflammatory factors. Previous studies have found that NAD... + It can effectively treat multi-organ damage and uncontrollable inflammation caused by sepsis.

[0004] Although NAD is an endogenous molecule + Side effects are very low, but NAD +The hydrophilic nature and negative charge of these molecules prevent their effective uptake by cells, limiting their efficacy and application in the anti-inflammatory field. Inflammation and oxidative stress are two closely linked and mutually amplifying core pathological processes in the body, jointly promoting a vicious cycle in many chronic diseases. Therefore, developing drugs, excipients, and delivery systems that can simultaneously inhibit inflammation and oxidative stress is of great significance. Amphiphilic molecules can autonomously assemble into nanomicelles in the aqueous phase, achieving stability in blood circulation and enhancing cellular uptake through endocytosis. (Construction of NAD...) + Amphiphilic conjugates with hydrophobic antioxidant molecules have advantages such as long circulation, high drug loading, and efficient cellular uptake. Moreover, by designing responsive linkages, the active molecules can be released at the site of inflammation, which has important theoretical significance and translational value for the treatment of various inflammatory diseases. Summary of the Invention

[0005] To address the above technical problems, this invention provides an amphiphilic nicotinamide adenine dinucleotide conjugate, nanomicelles, and their applications. The amphiphilic nicotinamide adenine dinucleotide conjugate (abbreviated as amphiphilic NAD) + Couplings or NAD + (Conjugate). This conjugate can be used alone or in combination for the treatment of oxidative stress and / or inflammation-related diseases. NAD+ can also be utilized. + Self-assembling nanomicelles of conjugates enable target cells to target NAD + The efficient uptake and utilization of excess reactive oxygen species in the inflammatory microenvironment to achieve NAD + In situ release of hydrophobic antioxidant molecules, and the interaction between antioxidant molecules and NAD+. + Co-delivery. Using amphiphilic NAD. + The self-assembly of conjugate nanomicelles enables the treatment of osteoarthritis using amphiphilic NAD. + The self-assembled nanomicelles of coupling compounds physically encapsulate various active molecules and have broad application value.

[0006] In a first aspect, the present invention provides an amphiphilic nicotinamide adenine dinucleotide (NAD) + Couplers have the following general structural formula: ; Among them, NAD + R is a hydrophilic group, R is a hydrophobic antioxidant group, and the phenylboronic acid derivative is a linking bond; R is selected from free radical scavengers, small molecule organoselenic compounds that mimic the function of glutathione peroxidase 4 (GPX4), iron chelators, 7-dehydrocholesterol, royal jelly acid (10-hydroxy-2-decenoic acid), α-lipoic acid, oleic acid, or derivatives of the above molecules.

[0007] Furthermore, the free radical scavengers in the hydrophobic antioxidant groups are vitamin E / VE, Ferrostatin-1 / Fer-1, idebenone, SRS11-92 / AA9, SRS16-86, Liproxstatin-1, phenoxazine / Pnx, phenothiazine or their derivatives. Small molecule organic selenium compounds that mimic the function of glutathione peroxidase 4 are ebuselenline / Ebs or their derivatives. The iron chelating agent is deferoxone, deferoxamine, or derafloxacin or its derivatives.

[0008] Further, VE, Fer-1, idebenone, AA9, Pnx, Ebs, and derivatives thereof are preferred, more preferably VE, Fer-1, idebenone, or their derivatives. VE is the most preferred.

[0009] Furthermore, when the hydrophobic antioxidant group is VE, the structural formula of the amphiphilic nicotinamide adenine dinucleotide conjugate is: ; When the hydrophobic antioxidant group is Ferrostatin-1 / Fer-1, the structural formula of the amphiphilic nicotinamide adenine dinucleotide conjugate is: ; When the hydrophobic antioxidant group is idebenone, the structural formula of the amphiphilic nicotinamide adenine dinucleotide conjugate is: .

[0010] Furthermore, under oxidative stress, the phenylboronic acid linkage can be broken by free radicals, releasing NAD in situ. + And hydrophobic antioxidant molecules (e.g., VE, Fer-1 and idebenone).

[0011] Secondly, the present invention provides a nanomicelle in which one or more of the above-mentioned amphiphilic nicotinamide adenine dinucleotide conjugates self-assemble into the nanomicelle, or the above-mentioned amphiphilic nicotinamide adenine dinucleotide conjugates are co-assembled with other biocompatible amphiphilic molecules into the nanomicelle.

[0012] Furthermore, the other biocompatible amphiphilic molecules include small molecule surfactants, high molecule surfactants, phospholipids, and amphiphilic polyethylene glycol derivatives.

[0013] Furthermore, the amphiphilic polyethylene glycol derivative includes phospholipid-polyethylene glycol, more preferably distearate-phosphatidylethanolamine-polyethylene glycol (DSPE-PEG); wherein the polyethylene glycol (PEG) has a molecular weight of 400-3000 Da, preferably 750-2000 Da, more preferably 2000 Da; and the PEG end group is carboxyl, amino, mercapto, maleimide, or succinimide.

[0014] Furthermore, the PEG end group is covalently linked to the target molecule, which includes small molecules, monosaccharides, peptides, antibodies, and nanobodies, thereby endowing the nanomicelles with active targeting properties.

[0015] Thirdly, the present invention provides the use of the above-mentioned amphiphilic nicotinamide adenine dinucleotide conjugate or the above-mentioned nanomicelles in the preparation of a medicament for treating inflammatory and / or oxidative stress-related diseases, including: ① Orthopedic diseases: osteoarthritis, rheumatoid arthritis, osteoporosis; ② Ophthalmic diseases: Age-related macular degeneration / AMD, dry eye syndrome, guttate keratosis / Fuchs' endothelial dystrophy, neurotrophic keratitis; ③ Cardiovascular diseases: anthracycline-induced cardiotoxicity, ischemic heart disease, atherosclerosis, and myocardial infarction; ④ Kidney diseases: kidney injury induced by platinum-based drugs, acute kidney injury, chronic kidney disease, polycystic kidney disease, and diabetic nephropathy; ⑤ Liver diseases: drug-induced liver injury, acute liver failure, autoimmune liver disease, alcoholic liver disease, non-alcoholic fatty liver disease, liver fibrosis; ⑥ Neurological diseases: Parkinson's disease, Alzheimer's disease, ischemic stroke, traumatic brain injury, epilepsy, amyotrophic lateral sclerosis, Huntington's disease; ⑦ Lung diseases: chronic obstructive pulmonary disease, asthma, acute lung injury, pulmonary fibrosis; ⑧ Organ damage caused by ischemia-reperfusion, multiple organ dysfunction, sepsis, uncontrollable inflammation, neuropathic pain caused by chemotherapy, tissue and organ damage caused by radiotherapy, antiviral infection, cerebral infarction, iron overload disease, and biliary tract disease.

[0016] Furthermore, in application, the nanomicelles encapsulate active drug molecules, including anthracycline drugs, platinum-based drugs, and active molecules for treating neurodegenerative diseases, ophthalmic diseases, and orthopedic diseases; the routes of administration include oral administration, intravenous administration, nasal administration, pulmonary administration, intra-articular injection, transdermal administration, ocular administration, intraperitoneal administration, and mucosal administration.

[0017] Fourthly, the present invention provides a drug delivery system for treating inflammation and / or oxidative stress-related diseases, the drug delivery system comprising the above-described amphiphilic nicotinamide adenine dinucleotide conjugate or the above-described nanomicelles encapsulating active drug molecules; The inflammation and / or oxidative stress-related diseases include: ① Orthopedic diseases: osteoarthritis, rheumatoid arthritis, osteoporosis; ② Ophthalmic diseases: Age-related macular degeneration / AMD, dry eye syndrome, guttate keratosis / Fuchs' endothelial dystrophy, neurotrophic keratitis; ③ Cardiovascular diseases: anthracycline-induced cardiotoxicity, ischemic heart disease, atherosclerosis, and myocardial infarction; ④ Kidney diseases: kidney injury induced by platinum-based drugs, acute kidney injury, chronic kidney disease, polycystic kidney disease, and diabetic nephropathy; ⑤ Liver diseases: drug-induced liver injury, acute liver failure, autoimmune liver disease, alcoholic liver disease, non-alcoholic fatty liver disease, liver fibrosis; ⑥ Neurological diseases: Parkinson's disease, Alzheimer's disease, ischemic stroke, traumatic brain injury, epilepsy, amyotrophic lateral sclerosis, Huntington's disease; ⑦ Lung diseases: chronic obstructive pulmonary disease, asthma, acute lung injury, pulmonary fibrosis; ⑧ Organ damage caused by ischemia-reperfusion, multiple organ dysfunction, sepsis, uncontrollable inflammation, neuropathic pain caused by chemotherapy, tissue and organ damage caused by radiotherapy, antiviral infection, cerebral infarction, iron overload disease, and biliary tract disease.

[0018] The advantages and positive effects of the amphiphilic nicotinamide adenine dinucleotide conjugate, nanomicelles, and their applications described in this invention are as follows: 1. The amphiphilic nicotinamide adenine dinucleotide conjugate NAD described in this invention + Self-assembling into nanomicelles allows for the in-situ release of antioxidant molecules and NAD at sites of oxidative stress or inflammation. + It can simultaneously inhibit oxidative stress and inflammatory response, and has good clinical application value.

[0019] 2. NAD in this invention + Self-assembled nanomicelles of coupling compounds have high NAD + It has a high load capacity and can physically encapsulate various hydrophobic drugs to achieve synergistic effects.

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0021] Figure 1The amphiphilic conjugate VE-NAD in Example 1 of this invention + The proton nuclear magnetic resonance spectrum; Figure 2 The above are the 1H NMR spectra of the amphiphilic conjugates in Examples 2-3 of this invention, where (A) is Fer1-NAD. + The proton NMR spectrum, (B) is IDB-NAD + The proton nuclear magnetic resonance spectrum; Figure 3 The amphiphilic conjugate VE-NAD in Example 4 of this invention + Absorption spectrum, hydrodynamic particle size and morphology, critical micelle concentration and kinetic stability of nanomicelles (n = 3), where A is the absorption spectrum, B is the hydrodynamic particle size and morphology, C is the critical micelle concentration and D is the kinetic stability. Figure 4 VE-NAD in Embodiment 5 of the present invention + ROS scavenging ability, ROS responsiveness and NAD of micelles + Uptake capacity was characterized, where A represents ROS scavenging capacity, B represents ROS responsiveness, C represents the uptake capacity of different samples in RAW 264.7 cells, and D represents the uptake capacity of different samples in rat primary chondrocytes; ns indicates no statistically significant difference. express p <0.05, express p <0.01, express p <0.001; Figure 5 This invention illustrates the effect of co-incubation of four samples with the ferroptosis inducer RSL3 (0.2 μM) on the cell viability of RAW264.7 macrophages in Example 6 of this invention. In this example, A represents free vitamin E; B represents free NAD+. + C represents free vitamin E and NAD. + A physical mixture; D is VE-NAD + Nanomicelles, ns indicates no statistically significant difference. express p <0.05, express p <0.001; Figure 6 This invention illustrates the effect of co-incubation of four samples with the ferroptosis inducer RSL3 (0.2 μM) on the cell viability of primary rat chondrocytes in Example 6 of this invention. In this example, A represents free vitamin E; B represents free NAD+.+ C represents free vitamin E and NAD. + A physical mixture; D is VE-NAD + Nano micelles. express p <0.001; Figure 7 The present invention is VE-NAD + Analysis of the ability of conjugated nanomicelles to inhibit lipid peroxidation in lipopolysaccharide (LPS, 1 μg / mL)-induced inflammatory RAW264.7 macrophages. A shows confocal microscopy images of cells stained with the Liperfluo probe; B shows quantitative analysis of the fluorescence intensity of the Liperfluo probe. ns indicates no statistically significant difference. express p <0.001; Figure 8 The present invention is VE-NAD + Analysis of the inhibitory and antioxidant capacity of conjugated nanomicelles in interleukin-1β (IL-1β, 10 ng / mL) inflammatory rat primary chondrocytes. In the image, A shows a confocal microscopy image of cells stained with the DCF probe; B shows the quantitative analysis of the fluorescence intensity of the DCF probe; and C shows a confocal microscopy image of cells stained with the Liperfluo probe. In the image, B shows the quantitative analysis of the fluorescence intensity of the Liperfluo probe. ns indicates no statistically significant difference. express p <0.05, express p <0.001; Figure 9 The present invention is VE-NAD + Analysis of anti-inflammatory and lipid peroxidation inhibitory markers in LPS (1 μg / mL)-induced inflammatory RAW264.7 macrophages using conjugated nanomicelles, where A is malondialdehyde (MDA), B is tumor necrosis factor-α (TNF-α), and C is nitrite (NO2). - ), ns indicates no statistical difference, express p <0.05, express p <0.01, express p <0.001; Figure 10 The present invention is VE-NAD + Analysis of the antioxidant and lipid peroxidation inhibitory markers of conjugated nanomicelles in rat primary chondrocytes, where A is 4-hydroxynonenal (4-HNE), B is malondialdehyde (MDA), C is thioredoxin (Trx), and D is glutathione (GSH). ns indicates no statistically significant difference. express p <0.05, express p <0.01, express p <0.001; Figure 11 The present invention is VE-NAD + Analysis of anti-inflammatory markers in conjugated nanomicelles in primary rat chondrocytes induced by interleukin-1β (IL-1β, 10 ng / mL) inflammation: A represents tumor necrosis factor-α (TNF-α), B represents interleukin-6 (IL-6), C represents interleukin-4 (IL-4), D represents inducible nitric oxide synthase (iNOS), E represents cyclooxygenase-2 (COX-2), F represents prostaglandin E2 (PGE2), G represents matrix metalloproteinase-3 (MMP-3), H represents matrix metalloproteinase-13 (MMP-13), and I represents the chemokine monocyte chemoattractant protein-1 (MCP-1). ns indicates no statistically significant difference. express p <0.05, express p <0.01, express p <0.001; Figure 12 The present invention is VE-NAD + Staining analysis of the knee joints of aged OA rats treated with conjugated nanomicelles and a reference formulation. A shows H&E staining images, B shows synovitis scores, C shows SOFG staining images, and D shows quantitative analysis of cartilage matrix. ns indicates no statistically significant difference. express p <0.01, express p <0.001; Figure 13 The present invention is VE-NAD +Analysis of the cartilage matrix synthesis and degradation capacity of knee joint cartilage tissue in aged OA rats treated with conjugated nanomicelles and a reference formulation. In the table, A shows staining images of proteoglycan (ACAN), type II collagen (Col II), and matrix metalloproteinase-3 (MMP-3) in the cartilage tissue; B shows quantitative analysis of ACAN staining images; C shows quantitative analysis of Col II staining images; and D shows quantitative analysis of MMP-3 staining images. ns indicates no statistically significant difference. express p <0.05, express p <0.01, express p <0.001. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0024] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0025] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0026] The conjugate in this invention consists of three modules: a hydrophobic antioxidant molecule, a responsive linker, and a hydrophilic nicotinamide adenine dinucleotide (NADP), covalently linked together. The responsive linker enables the conjugate to release the hydrophobic antioxidant molecule and NADP in situ under stimulation from free radicals in the disease microenvironment, such as inflammation, thereby exerting their respective pharmacological effects. The conjugate and its self-assembled nanomicelles possess functions such as reducing oxidative stress, inhibiting inflammation, promoting DNA repair, maintaining cellular homeostasis, and delaying aging. Due to the hydrophilicity and negative charge of NADP, its cellular uptake is weak; the conjugate nanomicelles can increase NADP uptake through endocytosis. These nanomicelles can simultaneously inhibit inflammation and oxidative stress and can physically encapsulate hydrophobic active drugs, showing broad application prospects in inflammatory diseases, age-related diseases, organ damage, and the prevention and treatment of chemotherapy drug toxicity.

[0027] The following examples provide a detailed explanation.

[0028] Example 1 This example illustrates how to prepare a reactive oxygen species-responsive conjugate, VE-NAD. + The specific synthesis steps are as follows.

[0029] (1) Weigh D α-Tocopherol (VE, 297.2 mg, 0.69 mmol) and dry sodium hydride (NaH, 24.8 mg, 1.04 mmol) were placed in a dry reaction flask, and 6 mL of anhydrous sodium hydride was added. N,N Dimethylformamide (DMF) was stirred at room temperature for 1 hour under nitrogen protection. Then, 4-bromophenylboronic acid (BrPBA, 163.3 mg, 0.76 mmol) was dissolved in 5 mL of anhydrous DMF and added to the reaction system. The reaction was continued at room temperature for another 4 hours. After the reaction was complete, saturated brine was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by preparative thin-layer chromatography using petroleum ether / ethyl acetate (2:1, v / v) as the developing solvent to obtain the intermediate VE-PBA.

[0030] (2) Weigh out the synthesized VE-PBA (53.5 mg, 0.09 mmol) and NAD. +(59.7 mg, 0.09 mmol) was added to a dry reaction flask, and 15 mL of anhydrous dimethyl sulfoxide (DMSO) was added under argon protection. After adding molecular sieves, the reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed, the reaction solution was slowly added dropwise to alkaline distilled water adjusted to pH 9 with ammonia while stirring. The resulting solution was transferred to a dialysis bag with a molecular weight cutoff of 500 Da, and dialyzed against alkaline distilled water at pH 9 for 12 hours, followed by dialyzing with pure distilled water for another 12 hours. The dialyzed solution was freeze-dried to obtain a pale yellow solid powder, which was VE-NAD. + The coupling compound, its NMR spectrum is shown in [reference needed]. Figure 1 .

[0031] VE-NAD + of 1 H NMR (600 MHz, DMSO- d 6) δ 9.59 (s, 1H, ArH), 9.39 (d, J = 6.2Hz, 1H, ArH), 8.94 (d, J = 8.3 Hz, 1H, ArH), 8.56-8.48 (m, 2H, ArH), 8.47 (s, 1H, ArH), 8.33 (s, 2H, NH2), 8.17 (s, 2H, NH2), 7.81 (d, J = 7.6 Hz, 2H, ArH),7.43 (d, J = 7.7 Hz, 2H, ArH), 5.93 (dd, J = 12.2, 5.5 Hz, 2H, CH), 4.61 (s, 2H, CH2), 4.57-4.47 (m, 2H, CH, OH), 4.35 (d, J = 4.3 Hz, 1H, OH), 4.32-4.27 (m,2H, CH), 4.18 (s, 1H, OH), 4.10-3.99 (m, 5H, CH, CH2), 3.96 (d, J = 7.5 Hz, 2H,CH), 2.57 (d, J = 1.2 Hz, 2H, CH2), 2.09 (d, J= 17.5 Hz, 6H, CH3), 2.00 (s, 3H, CH3), 1.76-1.69 (m, 2H, CH2), 1.54-1.46 (m, 3H, CH, CH2), 1.41-1.32 (m, 4H, CH, CH2), 1.30-1.19 (m, 8H, CH2), 1.18 (s, 3H, CH3), 1.12-0.99 (m, 6H, CH2), 0.88-0.76 (m, 12H, CH3). HRMS (ESI): m / z calculation results: C 57 H 80 BN7O 16 P2[MH] + 1190.52; Analysis of measured results: 1190.32.

[0032] Example 2 This example illustrates how to prepare the reactive oxygen species-responsive conjugate Fer-1-NAD. + The specific synthesis steps are as follows.

[0033] (1) Weigh 89 mg (0.3 mmol) of triphosgene (BTC) into a 25 mL round-bottom flask, add 2 mL of anhydrous dichloromethane (DCM) to dissolve it, add 20 μL of pyridine, and stir at room temperature for 30 min under argon protection. Weigh 144 mg (0.615 mmol) of pinacol 4-hydroxymethylphenylboronic acid (PBAE) and dissolve it in 2 mL of anhydrous DCM. Add the solution dropwise to the above triphosgene solution and continue stirring at room temperature for 30 min. Weigh 50 mg (0.2 mmol) of Fer-1 into a 25 mL round-bottom flask, add 2 mL of anhydrous dichloromethane (DCM) to dissolve it, add 20 μL of pyridine, and stir at room temperature for 10 min under argon protection. Then slowly add the mixture of triphosgene and pinacol 4-hydroxymethylphenylboronic acid (PBAE) solution dropwise to the mixture and stir at room temperature for 4 h. The solvent was removed under reduced pressure, and the product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, PE / EA = 4:1, v / v). After rotary evaporation, a brown oily substance was obtained, which is Fer-1-PBAE.

[0034] (2) Weigh 53 mg (0.1 mmol) of Fer-1-PBAE into a 25 mL round-bottom flask, add 2 mL of acetone and 2 mL of 0.02 M hydrochloric acid solution to dissolve it. Weigh 64 mg (1 mmol) of methylboric acid, add 1 mL of acetone and 1 mL of 0.02 M hydrochloric acid to dissolve it, and then add this methylboric acid solution dropwise to the above Fer-1-PBAE solution under stirring. React at room temperature for 12 hours under argon protection. After the reaction is complete, add 10 mL of ethyl acetate, extract, dry the organic phase with anhydrous magnesium sulfate, remove the solvent under reduced pressure, purify by silica gel column chromatography (PE / EA=2:1, v / v), and obtain a white powder after rotary evaporation, which is Fer-1-PBA.

[0035] (3) Weigh 44 mg (0.1 mmol) of Fer-1-PBA into a 25 mL round-bottom flask, add 2 mL of anhydrous dimethyl sulfoxide (DMSO) to dissolve it, then add 14 μL (0.1 mmol) of triethylamine. Add 100 mg of grade 4A molecular sieve to the mixture, and stir at room temperature for 6 h under argon protection. Weigh 66 mg (0.1 mmol) of NAD. + Dissolve the DMSO in 6 mL of anhydrous DMSO and slowly add it to the Fer-1-PBA solution. Continue stirring at room temperature for 24 h. Add 12 mL of distilled water to a 50 mL round-bottom flask and adjust the pH to 9 with triethylamine. Add the reacted solution dropwise to the distilled water at pH 9 and stir for 20 min. After the reaction is complete, place the reaction solution in a dialysis bag (molecular weight cutoff: 500 Da). Dialyze with distilled water at pH 9 (adjusted with ammonia) for 12 h, then dialyze with distilled water for 12 h. Transfer the liquid in the dialysis bag to a centrifugal ultrafiltration tube (molecular weight cutoff: 3000 Da) for ultrafiltration. Collect the liquid in the cutoff chamber and freeze-dry. Purify by silica gel column chromatography (MeOH / H2O = 6:1, v / v), freeze-dry, and obtain the amphiphilic conjugate Fer-1-NAD. + Its 1H NMR spectrum is shown in Figure 2 (A)

[0036] Fer-1-NAD + of 1 H NMR (DMSO- d 6,400MHz): δ 9.66 (s, 1H), 8.95 (s, 1H), 8.51 (s, 7H), 8.15 (d, J = 3.1 Hz, 8H), 7.79 (d, J = 7.8 Hz, 3H), 7.75 (s, 1H), 7.60 (dd,J = 8.7, 2.1 Hz, 1H), 7.38 (s, 1H), 7.36 (s, 1H), 6.69 (d, J = 8.7 Hz, 1H), 5.92 (d, J = 5.1 Hz, 6H), 5.14 (s, 2H), 4.92 (d, J = 4.8 Hz, 2H), 4.69 (d, J =12.4 Hz, 2H), 4.49 (d, J = 13.7 Hz, 3H), 4.33 (s, 2H), 4.21 (q, J = 7.1 Hz, 3H), 4.08 – 4.03 (m, 7H), 3.84 (s, 1H), 1.89 (d, J = 11.7 Hz, 3H), 1.69 (d, J = 12.8Hz, 2H), 1.57 (s, 1H), 1.34 (d, J = 11.0 Hz, 1H), 1.30 – 1.11 (m, 11H), 1.05 (t, J = 6.9 Hz, 1H), 0.84 (d, J = 7.1 Hz, 1H). HRMS (ESI): m / z calculation result: C 44 H 52 BN9O 18 P2[M+H] + 1068.30; Analytical measurement result: 1068.00.

[0037] Example 3 This example illustrates how to prepare the reactive oxygen species-responsive conjugate IDB-NAD. + The specific synthesis steps are as follows.

[0038] (1) Weigh 229.64 mg of Na2CO3 and 79.127 mg of triphosgene (BTC) (molar ratio Na2CO3:BTC = 8.08:1) into a 25 mL round-bottom flask, add 8 mL of anhydrous tetrahydrofuran, and react at 0 °C for about 2 h under N2 / Ar protection. Then, weigh 198.98 mg of idebenone (IDB) (molar ratio Na2CO3:BTC:IDB = 16:1:2), dissolve it in 2 mL of anhydrous tetrahydrofuran, inject it into a round-bottom flask, and react at room temperature overnight. After the reaction is complete, the crude product is purified by silica gel column chromatography with eluent ratios of PE:EA = 20:1, 15:1, and 10:1, respectively. The organic phase is removed by rotary evaporation to obtain an orange oily substance, which is IDB-Cl.

[0039] (2) Weigh 156 mg of IDB-Cl and 160.32 mg of PBAE (molar ratio IDB-Cl:PBAE = 1:1.7) into a 25 mL round-bottom flask, add 13 mL of anhydrous dichloromethane to dissolve them, and then slowly add 100 μL of anhydrous pyridine. Under N2 / Ar protection, react at room temperature for 4-6 h. After the reaction is complete, the crude product is purified by thin-layer chromatography with a developing solvent ratio of PE:EA = 4:1. The organic phase is removed by rotary evaporation to obtain an orange oily substance, which is IBD-PBAE.

[0040] (3) Weigh 226.7 mg of IDB-PBAE and 242.98 mg of NaIO4 (molar ratio of IDB-PBAE:NaIO4 = 1:3) into a 25 mL round-bottom flask, add 25 mL of a mixed solution of THF and water (volume ratio of THF:H2O = 4:1), react at room temperature for 2 h, then add 1.1 mL of 1 M HCl and continue the reaction for another 2 h. After the reaction is complete, add 10 mL of water to terminate the reaction and extract with ethyl acetate (3 × 15 mL). The combined organic layers are washed twice with saturated sodium carbonate and saturated brine, respectively. Then, add anhydrous sodium sulfate to the organic phase to dry it, filter it, and rotary evaporate the filtrate. The crude product is purified by thin-layer chromatography with a developing solvent ratio of PE:EA = 2:1. Rotary evaporation removes the organic phase, yielding an orange oily substance, which is IDB-PBAE.

[0041] (4) Weigh 50 mg of IDB-PBA into a 25 mL round-bottom flask, add an equimolar amount of triethylamine and 5 mL of anhydrous DMSO, and stir at room temperature for 12 h. Then weigh 64.2 mg of NAD +Dissolved in 10 mL anhydrous DMSO, the solution was added dropwise to a reaction flask and reacted at room temperature for 24 h. After the reaction, the reaction solution was added dropwise to an equal volume of distilled water and transferred to a dialysis bag (molecular weight cutoff: 500 Da). The mixture was treated with distilled water at pH 9 for 12 h, followed by dialyzing with distilled water for another 12 h. The final product, IDB-NAD, was obtained by freeze-drying. + Its 1H NMR spectrum is shown in Figure 2 (B)

[0042] IDB-NAD + of 1 H NMR (DMSO- d6 , 600 MHz): δ 9.37 (s, 1H), 8.96 (s, 2H), 8.46 (s, 3H), 8.19 (s, 4H), 7.78 (d, J = 7.4 Hz, 2H), 7.33 (d, J = 7.6 Hz, 2H), 6.11 (d, J = 19.9 Hz, 2H), 5.13 (s, 2H), 4.94 (s, 2H), 4.26 (s, 3H), 4.09 (s, 6H), 4.05 (s, 4H), 3.87 (s, 6H), 2.38 (d, J = 7.2 Hz, 2H), 1.93 (s, 3H), 1.58 (s, 2H), 1.25 (d, J = 15.5 Hz, 14H). HRMS (ESI): m / z calculation result: C 48 H 60 BN7O 21 P2[M+H] + 1144.34; Analysis of measured results: 1144.70.

[0043] Example 4 This example illustrates how to prepare VE-NAD. + Micelles were analyzed, and their spectral properties, hydrodynamic particle size, microstructure, stability, and critical polymerization micelle concentration were characterized.

[0044] Weigh out VE-NAD +20.0 mg of the conjugate was dissolved in 10 mL of DMSO, and the solution was transferred to a dialysis bag with a molecular weight cutoff of 500 Da. The dialysis bag was first placed in an alkaline aqueous solution at pH 9.0 and dialyzed at room temperature for 24 hours, during which the dialysis fluid was changed three times. Subsequently, the dialysis bag was transferred to ultrapure water and dialyzed for another 12 hours to completely remove organic solvents and free small molecules. After dialysis, the solution in the bag was transferred to an ultrafiltration concentrator with a molecular weight cutoff of 3000 Da and concentrated at 2,500 mL / min. g The solution was concentrated by centrifugation for 10 minutes. Finally, the concentrated micelle solution was filtered through a 0.22 μm microporous membrane to obtain VE-NAD. + Micellar stock solution.

[0045] VE-NAD + The VE-NAD precursors were dissolved in a 1:1 v / v mixed solvent of DMSO / water to prepare 100 μM solutions. The absorption spectra of each solution were scanned and recorded in the wavelength range of 250 nm–350 nm using a UV-Vis spectrophotometer to analyze the VE-NAD. + The conjugated structure characteristics were observed. The prepared micelle stock solution was diluted with ultrapure water to a concentration of 0.1 mg / mL. The hydrodynamic particle size of the micelle solution was determined at room temperature using a dynamic light scattering instrument. Freshly prepared VE-NAD... + The micelle solution was adjusted to a concentration of 2 mg / mL. 5 μL of the micelle solution was pipetted onto a 300-mesh copper mesh and allowed to air dry at room temperature. A 2% (w / v) phosphotungstic acid aqueous solution was prepared, and its pH was adjusted to 7.4 with sodium hydroxide solution. 20 μL of phosphotungstic acid negative staining solution was added to the dried sample, and after staining for 1 minute, excess staining solution was absorbed from the edge of the copper mesh with filter paper. The sample was dried overnight at room temperature in the dark, and then observed and imaged using a transmission electron microscope to analyze the morphology and particle size of the micelles. Simultaneously, VE-NAD... + The micelle solutions were diluted to 1 mg / mL with ultrapure water and complete DMEM medium containing 10% fetal bovine serum (FBS), respectively. The diluted samples were placed at a constant temperature of 25°C, and samples were taken at the set time points. The hydrodynamic particle size was determined by the DLS method described above to evaluate the short-term stability of the micelles in different media.

[0046] VE-NAD was determined using the pyrene fluorescent probe method. + Critical micelle concentration (CMC). First, prepare a VE-NAD solution with a concentration of 1 mg / mL using ultrapure water. +Micellar stock solutions were prepared and serially diluted to obtain a series of micelle suspensions with concentrations ranging from 0.1 to 1.0 µg / mL. 40 μL of a 50 μM pyrene-acetone solution was added to each centrifuge tube, and the acetone was allowed to evaporate completely at room temperature in the dark. Subsequently, 4 mL of the corresponding concentration of VE-NAD was added to each centrifuge tube. + A micelle suspension was prepared to achieve a final pyrene concentration of 0.5 μM. The sample was sonicated for 10 minutes, incubated in a 65°C water bath for 2 hours, and then equilibrated overnight at room temperature in the dark. The fluorescence spectrum of the sample was recorded using a fluorescence spectrophotometer with an excitation wavelength of 333 nm and an emission spectrum scanning range of 350-450 nm. The third vibrational peak of pyrene (I0.05) was used as the starting point for fluorescence analysis. 384 ) and the first vibration peak (I 373 The ratio of fluorescence intensity (I) 384 / I 373 ( ) as the ordinate, plotted with VE-NAD + Plot the logarithm of the concentration on the x-axis; the concentration corresponding to the intersection of the two linear trend lines is the VE-NAD concentration. + CMC results ( Figure 3 This indicates that VE-NAD + Nanomicelles have small hydrodynamic particle size (<100 nm) and critical micelle concentration, and exhibit good stability in different media.

[0047] Example 5 This example illustrates VE-NAD. + Micelles' ability to scavenge hydroxyl radicals, ROS-responsive disassembly, and cellular uptake performance.

[0048] The hydroxyl radical scavenging activity of micelles was evaluated using the methylene blue (MB) bleaching method. All solutions were prepared using ultrapure water. The test reaction system consisted of: 300 μL ferrous ammonium sulfate aqueous solution (3.33 mM), 300 μL MB aqueous solution (0.67 mM), and VE-NAD... + Micellar suspension (100 μL, 3 mM) and aqueous H2O2 solution (300 μL, 6.67 mM). Control group: VE-NAD replaced with 100 μL of ultrapure water. + The micelle suspension was identical to the test group in all other components. After all components were mixed and reacted at room temperature for 15 minutes, the absorbance spectrum was scanned and recorded using a microplate reader in the wavelength range of 400 nm to 800 nm. The hydroxyl radical scavenging activity was assessed by the absorbance change at the MB characteristic absorption peak (approximately 665 nm).

[0049] By examining VE-NAD +The change in micelle size after ROS stimulation under different pH conditions was investigated to verify the ROS responsiveness of phenylboronic acid ester linkages. The specific steps are as follows: 30% H2O2 stock solution was diluted to 200 μM using phosphate-buffered saline (PBS) at pH 6.5 or 7.4. Simultaneously, 3 mg of vitamin E-NAD was weighed... + Micelles were prepared by dispersing them in PBS at the appropriate pH to form a 200 μM micelle dispersion. An equal volume of H2O2 solution was mixed with the micelle dispersion and incubated at room temperature for 6 hours. After incubation, the hydrodynamic particle size of the mixture was measured at 25 °C using dynamic light scattering and compared with a control group without H2O2 treatment to evaluate the ROS-responsive dissociation behavior of the micelles.

[0050] Using WST-8 based NAD + The NADH assay kit was used to quantitatively measure intracellular NAD using RAW264.7 macrophages and primary chondrocytes as cell models, according to the manufacturer's instructions. + Content. The specific steps are as follows: Cells are seeded in six-well plates. When the cell confluence reaches 60%, the medium is replaced with complete culture medium (control group) and 40 µM free NAD+. + Or 40 µMVE-NAD + The cells were cultured in micelle medium for another 24 hours. After treatment, the cells were washed twice with pre-cooled PBS, 1 × 10⁶ cells per wash. 6 Add 200 µL NAD to each cell + Lysis was performed using NADH extraction buffer. The lysate was incubated on ice and then incubated at 4°C with 12,000 mL of NADH extract buffer. g Centrifuge for 10 minutes and collect the supernatant for analysis. Simultaneously, determine the total protein concentration of the sample using the Bradford method. Intracellular NAD+ + Concentration via total NAD + The difference between the NADH content and the total protein concentration was calculated and normalized using the total protein concentration.

[0051] result( Figure 4 This indicates that VE-NAD + Nanomicelles possess excellent antioxidant capabilities and can disassemble under the influence of reactive oxygen species. Furthermore, nanomicelles can significantly enhance cellular NAD+ uptake. + Intake.

[0052] Example 6 This example illustrates how to evaluate VE and NAD. + A physical mixture of the two (VE + NAD) + ) and VE-NAD +Micelles inhibit oxidative stress and ferroptosis, thus protecting cell viability. Specifically, using RAW264.7 macrophages and primary chondrocytes as cell models, the MTT assay was used to evaluate the inhibitory effects of each formulation on ferroptosis-inducing cell death.

[0053] Log-phase RAW264.7 macrophages (8000 cells / well) or primary rat chondrocytes (4500 cells / well) were seeded into 96-well plates and cultured adherently for 24 hours. Then, ferroptosis was induced by pretreatment with the classic ferroptosis inducer RSL3 (0.2 μM) for 2 hours. The medium containing the inducer was then discarded, and the medium was replaced with a mixture containing 0.2 μM RSL3 and different concentrations of the target compounds (VE, NAD). + VE and NAD + Physical mixtures or VE-NAD + The micelles were cultured in fresh medium for 24 hours. After treatment, 100 μL of medium solution containing 0.5 mg / mL MTT was added to each well, and the cells were cultured for another 4 hours. After the culture medium was aspirated, 100 μL of DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader, and the relative cell viability was calculated to evaluate the ferroptosis inhibitory efficacy of each formulation.

[0054] result( Figure 5-6 This indicates that, in addition to free NAD, + In addition, all other samples reversed the RSL3-induced decrease in cell activity in both model cells, and this protective effect was dose-dependent.

[0055] Example 7 This example illustrates how to detect lipid peroxides and reactive oxygen species levels in RAW264.7 macrophages using Liperfluo and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probes to assess vitamin E and NAD+ levels. + The physical mixture of the two and VE-NAD + The ability of conjugated nanomicelles to inhibit lipid peroxidation and oxidative stress under inflammatory conditions.

[0056] The specific steps are as follows: RAW264.7 macrophages were cultured at a rate of 3 × 10⁻⁶. 5 Cells were seeded at a density of 100 cells / plate in 20 mm confocal culture dishes and cultured for 24 hours to allow them to adhere. Subsequently, cells were stimulated with 1 μg / mL lipopolysaccharide (LPS) for 2 hours to induce inflammation. Afterward, the cells were replaced with solutions containing 1 μg / mL LPS and 40 μM of different drugs (free vitamin E, free NAD). + VE and NAD +Physical mixtures or VE-NAD + Cells were treated with fresh culture medium containing coupled micelles for 24 hours. After treatment, cells were washed twice with PBS, and 5 μM Liperfluo fluorescent probe (stock solution was 0.5 mM DMSO solution) diluted with DMEM was added. Cells were incubated at 37°C in the dark for 30 minutes. After washing cells three more times with PBS, imaging was performed using a confocal laser scanning microscope with an excitation wavelength of 514 nm and an emission collection range of 530-600 nm. For primary chondrocytes, cells were seeded in confocal culture dishes and stimulated with 10 ng / mL interleukin-1β (IL-1β) for 2 hours to induce inflammation. Cells were then treated with the same concentration of the aforementioned drug preparation (40 μM each, containing 10 ng / mL IL-1β) for 24 hours. Lipid peroxide detection was performed using the same Liperfluo staining and confocal imaging method as for RAW264.7 cells. Intracellular ROS was detected using the DCFH-DA probe. Cells were treated as described above, washed three times with PBS, and then incubated at 37°C in the dark with 10 μM DCFH-DA probe, which was freshly diluted with DMEM, for 30 minutes. After incubation, the cells were washed three more times with PBS, and fluorescence images were observed and recorded using a confocal laser scanning microscope. The excitation wavelength was 488 nm, and the emission light collection range was 520-590 nm. The fluorescence intensity of the fluorescence images was semi-quantitatively analyzed using ImageJ software.

[0057] result( Figure 7-8 This indicates that free vitamin E, vitamin E and NAD + Physical mixtures, and VE-NAD + Both conjugated nanomicelles and free NAD+ effectively inhibit LPS-induced lipid peroxidation and oxidative stress. + No significant antioxidant and anti-lipid peroxidation effects were observed.

[0058] Example 8 This example illustrates how to detect MDA levels, inflammatory cytokine content, and nitric oxide metabolites in RAW264.7 macrophages using colorimetric methods, enzyme-linked immunosorbent assay (ELISA), and Griess reaction to assess vitamin E and NAD+ levels. + The physical mixture of the two and VE-NAD + Anti-inflammatory and antioxidant stress effects of coupled micelles under inflammatory conditions.

[0059] The specific steps are as follows: RAW264.7 macrophages were cultured at a rate of 4 × 10⁻⁶. 6After seeding at a density of 10 cells / dish and pretreating with 1 μg / mL LPS for 2 hours, the cells were replaced with cells containing 1 μg / mL LPS and 40 μM of different drugs (free vitamin E, free NAD). + VE and NAD + Physical mixtures or VE-NAD + The cells were cultured in fresh culture medium containing conjugated nanomicelles for 24 hours. Intracellular MDA content was determined using a thiobarbituric acid (TBA)-based colorimetric method: After drug treatment, cells were collected and lysed, and the supernatant was obtained by centrifugation. The cell lysate supernatant was mixed with TBA detection solution, heated at 100°C for 15 minutes, cooled, and then incubated at 5,000 mL / min. g Centrifuge for 10 minutes, collect the supernatant, and measure the absorbance at 532 nm. Calculate the MDA content based on the standard curve, and finally normalize using the total protein concentration determined by the Bradford method. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of intracellular inflammatory factors: After drug treatment, cells were collected and lysed, centrifuged to obtain the supernatant, and the TNF-α content in the cell lysate was measured using a mouse tumor necrosis factor-α (TNF-α) specific ELISA kit, following the instructions. The TNF-α content was then normalized using the total protein concentration. The Griess reaction method was used to determine the nitrite (NO2) content in the supernatant of RAW264.7 macrophage culture. - To assess NO levels: After cell treatment with the drug, the culture medium was collected, and the supernatant was obtained by centrifugation. A commercial nitrate / nitrite assay kit was used to plot a standard curve with sodium nitrite standards (0-100 μM). 50 μL of sample or standard was added to each well of a 96-well plate, followed by 50 μL of Griess reagent I and 50 μL of Griess reagent II. After incubation at room temperature for 10 minutes, the absorbance was measured at 540 nm. The nitrite concentration was calculated based on the standard curve and normalized to the total protein content of the corresponding cell lysate.

[0060] result( Figure 9 This indicates that VE-NAD + The conjugated nanomicelles can effectively reduce key inflammatory and lipid peroxidation markers in LPS-induced RAW264.7 macrophages.

[0061] Example 9 This example illustrates how to assess the degree of cellular lipid peroxidation damage and endogenous antioxidant defense capacity by detecting the levels of MDA, 4-hydroxynonenal (4-HNE), glutathione (GSH), and thioredoxin (Trx) in primary rat chondrocytes, in order to evaluate vitamin E and NAD. + The physical mixture of the two and VE-NAD+ Antioxidant stress effect of conjugated nanomicelles under inflammatory conditions.

[0062] The specific procedure is as follows: Primary chondrocytes are cultured at a concentration of 2×10⁻⁶. 6 Cells were seeded at a density of 10 cells / dish and cultured for 24 hours. Then, they were pretreated with IL-1β (10 ng / mL) for 2 hours to induce inflammation. Subsequently, the solutions were replaced with different drugs (free vitamin E, free NAD) containing 10 ng / mL IL-1β and 40 μM of each. + VE and NAD + Physical mixtures or VE-NAD + The cells were cultured in fresh medium containing conjugated nanomicelles for 24 hours. Intracellular MDA content was determined using a thiobarbituric acid (TBA)-based colorimetric method, and 4-HNE levels were detected using ELISA (procedure as before). GSH and Trx are endogenous cellular antioxidants that neutralize reactive oxygen species and protect chondrocytes from oxidative damage; higher antioxidant levels reflect enhanced cellular antioxidant capacity. Intracellular GSH levels were determined using a colorimetric glutathione assay kit: After drug treatment, cells were washed with PBS, and 400 μL of pre-chilled lysis reagent (Reagent 1) was added to each sample for cell lysis on ice. The lysis buffer was centrifuged (8,000 rpm). g After 10 minutes, the supernatant was collected. 20 μL of GSH standard solution (0-100 μg / mL) or sample was added sequentially to a 96-well plate, followed by 140 μL of Reagent2 and 40 μL of Reagent3. After incubation at room temperature for 2 minutes, the absorbance was measured at 412 nm. The GSH concentration was calculated based on the standard curve and normalized using the total protein content determined by the BCA method. The thioredoxin content in chondrocyte lysate was determined using the 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) method. First, a 10 mg / mL GSH stock solution was serially diluted with ultrapure water to prepare 0-100 μg / mL GSH standard solutions for plotting a standard curve. Cells were lysed on ice for 40 minutes with Western lysis / IP lysis buffer, and the lysis buffer was incubated at 10,000 mL. g Centrifuge for 10 minutes and collect the supernatant. Then, ultracentrifuge the collected supernatant (molecular weight cutoff: 3000 Da, 4000 Da). gAfter 30 minutes, the concentrated lysate was analyzed, and the total protein concentration was determined using the BCA method. 10 μL of sample or standard was mixed with 90 μL of DTNB solution (1 mM, dissolved in 50 mM Tris-HCl buffer containing 6 M guanidine hydrochloride, pH 8.0). After incubation at room temperature for 5 minutes, the absorbance was measured at 412 nm using a microplate reader. The thioredoxin concentration was calculated based on the GSH standard curve and normalized to the total protein content.

[0063] result( Figure 10 This indicates that VE-NAD + The conjugated nanomicelles effectively reduced key lipid peroxidation markers (MDA and 4-HNE) in LPS-induced rat primary chondrocytes and significantly increased the antioxidant coenzymes GSH and Trx. Furthermore, VE-NAD... + The efficacy of conjugated nanomicelles is significantly better than that of vitamin E and NAD. + A physical mixture.

[0064] Example 10 This example illustrates how to use ELISA to detect the expression levels of a series of key inflammatory factors, enzymes, and mediators in primary chondrocytes to systematically assess VE and NAD. + The physical mixture of the two and VE-NAD + The anti-inflammatory effects of conjugated nanomicelles and their influence on cartilage degeneration-related pathways.

[0065] The specific steps are as follows: Primary chondrocytes are cultured at 2×10⁻⁶... 6Cells were seeded at a density of 10 cells / dish and stimulated with 10 ng / mL IL-1β for 2 hours to induce inflammation. They were then treated with fresh culture medium containing 10 ng / mL IL-1β and 40 μM of different compounds for 24 hours. After treatment, cell lysates were prepared, and total protein concentration was determined using the BCA method. Using a rat-specific ELISA kit, the levels of pro-inflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), anti-inflammatory factors interleukin-4 (IL-4), key cartilage degradation enzymes matrix metalloproteinase-3 (MMP-3) and MMP-13, chemokine monocyte chemoattractant protein-1 (MCP-1), key inflammatory inducible enzymes inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and their downstream inflammatory mediator prostaglandin E2 (PGE2) were measured sequentially in the cell lysates. During the assay, standards and samples were added to antibody-pre-coated 96-well plates, followed by the sequential addition of biotinylated detection antibody, streptavidin-horseradish peroxidase, and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution for incubation. After the reaction, sulfuric acid stop solution was added, and absorbance was measured at 450 nm. The concentrations of each indicator were calculated based on the standard curve and normalized to the total protein concentration.

[0066] result( Figure 11 This indicates that VE-NAD + The conjugated nanomicelles effectively reduced key pro-inflammatory cytokines induced by IL-1β in primary rat chondrocytes, increased anti-inflammatory cytokines, and decreased matrix metalloproteinases (MMP-3 and MMP-13). Furthermore, VE-NAD... + The efficacy of conjugated nanomicelles is significantly better than that of vitamin E and NAD. + A physical mixture.

[0067] Example 11 This example illustrates how to construct and evaluate a spontaneous osteoarthritis (OA) model using premature aging SAMP8 mice, and how to evaluate VE-NAD through intra-articular injection. + The therapeutic effect of coupled micelles on OA progression.

[0068] The specific steps are as follows: All animal experimental procedures were approved by the Tianjin Municipal Committee for the Use and Management of Laboratory Animals. The efficacy was evaluated using 14-week-old male premature aging SAMP8 mice (this strain spontaneously develops osteoarthritis-like pathological changes with age and is widely used as an age-related OA model). The 14-week-old SAMP8 mice were randomly divided into five groups (n=3 per group): saline group, free VE group (vitamin E polyethylene glycol succinate), free NAD+ group, and free NAD+ group. + Groups, VE and NAD +Physical mixture group and VE-NAD + Conjugate nanomicelles were used. All groups underwent intra-articular injections of 10 μL per joint (5.0 mg / kg) into the bilateral hind limb knee joints weekly for four consecutive weeks. At the end of the experiment, mice were euthanized, the knee joints were carefully dissected, and muscles and surrounding soft tissues were removed, preserving the joint structure intact for subsequent histological and molecular biological analysis.

[0069] Hematoxylin-eosin staining was used to assess the overall histological structure and cell morphology of the knee joint. The specific steps were as follows: Mouse knee joints were rinsed with sterile PBS, fixed in 4% paraformaldehyde for 48 hours, and then immersed in 10% EDTA decalcification solution, with the decalcification solution changed every other day for 4 weeks. The decalcified tissue was dehydrated with a gradient of ethanol (70% to 100%), embedded in paraffin, and sectioned serially along the sagittal plane to a thickness of 5 µm. The sections were baked at 65°C for 2 hours, dewaxed in xylene, and then rehydrated with a gradient of ethanol. For staining, the sections were first stained with hematoxylin for 2 minutes, differentiated with 1% acid ethanol, and then stained with eosin for 2 minutes. Finally, the sections were dehydrated with a gradient of ethanol, cleared with xylene, mounted with neutral resin, and scanned using a digital slide scanner. The histopathological scoring of synovitis severity (based on HE-stained sections) referenced the internationally accepted histopathological scoring system for synovitis, and was semi-quantitatively assessed based on the following three pathological features of the synovial tissue in HE-stained sections: degree of synovial lining cell proliferation: assessing the number of cell layers in the synovial lining; density of inflammatory cell infiltration: assessing the degree of infiltration of inflammatory cells such as lymphocytes and plasma cells in the synovial interstitium; degree of synovial fibrosis / angiogenesis: assessing the fibrosis and neovascularization of the synovial interstitium. Each feature was scored from 0 to 3 points according to severity (0: normal; 1: mild; 2: moderate; 3: severe). The sum of the three scores was the total synovitis score for the sample (total score range 0-9 points). Three researchers unaware of the experimental grouping independently observed and scored the sections.

[0070] A modified Safranin O / Fix Green staining kit was used to assess cartilage matrix content and structural integrity through specific staining of proteoglycans. The specific steps were as follows: After dewaxing and rehydration, knee joint sections were first stained with Weigert hematoxylin for 5 minutes, rinsed three times with distilled water (5 minutes each time), then differentiated with acidic differentiation solution for 15 seconds, and washed with distilled water for 10 minutes. Subsequently, the sections were immersed in Fast Green staining solution for 5 minutes, quickly rinsed with a weak acid solution for 15 seconds to remove residual dye, and air-dried at room temperature. The sections were then immersed in Safranin O staining solution for 15 minutes, rinsed with anhydrous ethanol to remove excess dye. Finally, the sections were dehydrated with graded ethanol, cleared with xylene, mounted with neutral resin, and scanned in their entirety for subsequent digital analysis.

[0071] The expression and distribution of proteoglycans (ACAN), type II collagen (ColII), and matrix metalloproteinase-3 (MMP-3) in cartilage tissue were assessed by immunohistochemical staining. The specific steps were as follows: Paraffin-embedded sections of the knee joint were dewaxed and hydrated. Antigen retrieval was performed using a pepsin-based antigen retrieval kit. The sections were incubated in pepsin antigen retrieval solution at 37°C for 15 minutes, followed by washing three times with PBS (5 minutes each time). Endogenous peroxidase activity was quenched by treatment with 3% H2O2 for 10 minutes, followed by washing again with PBS (three times, 5 minutes each time). Non-specific binding sites were blocked with 10% goat serum at room temperature for 1 hour. Subsequently, the sections were incubated overnight at 4°C and reacted with mouse-derived monoclonal primary antibodies (1:200 dilution) to label key synthetic components of the cartilage matrix (ACAN, ColII) and key cartilage degradation enzyme (MMP-3). After thorough washing with PBS, the sections were incubated with horseradish peroxidase-labeled secondary antibody at room temperature for 1 hour. 3,3′-Diaminobenzidine (DAB) was used as the chromogenic substrate for signal development, followed by counterstaining of cell nuclei with hematoxylin for 1 minute and washing with PBS. After dehydration, clearing, and mounting, the sections were scanned in their entirety, and semi-quantitative analysis of the positively stained areas was performed using ImageJ software.

[0072] Histological staining and results ( Figure 12-13 This indicates that VE-NAD + The conjugate nanomicelles can effectively inhibit cartilage damage, increase the content of proteoglycans and type II collagen in cartilage tissue, and significantly reduce the concentration of matrix metalloproteinase-3, demonstrating excellent OA treatment effects.

[0073] Therefore, the amphiphilic nicotinamide adenine dinucleotide conjugate in this invention consists of three modules: a hydrophobic antioxidant molecule, a responsive linker, and a hydrophilic nicotinamide adenine dinucleotide, which are covalently linked. The responsive linker enables the conjugate to release the hydrophobic antioxidant molecule and nicotinamide adenine dinucleotide in situ under the stimulation of free radicals in the disease microenvironment, such as inflammation, thereby exerting their respective pharmacological effects. The conjugate and its self-assembled nanomicelles have functions such as reducing oxidative stress, inhibiting inflammation, promoting DNA repair, maintaining cellular homeostasis, and delaying aging. Due to the hydrophilicity and negative charge of nicotinamide adenine dinucleotide, its cellular uptake is weak. The conjugate nanomicelles can increase the cellular uptake of nicotinamide adenine dinucleotide through endocytosis. The nanomicelles can simultaneously inhibit inflammation and oxidative stress and can physically encapsulate hydrophobic active drugs, showing broad application prospects in inflammatory diseases, age-related diseases, organ damage, and the prevention and treatment of toxic side effects of chemotherapy drugs.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. An amphiphilic nicotinamide adenine dinucleotide conjugate, characterized in that: It has the following general structural formula: ； Among them, NAD + R is a hydrophilic group, R is a hydrophobic antioxidant group, and the phenylboronic acid derivative is a linking bond; R is selected from free radical scavengers, small molecule organoselenic compounds that mimic the function of glutathione peroxidase 4 (GPX4), iron chelators, 7-dehydrocholesterol, royal jelly acid (10-hydroxy-2-decenoic acid), α-lipoic acid, oleic acid, or derivatives of the above molecules.

2. The amphiphilic nicotinamide adenine dinucleotide conjugate according to claim 1, characterized in that: Among the hydrophobic antioxidant groups, the free radical scavengers are vitamin E / VE, Ferrostatin-1 / Fer-1, idebenone, SRS11-92 / AA9, SRS16-86, Liproxstatin-1, phenoxazine / Pnx, phenothiazine or their derivatives; Small molecule organic selenium compounds that mimic the function of glutathione peroxidase 4 are ebuselenline / Ebs or their derivatives. The iron chelating agent is deferoxone, deferoxamine, or derafloxacin or its derivatives.

3. The amphiphilic nicotinamide adenine dinucleotide conjugate according to claim 2, characterized in that: When the hydrophobic antioxidant group is vitamin E, the structural formula of the amphiphilic nicotinamide adenine dinucleotide conjugate is: ； When the hydrophobic antioxidant group is Ferrostatin-1 / Fer-1, the structural formula of the amphiphilic nicotinamide adenine dinucleotide conjugate is: ； When the hydrophobic antioxidant group is idebenone, the structural formula of the amphiphilic nicotinamide adenine dinucleotide conjugate is: 。 4. A nanomicelle, characterized in that: The amphiphilic nicotinamide adenine dinucleotide conjugate according to any one of claims 1-3 self-assembles into the nanomicelles, or the amphiphilic nicotinamide adenine dinucleotide conjugate according to any one of claims 1-3 co-assembles with other biocompatible amphiphilic molecules into the nanomicelles.

5. The nanomicelles according to claim 4, characterized in that: Other biocompatible amphiphilic molecules include small molecule surfactants, high molecule surfactants, phospholipids, and amphiphilic polyethylene glycol (PEG) derivatives.

6. The nanomicelles according to claim 5, characterized in that: The amphiphilic polyethylene glycol derivatives include phospholipid-polyethylene glycol, with a polyethylene glycol molecular weight of 400-3000 Da and PEG end groups of carboxyl, amino, thiol, maleimide, or succinimide.

7. The nanomicelles according to claim 6, characterized in that: The PEG end group is covalently linked to the target molecule, which includes small molecules, monosaccharides, peptides, antibodies, and nanobodies, thus endowing the nanomicelles with active targeting properties.

8. The use of the amphiphilic nicotinamide adenine dinucleotide conjugate according to any one of claims 1-3 or the nanomicelles according to any one of claims 4-7 in the preparation of medicaments for treating inflammatory and / or oxidative stress-related diseases, characterized in that, Inflammation and / or oxidative stress-related diseases include: ① Orthopedic diseases: osteoarthritis, rheumatoid arthritis, osteoporosis; ② Ophthalmic diseases: Age-related macular degeneration / AMD, dry eye syndrome, guttate keratosis / Fuchs' endothelial dystrophy, neurotrophic keratitis; ③ Cardiovascular diseases: anthracycline-induced cardiotoxicity, ischemic heart disease, atherosclerosis, and myocardial infarction; ④ Kidney diseases: kidney injury induced by platinum-based drugs, acute kidney injury, chronic kidney disease, polycystic kidney disease, and diabetic nephropathy; ⑤ Liver diseases: drug-induced liver injury, acute liver failure, autoimmune liver disease, alcoholic liver disease, non-alcoholic fatty liver disease, liver fibrosis; ⑥ Neurological diseases: Parkinson's disease, Alzheimer's disease, ischemic stroke, traumatic brain injury, epilepsy, amyotrophic lateral sclerosis, Huntington's disease; ⑦ Lung diseases: chronic obstructive pulmonary disease, asthma, acute lung injury, pulmonary fibrosis; ⑧ Organ damage caused by ischemia-reperfusion, multiple organ dysfunction, sepsis, uncontrollable inflammation, neuropathic pain caused by chemotherapy, tissue and organ damage caused by radiotherapy, antiviral infection, cerebral infarction, iron overload disease, and biliary tract disease.

9. The application according to claim 8, characterized in that: In application, nanomicelles encapsulate active drug molecules, including anthracycline drugs, platinum-based drugs, and active molecules for treating neurodegenerative diseases, ophthalmic diseases, and orthopedic diseases. The routes of administration include oral administration, intravenous administration, nasal administration, pulmonary administration, intra-articular injection, transdermal administration, ocular administration, intraperitoneal administration, and mucosal administration.

10. A drug delivery system for treating inflammation and / or oxidative stress-related diseases, characterized in that: The drug delivery system comprises an amphiphilic nicotinamide adenine dinucleotide conjugate as described in any one of claims 1-3 or nanomicelles as described in any one of claims 4-7 encapsulating active drug molecules; The inflammation and / or oxidative stress-related diseases include: ① Orthopedic diseases: osteoarthritis, rheumatoid arthritis, osteoporosis; ② Ophthalmic diseases: Age-related macular degeneration / AMD, dry eye syndrome, guttate keratosis / Fuchs' endothelial dystrophy, neurotrophic keratitis; ③ Cardiovascular diseases: anthracycline-induced cardiotoxicity, ischemic heart disease, atherosclerosis, and myocardial infarction; ④ Kidney diseases: kidney injury induced by platinum-based drugs, acute kidney injury, chronic kidney disease, polycystic kidney disease, and diabetic nephropathy; ⑤ Liver diseases: drug-induced liver injury, acute liver failure, autoimmune liver disease, alcoholic liver disease, non-alcoholic fatty liver disease, liver fibrosis; ⑥ Neurological diseases: Parkinson's disease, Alzheimer's disease, ischemic stroke, traumatic brain injury, epilepsy, amyotrophic lateral sclerosis, Huntington's disease; ⑦ Lung diseases: chronic obstructive pulmonary disease, asthma, acute lung injury, pulmonary fibrosis; ⑧ Organ damage caused by ischemia-reperfusion, multiple organ dysfunction, sepsis, uncontrollable inflammation, neuropathic pain caused by chemotherapy, tissue and organ damage caused by radiotherapy, antiviral infection, cerebral infarction, iron overload disease, and biliary tract disease.

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