Use of lipoic acid nanoparticles in the preparation of drugs for the treatment of oxidative stress-related diseases

Abstract

The application discloses application of lipoic acid nanoparticles in preparation of an oxidative stress related disease treatment drug, and belongs to the technical field of biological medicines. The oxidative stress related disease treatment drug adopts lipoic acid nanoparticles as an active ingredient, and can treat one or more diseases by resisting oxidative stress. The treatment effect of the lipoic acid nanoparticles is obviously superior to that of lipoic acid monomers, and reaches or is superior to the treatment effect of a corresponding positive drug, thereby effectively overcoming the defect of poor treatment effect of an existing natural small molecule antioxidant on oxidative stress related diseases, achieving good treatment effect under the condition of a low dose, and having a clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nanomedicine for treating oxidative stress-related diseases. Technical Background

[0002] Reactive oxygen species (ROS) are a class of single-electron reduction products of oxygen in the body, including hydrogen peroxide (H₂O₂), hydroxyl radicals (·OH), and superoxide anions (O₂). ·- Under normal conditions, reactive oxygen species (ROS) produced by phagocytes such as macrophages and neutrophils exert their physiological functions by killing invading pathogens. However, excessive ROS production can cause oxidative stress, leading to tissue and cellular dysfunction and accelerating the development of many diseases, such as neurological diseases (ischemic stroke, Parkinson's disease, Alzheimer's disease, epilepsy, cerebrovascular disease, meningitis, and diseases of nerve roots / plexuses or peripheral nerves), circulatory system diseases (atherosclerosis, hypertension, endocarditis, pericarditis, cardiomyopathy, and heart failure), respiratory system diseases (pulmonary fibrosis, nasopharyngitis, bronchitis, and asthma), and genitourinary system diseases (premature ovarian failure, cervicitis, pelvic inflammatory disease, vaginitis, nephropathy, cystitis, and urethritis). Immune system diseases (lupus and vasculitis), visual system diseases (cataracts, conjunctivitis, keratitis, retinopathy, and glaucoma), digestive system diseases (non-alcoholic fatty liver disease, liver fibrosis, cirrhosis, liver failure, periodontitis, duodenal ulcers, cholecystitis, pancreatitis, peritonitis, and inflammatory bowel disease), endocrine disorders (diabetes), skin diseases (psoriasis, melasma, vitiligo, and herpes), musculoskeletal or connective tissue diseases (osteoarthritis, synovitis, tenosynovitis, and synovitis), and infectious diseases (colitis, gastritis, HIV / AIDS, viral hepatitis, and sepsis). Antioxidants are considered a reasonable strategy for treating oxidative stress-related diseases because they can counteract oxidative damage. Although natural antioxidants can reduce reactive oxygen species levels in tissues and cells to some extent, they can only function as health supplements and are far from meeting the requirements for clinical pharmaceutical treatment. To date, no antioxidant drugs have been found that meet the clinical requirements for treating oxidative stress-related diseases. Summary of the Invention

[0003] To address the above problems, this invention provides a nanomedicine for treating oxidative stress-related diseases, which exhibits excellent therapeutic effects and has promising clinical application prospects.

[0004] This invention includes the following technical solutions:

[0005] A nanomedicine for treating oxidative stress-related diseases, wherein the nanomedicine uses lipoic acid nanoparticles as the active ingredient, overcoming the drawback of poor therapeutic efficacy of lipoic acid monomers. The lipoic acid nanoparticles of this invention can slowly dissociate and release the active ingredient lipoic acid, exhibiting a strong antioxidant effect at significantly lower doses compared to lipoic acid monomers, thus meeting the clinical application needs for oxidative stress-related diseases.

[0006] Alternatively, the lipoic acid nanoparticles are nanoparticles formed from raw materials containing lipoic acid and / or lipoic acid derivatives, and the formation process may include various modifications or alterations to the nanoparticles. The lipoic acid derivatives include lipoic acid salts or pharmaceutically acceptable modifiers of lipoic acid obtained by non-substantial modifications that do not affect its core function (including, but not limited to, grafting functional groups onto the lipoic acid molecule).

[0007] Alternatively, the oxidative stress-related diseases may include: neurological diseases (ischemic stroke, Parkinson's disease, Alzheimer's disease, epilepsy, cerebrovascular disease, meningitis, and nerve root / plexus or peripheral nerve diseases); circulatory system diseases (atherosclerosis, hypertension, endocarditis, pericarditis, cardiomyopathy, and heart failure); respiratory system diseases (pulmonary fibrosis, nasopharyngitis, bronchitis, and asthma); genitourinary system diseases (premature ovarian failure, kidney disease, cervicitis, pelvic inflammatory disease, vaginitis, cystitis, and urethritis); and immune system diseases (lupus and...). Vasculitis), visual system diseases (cataracts, conjunctivitis, keratitis, retinopathy, and glaucoma), digestive system diseases (non-alcoholic fatty liver disease, liver fibrosis, cirrhosis, liver failure, periodontitis, duodenal ulcers, cholecystitis, pancreatitis, peritonitis, and inflammatory bowel disease), endocrine disorders (diabetes), skin diseases (psoriasis, melasma, vitiligo, and herpes), musculoskeletal or connective tissue diseases (osteoarthritis, synovitis, tenosynovitis, and synovitis), and infectious diseases (colitis, gastritis, AIDS, viral hepatitis, and sepsis).

[0008] Alternatively, the oxidative stress-related diseases may be diabetes and its complications. These complications include diabetic cataracts, diabetic nephropathy, diabetic peripheral neuropathy, diabetic foot, and diabetic retinopathy.

[0009] Alternatively, the nanomedicine for treating oxidative stress-related diseases may be an injectable or oral formulation.

[0010] Alternatively, in the aforementioned nanomedicines for treating oxidative stress-related diseases, the disulfide bonds in lipoic acid and / or lipoic acid derivatives are first broken, followed by self-crosslinking. The disulfide bonds in the five-membered rings of some lipoic acid and / or lipoic acid derivatives are broken, and the thiol groups between lipoic acid and / or lipoic acid derivative molecules crosslink with each other. Stable crosslinking can be achieved without introducing additional crosslinking molecules, resulting in a single, controllable drug component. The crosslinked nanomedicine structure is stable, which is beneficial for long-term circulation in the blood. In contrast, uncrosslinked lipoic acid nanoparticles will rapidly dissociate in vivo, causing premature release of the encapsulated drug and thus affecting efficacy.

[0011] Alternatively, the nanomedicine for treating oxidative stress-related diseases is a micelle, vesicle, or aggregate formed by the self-assembly of lipoic acid and / or lipoic acid derivatives. Lipoic acid and / or lipoic acid derivatives can form self-assemblies through hydrophilic-hydrophobic interactions, resulting in a simple preparation process and stable product structure.

[0012] As an alternative, in the above-mentioned nanomedicines for treating oxidative stress-related diseases, the particle size of the nanomedicine is 1-1000 nm, preferably 10-200 nm. Nanoparticles smaller than 10 nm are easily cleared by the kidneys, while nanoparticles larger than 200 nm tend to accumulate in the liver and spleen and pose a risk of activating the complement system.

[0013] Alternatively, in the aforementioned nanomedicines for treating oxidative stress-related diseases, the nanomedicine has a negative surface potential, which helps reduce binding to opsonins and enhance long-term circulation.

[0014] Alternatively, in the above-mentioned nanomedicines for treating oxidative stress-related diseases, the nanomedicines may also encapsulate other active pharmaceutical ingredients that have therapeutic effects on the above diseases.

[0015] Alternatively, in the above-mentioned nanomedicines for treating oxidative stress-related diseases, the nanomedicine is a lipoic acid nanovesicle loaded with vitamin C.

[0016] This invention also provides a method for preparing the above-mentioned nanomedicine for treating oxidative stress-related diseases, specifically including the following steps:

[0017] (1) A superamphilic molecular solution was prepared by mixing triazanonane and thioctic acid in an organic solvent;

[0018] (2) Under ultrasonic conditions, the solution in (1) was added dropwise to deionized water to form uncrosslinked lipoic acid nanovesicles constructed from lipoic acid and triazanonane;

[0019] (3) Crosslinked thioctic acid nanovesicles were obtained by irradiating them with ultraviolet light and removing triazanonane.

[0020] This invention also provides a method for preparing the above-mentioned nanomedicine for treating oxidative stress-related diseases, specifically including the following steps:

[0021] (1) N,N-dimethylformamide was used to dissolve lipoic acid to obtain lipoic acid mother liquor;

[0022] (2) The lipoic acid mother liquor in (1) was subjected to photocrosslinking to obtain lipoic acid nano-aggregates.

[0023] This invention also provides a method for preparing the above-mentioned nanomedicine for treating oxidative stress-related diseases, specifically including the following steps:

[0024] (1) Add lipoic acid to an alkaline aqueous solution, then adjust the pH to neutral with an acid solution, and ultrasonically emulsify to form lipoic acid nanoparticles.

[0025] (2) The nanoparticle solution obtained above was subjected to photocrosslinking to obtain thioctic acid nanomicelles.

[0026] As an alternative, in the above preparation method, during the photo-crosslinking process, ultraviolet light is used to break a portion of the disulfide bonds in the sodium thiooctanoate five-membered ring and induce partial self-crosslinking of the thiol groups formed after the disulfide bonds are broken.

[0027] Alternatively, in the above preparation method, other active pharmaceutical ingredients can be encapsulated in thioctic acid nanoparticles.

[0028] The present invention also provides the application of the above-mentioned thioctic acid nanoparticles in the preparation of drugs for the treatment of oxidative stress diseases.

[0029] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0030] The beneficial effects of this invention are:

[0031] The nanomedicine described in this invention effectively overcomes the shortcomings of existing natural small-molecule antioxidants in treating oxidative stress-related diseases, exhibiting strong antioxidant effects at significantly lower doses. It effectively treats diseases of the nervous system, circulatory system, respiratory system, genitourinary system, immune system, visual system, digestive system, endocrine system, musculoskeletal system, connective tissue, and infectious diseases. Specifically, the therapeutic effect of the nanomedicine is approximately 1.5-3 times that of lipoic acid monomers, and reaches or exceeds that of corresponding positive control drugs. Furthermore, when the nanomedicine is used in combination with other active ingredients of drugs for oxidative stress-related diseases, it has a synergistic effect of "1+1>2", further enhancing the therapeutic effect. Attached image description:

[0032] Figure 1 A schematic diagram illustrating the use of lipoic acid nanoparticles for the treatment of oxidative stress-related diseases;

[0033] Figure 2 The particle size of different nanoparticles in Example 1, and the stability of cross-linked and uncross-linked lipoic acid nanovesicles;

[0034] Figure 3 The particle size of the nanoparticles loaded with different antioxidants in Example 2, and the potential of the nanovesicles loaded with or without vitamin C lipoic acid;

[0035] Figure 4 The antioxidant results of different lipoic acid nanoparticles in Example 3;

[0036] Figure 5 The results of scavenging 1,1-diphenyl-2-picrylhydrazine, superoxide anion, hydrogen peroxide and hydroxyl radicals by lipoic acid nanovesicles and vitamin C-loaded lipoic acid nanovesicles in Example 4;

[0037] Figure 6 The results are experimental findings on the in vitro antioxidant activity of lipoic acid monomer, lipoic acid nanovesicles, and vitamin C-loaded lipoic acid nanovesicles in Example 5.

[0038] Figure 7 The results of the experiment in Example 6 on the inhibition of the lipid peroxide malondialdehyde by lipoic acid monomer, lipoic acid nanovesicles and vitamin C-loaded lipoic acid nanovesicles.

[0039] Figure 8 The experimental results of lipoic acid monomer, lipoic acid nanovesicles and vitamin-loaded lipoic acid nanovesicles inhibiting inflammatory factors at the cellular level in Example 7;

[0040] Figure 9 The in vivo results of the treatment of atherosclerosis with lipoic acid monomer, lipoic acid nanovesicles, lipoic acid monomer + vitamin C and vitamin C-loaded lipoic acid nanovesicles in Example 8 are shown.

[0041] Figure 10 The in vivo results of the treatment of ischemic stroke with lipoic acid monomer and lipoic acid nanovesicles in Example 9;

[0042] Figure 11 The in vivo results of lipoic acid monomer and lipoic acid nanovesicles in treating pulmonary fibrosis in Example 10;

[0043] Figure 12 The in vivo results of the treatment of non-alcoholic fatty liver disease with lipoic acid monomer and lipoic acid nanovesicles in Example 11;

[0044] Figure 13 The in vivo results of the treatment of premature ovarian failure with lipoic acid monomer and lipoic acid nanovesicles in Example 12;

[0045] Figure 14 A schematic diagram illustrating the mechanism of lipoic acid nanoparticles in treating atherosclerosis. Detailed implementation method:

[0046] The following detailed description of specific embodiments further illustrates the above-described content of the present invention. However, this should not be construed as limiting the scope of the subject matter of the present invention to the following examples. Any modifications made without departing from the spirit and principles of the present invention, as well as equivalent substitutions or improvements made based on ordinary technical knowledge and common practice in the art, should be included within the scope of protection of the present invention.

[0047] Example 1: Preparation of different lipoic acid nanoparticles

[0048] Preparation of thioctic acid nanomicelles

[0049] 300 mg of lipoic acid (LA) was added to 150 mL of deionized water. Under stirring, 1 M NaOH aqueous solution was added dropwise until LA was completely dissolved. The LA solution was then titrated with 1 M HCl solution until neutral. The solution was freeze-dried to obtain sodium lipoate powder. 41.2 mg of sodium lipoate was weighed and dissolved in 1 mL of deionized water, then ultrasonically emulsified to form lipoic acid nanoparticles. The obtained nanoparticles were subjected to 365 nm ultraviolet light to induce self-crosslinking of the disulfide bonds in lipoic acid. After 2.5 h of reaction and dialysis for 48 h, crosslinked lipoic acid nanomicelles with a size of approximately 15 nm were obtained. The results are as follows: Figure 2 As shown in a.

[0050] Preparation of lipoic acid nanovesicles

[0051] 210 mg of lipoic acid (LA) and 43.5 mg of the template molecule 1,4,7-triazanonane were dissolved in 3.5 mL of dimethyl sulfoxide (DMSO) and shaken for 4 h to form a superamphilic solution. Under ultrasonic conditions, the superamphilic solution was slowly added dropwise to 300 mL of deionized water to form uncrosslinked lipoic acid nanovesicles constructed from LA and 1,4,7-triazanonane, with a particle size of [missing information]. Figure 2 As shown in Figure a. The uncrosslinked lipoic acid nanovesicles were irradiated with 365 nm UV light for 4 h to induce self-crosslinking of the disulfide bonds in lipoic acid. The pH was adjusted to approximately 9.0 with NaOH, and the solution was extracted three times with dichloromethane to remove 1,4,7-triazanonane. The supernatant was then adjusted to neutral with dilute HCl and dialyzed against deionized water for 48 h (spectrum / pore, MWCO 2000) to prepare crosslinked lipoic acid nanovesicles with a particle size of approximately 130 nm. The results are shown in Figure a. Figure 2 As shown in a.

[0052] Stability test: 4.5 mL of the above-mentioned cross-linked lipoic acid nanovesicles and uncross-linked lipoic acid nanovesicles were incubated with 0.5 mL of fetal bovine serum for 2 h, and the particle size changes before and after incubation were measured. The results are as follows: Figure 2 As shown in b, the size of cross-linked nanovesicles remained unchanged at around 130 nm, while the size of uncross-linked nanovesicles decreased from 100 nm to only tens of nanometers, indicating that cross-linked lipoic acid nanovesicles are more stable than uncross-linked lipoic acid nanovesicles.

[0053] Preparation of lipoic acid nanoaggregates

[0054] 41.2 mg of lipoic acid was dissolved in 1 mL of DMF and shaken on a shaker for 2 h to obtain a 0.2 M lipoic acid stock solution. 50 μL of this stock solution was added to 5 mL of deionized water under ultrasonic conditions to obtain lipoic acid nanoparticles. The obtained nanoparticles were irradiated with 365 nm UV light for 2.5 h to induce self-crosslinking of the disulfide bonds in lipoic acid. After dialyzing for 48 h, crosslinked lipoic acid aggregates with a size of approximately 80 nm were obtained. The results are as follows. Figure 2 As shown in a.

[0055] Example 2: Preparation of lipoic acid nanoparticles loaded with different antioxidants

[0056] Preparation of hydrophilic antioxidant vitamin C lipoic acid nanovesicles

[0057] 10g of vitamin C (VC) was dissolved in 50mL of lipoic acid empty vesicle solution. After complete dissolution, the solution was allowed to stand at 4℃ for 13h, and then dialyzed in the dark for 16h to prepare vitamin C-loaded lipoic acid nanovesicles (VC@cLAVs). The results are as follows: Figure 3 As shown in a and b, the particle size of VC@cLAVs is approximately 200 nm, and the potential is approximately -6 mV.

[0058] Preparation of lipophilic acid nanomicelles loaded with vitamin E, a lipophilic antioxidant

[0059] 200 mg of sodium lipoate and 50 mg of vitamin E (VE) were dissolved in 5 mL of deionized water and ultrasonically emulsified to form lipoic acid nanoparticles. The obtained nanoparticles were then irradiated with 365 nm ultraviolet light for 2.5 h to induce self-crosslinking of the disulfide bonds in lipoic acid. After dialysis for 48 h, crosslinked lipoic acid nanomicelles with a size of approximately 30 nm were obtained. The results are as follows... Figure 3 As shown in a.

[0060] Preparation of lipoic acid nanoaggregates loaded with the lipophilic antioxidant coenzyme Q10

[0061] 200 mg of lipoic acid and 50 mg of coenzyme Q10 were dissolved in 5 mL of DMF and shaken on a shaker for 2 h to obtain a 0.2 M lipoic acid stock solution. 50 μL of this stock solution was added to 5 mL of deionized water under ultrasonic conditions to obtain lipoic acid nanoparticles. Self-crosslinking of the disulfide bonds in lipoic acid was initiated by irradiation with 365 nm UV light for 2.5 h. After dialysis for 48 h, crosslinked lipoic acid nanoaggregates with a size of approximately 110 nm were obtained. The results are as follows. Figure 3 As shown in a.

[0062] Preparation of atorvastatin-loaded lipoic acid nanovesicles

[0063] 200 mg of lipoic acid nanovesicles and 20 mg of atorvastatin were weighed and dissolved in 50 mL of deionized water. After stirring for 5 h, the mixture was dialyzed for 24 h to prepare lipoic acid nanovesicles loaded with atorvastatin (AVT@cLAVs). The particle size was about 180 nm and the potential was about -15 mV.

[0064] Example 3 Antioxidant activity of different lipoic acid nanoparticles

[0065] Antioxidant activity of different lipoic acid nanoparticles: RAW264.7 cells in logarithmic growth phase were seeded in 96-well plates (clear bottom, black bottom) and cultured for 12 h. The cells were then starved for 18 h. Control group cells received no treatment. The three experimental groups were incubated with 8 μg / mL of the three types of lipoic acid nanoparticles prepared in Example 1 for 6 h. After discarding the culture medium, the model group and experimental groups were treated with serum-free LPS (500 ng / mL) for 12 h. Then, all groups were incubated with serum-free DCFH-DA probe (30 μM) for 30 min. Intracellular ROS were detected using a fluorescence microplate reader; the fluorescence intensity reflected the intensity of the detected ROS. Figure 4As shown in Figure a: Compared with the model group, the fluorescence intensity of the three lipoic acid nanoparticles was significantly reduced (P < 0.01), and the difference in fluorescence intensity among the three nanoparticles was small (P > 0.05), indicating that the three nanoparticles have strong antioxidant activity and there is no significant difference in antioxidant activity among the nanoparticles.

[0066] Antioxidant activity of lipoic acid nanoparticles loaded with different antioxidants: RAW264.7 cells in logarithmic growth phase were seeded in 96-well plates (clear bottom, black bottom) and cultured for 12 h. The cells were then starved for 18 h. Control group cells received no treatment. The three experimental groups were incubated with 20 μg / mL of the three types of lipoic acid nanoparticles loaded with different antioxidants prepared in Example 2 for 6 h. After discarding the culture medium, the model group and experimental groups were treated with serum-free LPS (500 ng / mL) for 12 h. Then, all groups were incubated with serum-free DCFH-DA probe (30 μM) for 30 min. Intracellular ROS were detected using a fluorescence microplate reader. The results are shown below. Figure 4 As shown in b, the fluorescence intensity of the antioxidant-loaded nanoparticles was significantly lower than that of the corresponding unloaded empty nanoparticles (P < 0.05), indicating that the antioxidant capacity of the nanoparticles was further enhanced after being loaded with antioxidants.

[0067] Example 4: The ability of lipoic acid nanovesicles and vitamin C-loaded lipoic acid nanovesicles to scavenge 1,1-diphenyl-2-picrylhydrazine, superoxide anions, hydrogen peroxide, and hydroxyl radicals.

[0068] The ability of lipoic acid nanoparticles to scavenge reactive oxygen species was evaluated by the scavenging rate.

[0069] Clearance rate (%) = (1 - A / A0) × 100

[0070] 1. Scavenging of 1,1-diphenyl-2-picrylhydrazine (DPPH)

[0071] (1) Preparation of DPPH test solution

[0072] Dissolve 10 mg of DPPH in 200 mL of anhydrous ethanol, sonicate for 5 min, and mix thoroughly. Take 1 mL of DPPH solution and measure its UV absorbance (A) at 519 nm. The optimal absorbance (A) is between 1.2 and 1.3.

[0073] (2) Preparation of cLAVs and VC@cLAVs sample solutions

[0074] Sample gradient solutions with concentrations of 0, 5, 10, 15, 20, 25, 30 and 35 μg / mL were prepared using anhydrous ethanol.

[0075] (3) Measurement

[0076] Take 2 mL of the DPPH test solution from (1) and add it to a glass bottle. Add 200 μL of anhydrous ethanol, mix thoroughly, and measure the A value. This A value is A0. Take 2 mL of the DPPH test solution from (1) and add it to 200 μL of the sample gradient solution from (2). Mix well, let stand for 30 min, and then measure the A value. Set up three parallel controls for each concentration. The results are as follows: Figure 5 As shown in Figure a, 35 μg / mL cLAVs can generate approximately 71% DPPH clearance, while 35 μg / mL VC@cLAVs can generate approximately 86% DPPH clearance.

[0077] 2. Superoxide anion (O2) ·- ) clearing

[0078] (1) Preparation of pyrogallol / tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) test solution

[0079] Dissolve 100 mg of pyrogallol in 20 mL of PBS and sonicate to prepare a 5 mg / mL pyrogallol solution; add 1.21 g of Tris to 100 mL of distilled water and adjust the pH to 8.2 with HCl to obtain a Tris-HCl solution.

[0080] (2) Preparation of cLAVs and VC@cLAVs sample solutions

[0081] Sample gradient solutions with concentrations of 0, 5, 10, 15, 20, 25, 30, and 35 μg / mL were prepared using PBS.

[0082] (3) Measurement

[0083] Take 2950 μL of Tris-HCl solution from (1) and preheat it in a 25℃ water bath for 20 min. Add 1 mL of cLAVs and VC@cLAVs from (2) and 50 μL of pyrogallol solution from (1), respectively. Mix well and react at 25℃ for 5 min. Then add 1 mL of 8 mol / L HCl solution to terminate the reaction. Measure the UV absorbance at 325 nm as A. Replace the sample solution with 1 mL of PBS and measure the absorbance as A0. Set up three parallel controls for each concentration. The results are as follows: Figure 5 As shown in b, 35 μg / mL cLAVs produce approximately 79% O2. ·- Scavenging rate: 35 μg / mL VC@cLAVs can generate approximately 90% O2. ·- Clearance rate.

[0084] 3. Removal of hydrogen peroxide (H2O2)

[0085] (1) Preparation of H2O2 test solution

[0086] A 20 mM H2O2 solution was prepared by mixing 50 μL of 30% H2O2 with 25 mL of deionized water.

[0087] (2) Preparation of cLAVs and VC@cLAVs sample solutions

[0088] Sample gradient solutions with concentrations of 0, 5, 10, 15, 20, 25, 30, and 35 μg / mL were prepared using PBS.

[0089] (3) Measurement

[0090] Take 12 mL of H2O2 solution from (1) and mix it evenly with 1 mL of sample gradient solution from (2). Incubate at 37°C on a shaker for 24 h. Then, take 100 μL of the above mixed solution from each group and add it to an EP tube containing 1 mL of pH 4.0 standard solution. Add 20 μL of 3,3',5,5'-tetramethylbenzidine (TMB 5 mg / mL) and 20 μL of horseradish peroxidase (HRP, 0.2 μg / mL). After incubation for 5 min, measure the absorbance at 650 nm as A. Replace the sample solution with 100 μL of PBS and measure the absorbance as A0. Set up three parallel controls for each concentration. The results are as follows: Figure 5 As shown in c, cLAVs at a concentration of 35 μg / mL produced an H2O2 scavenging rate of approximately 68%, while VC@cLAVs at a concentration of 35 μg / mL produced an H2O2 scavenging rate of approximately 84%.

[0091] 4. Scavenging of hydroxyl radicals (·OH)

[0092] (1) Preparation of H2O2 and FeSO4 test solutions

[0093] Take 40 μL of 30% H2O2 into a 100 mL volumetric flask, dilute to volume with distilled water, and prepare an H2O2 solution with a concentration of 4 mmol / L; weigh 13.9 mg of FeSO4 crystals and place them in a 10 mL volumetric flask, dissolve and dilute to volume, and prepare a FeSO4 solution with a concentration of 5 mmol / L.

[0094] (2) Preparation of cLAVs and VC@cLAVs sample solutions

[0095] Sample gradient solutions with concentrations of 0, 5, 10, 15, 20, 25, 30, and 35 μg / mL were prepared using PBS.

[0096] (3) Measurement

[0097] Take 1 mL of each of the H2O2 and FeSO4 solutions from (1) and (2), mix well, let stand for 10 min, add 1 mL of methyl violet solution, incubate for 12 h, and measure the absorbance at 578 nm as A. Replace the sample solution with 1 mL of PBS and measure the absorbance A0. Set up three parallel controls for each concentration. The results are as follows. Figure 5 As shown in d, 35 μg / mL cLAVs produced approximately 65% ​​·OH scavenging, while 35 μg / mL VC@cLAVs produced approximately 83% ·OH scavenging.

[0098] The above results demonstrate that cLAVs can effectively scavenge ROS, and their antioxidant capacity is further enhanced after encapsulation with vitamin C. Comparatively, 0.4 mg / mL vitamin C can scavenge approximately 80-90% of H2O2 (1 mM), while 35 μg / mL cLAVs can scavenge approximately 68% of H2O2 (2 mM), and the scavenging rate reaches 83% (2 mM) after further encapsulation with vitamin C. These results confirm that lipoic acid nanomedicines can efficiently scavenge broad-spectrum ROS, which is beneficial for the treatment of oxidative stress-related diseases.

[0099] Example 5: In vitro antioxidant activity of lipoic acid nanovesicles and vitamin C-loaded lipoic acid nanovesicles

[0100] Mouse macrophages (RAW264.7) in the active logarithmic growth phase were seeded into 6-well plates and cultured for 12 h. Then, 8 μg / mL lipoic acid (LA), 8 μg / mL empty lipoic acid nanovesicles (cLAVs), and 20 μg / mL vitamin C-loaded lipoic acid nanovesicles (VC@cLAVs) were added and incubated for 3 h. Control and model groups were also established. After stimulation with 500 ng / mL LPS for 6 h, 30 μM of the DCFH-DA probe was added, and after incubation for 30 min, the in vitro antioxidant capacity was measured using confocal microscopy.

[0101] Mouse macrophages (RAW264.7) in the active logarithmic growth phase were seeded into 48-well plates and cultured for 12 h. Then, 8 μg / mL LA, 8 μg / mL cLAVs, and 20 μg / mL VC@cLAVs were added and incubated for 3 h, respectively. Control and model groups were set up, with 5 parallel controls for each concentration. Then, 500 ng / mL LPS was added and incubated for 6 h. After adding 30 μM DCFH-DA probe and incubating for 30 min, cells were collected, and their in vitro antioxidant capacity was quantitatively determined by flow cytometry. Results are as follows: Figure 6As shown, the fluorescence intensity of the LA monomer group decreased by 29.9% compared with the model group (P < 0.05), while the fluorescence intensity of the cLAVs group decreased by 60.6% compared with the model group (P < 0.01), which was twice that of the same dose of LA monomer (P < 0.05), indicating that the nanovesicles have a strong antioxidant effect. In addition, the fluorescence intensity of the VC@cLAVs group decreased by 67.7% compared with the model group (P < 0.001), which was 1.2 times that of cLAVs (P < 0.05), indicating that the antioxidant activity of the nanovesicles was further enhanced after loading with vitamin C.

[0102] Example 6: Inhibitory effect of lipoic acid nanovesicles and vitamin C-loaded lipoic acid nanovesicles on the lipid peroxide malondialdehyde (MDA).

[0103] Mouse macrophages (RAW264.7) in the active logarithmic growth phase were seeded into 48-well plates and cultured for 12 h. Then, they were incubated for 3 h with 8 μg / mL lipoic acid (LA), 8 μg / mL lipoic acid nanovesicles (cLAVs), and 20 μg / mL vitamin C-loaded lipoic acid nanovesicles (VC@cLAVs). Control and model groups were set up, with 5 replicates for each concentration. Then, 500 ng / mL LPS was added for stimulation for 12 h. After stimulation, cells were collected, lysed with cell lysis buffer, centrifuged at 15000 rpm for 20 min at 4°C, and the supernatant was collected. MDA production was measured using a malondialdehyde (MDA) detection kit. Results are as follows: Figure 7 As shown, compared with the model group, the MDA in the LA monomer group was reduced by 25.3% (P<0.05), while the MDA in the cLAVs group was reduced by 40.1% (P<0.01), which is 1.6 times that of the same dose of LA monomer (P<0.05); at the same time, the MDA in the VC@cLAVs group was reduced by 62.6% (P<0.001), which is 1.6 times that of cLAVs (P<0.05). These results indicate that nanovesicles significantly inhibit the production of lipid peroxide MDA, and the inhibitory effect is further enhanced after loading with vitamin C.

[0104] Example 7: Anti-inflammatory effects of lipoic acid nanovesicles and vitamin C-loaded lipoic acid nanovesicles

[0105] Mouse macrophages (RAW264.7) in the active logarithmic growth phase were seeded into 24-well plates and cultured for 12 h. Then, 8 μg / mL lipoic acid (LA), 8 μg / mL lipoic acid nanovesicles (cLAVs), and 20 μg / mL vitamin C-loaded lipoic acid nanovesicles (VC@cLAVs) were added and incubated for 3 h. Control and model groups were set up. Inflammatory cytokines in the supernatant, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and the anti-inflammatory factor interleukin-10 (IL-10), were detected using an enzyme-linked immunosorbent assay (ELISA) kit. Results are as follows: Figure 8 As shown: the LPS-induced model group significantly increased the secretion of pro-inflammatory cytokines and inhibited the secretion of anti-inflammatory cytokines; compared with the model group, the LA monomer group showed a decrease of 17.9%, 38.3%, and 26.4% in IL-6, TNF-α, and MCP-1, respectively (P < 0.01), while IL-10 increased by 30.6% (P < 0.01); while the cLAVs group showed a decrease of 26.9%, 32.9%, and 44.0% in IL-6, TNF-α, and MCP-1, respectively, while IL-10 increased by 35.3%, and IL-... 6. The MCP-1 reduction effect was approximately 1.5 times and 1.7 times that of the same dose of LA monomer, respectively (P < 0.05); at the same time, IL-6, TNF-α and MCP-1 in the VC@cLAVs group decreased by 33.2%, 55.9% and 45.7%, respectively, while IL-10 increased by 39.1%. The reduction effects of IL-6 and TNF-α were 1.2 times and 1.7 times that of cLAVs, respectively (P < 0.05). These results indicate that nanovesicles have a strong anti-inflammatory effect, and the anti-inflammatory effect is further enhanced after encapsulation with vitamin C.

[0106] Example 8: Effect of lipoic acid nanovesicles and vitamin C-loaded lipoic acid nanovesicles on reducing atherosclerotic plaques.

[0107] Fifty ApoE animals were fed a high-fat diet for one month. - / - Mice were randomly divided into 5 groups of 10 mice each, and treated with different therapeutic agents for 2 months. The model control group was treated with saline, while the other four groups received intravenous injections of 20 mg / kg lipoic acid monomer (LA), 20 mg / kg lipoic acid nanovesicles (cLAVs), 20 mg / kg lipoic acid monomer (LA) + 30 mg / kg vitamin C (VC), and 50 mg / kg vitamin C-loaded lipoic acid nanovesicles (VC@cLAVs), respectively. All formulations were administered intravenously twice within one week. After reaching the treatment time, ApoE... - / -Mice were euthanized. The aorta was excised and fixed with paraformaldehyde, then longitudinally dissected and stained with 0.3% Oil Red dye. The plaque area was then quantitatively analyzed. Results are as follows: Figure 9 As shown in the ad, there was no significant difference between the LA monomer group and the control group (P>0.05), while the positive area in the cLAVs group was reduced by 59.0% compared to the control group (P<0.01), and the positive area in the VC@cLAVs group was reduced by 73.7% compared to the control group (P<0.001). The aortic root was resected and fixed with paraformaldehyde. After fixation in 10% neutral buffered formalin, paraffin-embedded aortic root sections were prepared, and the plaques were stained with Oil Red. The results are as follows... Figure 9 As shown in c and e, the control group showed a high positive area (~21%) in the aortic root. There was no significant difference between the LA monomer group and the control group (P>0.05), while the cLAVs group showed a 46.2% reduction in positive area compared to the control group (P<0.01), and the VC@cLAVs group showed a 64.6% reduction in positive area compared to the control group (P<0.001). These quantitative results of the entire aorta and aortic root lesions indicate that cLAVs can effectively treat atherosclerosis, and its efficacy is further enhanced after encapsulation with vitamin C.

[0108] Example 9: In vivo results of lipoic acid nanovesicles for the treatment of ischemic stroke

[0109] First, a rat model of cerebral cerebral ischemia (MCAO) was established using a suture closure method. Specifically, male SD rats (250–280 g) were anesthetized with 1% pentobarbital, and their left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were separated. Then, nylon sutures were inserted from the ECA to the ICA to block blood supply to the middle cerebral artery. After a set time, the sutures were removed to achieve reperfusion. One hour before MCAO model establishment, 100 mg / kg of lipoic acid monomer (LA) and 50 mg / kg of lipoic acid vesicles (cLAVs) were administered intraperitoneally. DMSO and saline were used as controls (n=3). Cerebral vascular reperfusion was performed one hour after cerebral ischemia in the rats. Neurological function scores were assessed in the model mice at 0, 6, and 24 hours after reperfusion. Neurological function was assessed using a well-established five-point scale (scoring scale: 4 = spontaneous circling, 3 = circling to the left by tail pulling, 2 = decreased grip strength in the left forepaw, 1 = failure to extend the left forepaw, 0 = no defect). After neurological assessment, rats were euthanized, and brain tissue was immediately removed and frozen at -20°C for 5 min. Brain sections were prepared to 2 mm thickness and then stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) at 37°C for 30 min. Tissue sections were preserved in 4% paraformaldehyde solution. TTC-stained sections were photographed, and the percentage area of ​​the coronal section of the infarcted hemisphere was displayed as the infarct volume using Image Tool 3.0. Results are as follows: Figure 10As shown in Figure a, the neurological function scores of rats treated with cLAVs were significantly lower at 6 hours compared to the control group, and the cLAVs (50 mg / kg) group was superior to the group treated with twice the dose of LA monomer (100 mg / kg), indicating that cLAVs significantly improved neurological function. TTC sections of brain tissue at 24 hours showed that the white infarct areas in the brain tissue of rats treated with cLAVs were significantly smaller than those in the control group. Figure 10 b). By quantifying the area of ​​tissue infarction ( Figure 10 c) It was found that the infarct area in the brain tissue of rats treated with cLAVs was reduced by 6 times compared with the control group, while the infarct area in the LA group was only reduced by 2.8 times compared with the control group. In summary, cLAVs achieved a much better therapeutic effect than LA monomer at a dose of only half that of LA monomer, indicating the great potential of cLAVs in the treatment of ischemic stroke. More importantly, the solvent of LA monomer is DMSO, while cLAVs are soluble in physiological saline, the latter having greater clinical value.

[0110] Example 10: In vivo results of lipoic acid nanovesicles for the treatment of pulmonary fibrosis

[0111] Fifty male C57BL / 6 mice were randomly divided into five groups of 10 mice each, based on body weight: sham-operated group, model control group, thioctic acid vesicle group (100 mg / kg), thioctic acid monomer group (100 mg / kg), and bifenidone positive control group. Mice in each group were anesthetized with intraperitoneal injection of sodium phenobarbital (50 mg / kg). The neck skin was disinfected with ethanol, and the trachea was exposed by blunt dissection at the mid-neck. In the model group and the bronchodilator D group, 50 μL of bleomycin (3.5 mg / kg) was slowly injected into the tracheal cartilage rings. The control group received the same volume of physiological saline. Immediately after injection, the mice were rotated upright to ensure even distribution of the fluid in both lungs, and the neck skin was sutured. From day 7 of modeling, the designated drugs were administered intraperitoneally at a volume of 10 mL / kg once daily for 15 consecutive days. Mice in the sham-operated control group and model group received the same volume of physiological saline intraperitoneally daily. The mice were sacrificed 21 days after modeling. Tissue from the lower lobe of the right lung was fixed in 4% paraformaldehyde solution, routinely embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) to observe the lung tissue's morphology, cell degeneration, and fibrosis. Masson's trichrome staining was used to observe collagen fiber proliferation within the lung tissue. Separately, 30 mg of tissue from the upper lobe of the right lung was collected, and the hydroxyproline content was determined using the alkaline hydrolysis method according to the kit instructions.

[0112] Experimental results are as follows Figure 11As shown, after 21 days of bleomycin intratracheal infusion, the lung tissue structure of mice in the sham-operated group was clear, with alveoli appearing as polygonal sac-like bodies, no secretions in the alveolar cavities, intact alveolar epithelial structure, no thickening of the alveolar walls, no inflammatory cells in the alveolar septa, abundant capillaries, and only a small amount of blue collagen fiber deposition in the bronchiolar walls and alveolar septa. In the bleomycin model group, the alveolar structure was disordered, with significantly thickened alveolar walls, obvious infiltration of macrophages and lymphocytes in the alveolar septa, and a large number of blue-stained collagen fibers visible in the alveolar walls and septa. The hydroxyproline content in the lung tissue was significantly increased, indicating significant pulmonary fibrosis damage in the model group. Compared with the model group, the LA monomer group showed a 32.3% reduction in lung injury score (p<0.05), a 45.7% reduction in pulmonary fibrosis area (p<0.05), and a 28.8% reduction in hydroxyproline content in lung tissue (p<0.05), indicating that LA has a certain effect on pulmonary fibrosis. As a clinical drug for treating idiopathic pulmonary fibrosis, bifinidone treatment showed a 67.9% reduction in lung injury score (p<0.01), a 75.0% reduction in pulmonary fibrosis area (p<0.01), and a 48.4% reduction in hydroxyproline content in lung tissue (p<0.01). In contrast, the lipoic acid nanovesicle group showed a 78.0% reduction in lung injury score (p<0.01), a 77.0% reduction in pulmonary fibrosis area (p<0.01), and a 55.6% reduction in hydroxyproline content in lung tissue (p<0.01), with therapeutic effects 2.4, 1.7, and 1.9 times greater than those of the same dose of LA monomer (p<0.05), respectively, and no significant difference compared to bifinidone treatment (p>0.05). These results indicate the significant potential of lipoic acid nanovesicles in the treatment of pulmonary fibrosis.

[0113] Example 11: In vivo results of lipoic acid nanovesicles for the treatment of non-alcoholic fatty liver disease

[0114] Male C57BL / KsJ db / db mice, SPF grade, provided by Changzhou Branch of Jiangsu Jicui Yaokang Biotechnology Co., Ltd., Certificate No.: 202012449, initial weight 44±2g. Eighteen C57BL / KsJ db / db mice were randomly divided into three groups of six mice each: db / db model group, db / db + lipoic acid vesicle group (30mg / kg), and db / db + lipoic acid monomer group (30mg / kg); db / m mice served as the control group. Different doses of the drug were administered intraperitoneally at a volume of 10mL / kg, once daily for four consecutive weeks. The control and model groups received an equal volume of physiological saline intraperitoneally. One hour after drug administration at the end of the fourth week, the mice were weighed, then euthanized by enucleation and bloodletting. Serum samples were separated by centrifugation at 3000 rpm for 15 min at room temperature. The levels of ALT, AST, and FFA in the mouse serum were measured according to the kit instructions. Adipose tissue from the left lobe of the liver and epididymis was fixed with 4% paraformaldehyde, dehydrated, cleared, embedded in paraffin, and sectioned. Hematoxylin-eosin (HE) staining was used to observe liver tissue inflammation, cellular fatty degeneration, and epididymal adipocyte hypertrophy. Oil red O staining was used to observe lipid deposition in the liver tissue.

[0115] Experimental results are as follows Figure 12 As shown, in terms of improving liver tissue lesions, the db / m control group mice showed clear liver lobule structure and neatly arranged hepatocyte cords from the central vein outwards; the db / db model group mice showed loose cytoplasm and vacuolar degeneration of hepatocytes, with a large number of lipid droplets accumulating in the liver parenchyma, and a significant increase in liver tissue weight, indicating that the model group had obvious liver tissue lesions. Compared with the model group, the LA monomer group showed a 59.2% reduction in liver injury score (p<0.05), a 51.7% reduction in lipid deposition area (p<0.05), and a 13.0% reduction in liver weight (p<0.05), indicating that LA has a certain effect on non-alcoholic fatty liver disease. The lipoic acid nanovesicle group showed an 83.0% reduction in liver injury score (p<0.01), a 77.1% reduction in lipid deposition area (p<0.01), and a 35.3% reduction in liver weight (p<0.01), with therapeutic effects 1.4, 1.5, and 2.7 times that of the same dose of LA monomer, respectively (p<0.05). The above results demonstrate the excellent therapeutic effect of lipoic acid nanovesicles on non-alcoholic fatty liver disease tissue lesions.

[0116] Regarding the improvement of liver function, compared with the db / m control group, the serum ALT, AST, and FFA levels in the db / db model group mice were significantly increased (P<0.05), indicating that the model group had severe liver function damage. Compared with the db / db model group, the serum ALT content in the LA monomer group decreased by 30.3% (p<0.05), AST content decreased by 43.5% (p<0.05), and FFA content decreased by 17.6% (p<0.05), indicating that LA has a certain ameliorative effect on liver function in non-alcoholic fatty liver disease. The lipoic acid nanovesicle group showed a 44.2% reduction in serum ALT (p<0.01), a 59.1% reduction in AST (p<0.01), and a 38.2% reduction in FFA (p<0.01), with therapeutic effects 1.5, 1.4, and 2.2 times that of the same dose of LA monomer (p<0.05), respectively. There was no significant difference compared with the normal control group (p>0.05), indicating that lipoic acid nanovesicles have excellent therapeutic effects on liver function impairment in non-alcoholic fatty liver disease.

[0117] Example 12: In vivo results of lipoic acid nanovesicles for the treatment of premature ovarian failure

[0118] C57 mice raised to 28 weeks were randomly divided into 5 groups of 10 mice each: a normal control group, a model group, a lipoic acid monomer (20 mg / kg) group, a lipoic acid nanovesicle group (20 mg / kg), and a melatonin group (20 mg / kg). The normal control group received no treatment, while the other groups were used to establish a premature ovarian failure model via a single intraperitoneal injection of 120 mg / kg cyclophosphamide. Starting from the seventh day after membrane formation, the designated drugs were administered intraperitoneally at a volume of 10 mL / kg once daily for 21 consecutive days. The normal control and model groups received saline. After the drug administration was completed, blood samples were collected from the eyeballs to measure the serum levels of AMH (anti-Müllerian hormone) and FSH (follicle-stimulating hormone). The results are as follows: Figure 13 As shown, the AMH level in the model group treated with cyclophosphamide was significantly reduced, while the FSH level was significantly increased, indicating that ovarian function in mice was significantly impaired. Compared with the model group, there were no significant differences in the lipoic acid monomer group and the melatonin group (P>0.05); the lipoic acid nanovesicles showed a 40% increase in AMH and a 50.0% decrease in FSH compared with the model group (P<0.05), and no significant difference compared with the normal group (P>0.05), indicating the clinical potential of lipoic acid nanovesicles in the treatment of premature ovarian failure.

[0119] Example 13: Atorvastatin-loaded nanovesicles for the treatment of atherosclerosis

[0120] Fifty ApoE animals were fed a high-fat diet for one month. - / -Mice were randomly divided into 5 groups of 10 each, and treated with different therapeutic agents for 2 months. The model control group was treated with saline, while the other four groups received intravenous injections of 20 mg / kg of lipoic acid monomer (LA), 20 mg / kg of lipoic acid nanovesicles (cLAVs), 30 mg / kg of atorvastatin (AVT), and 50 mg / kg of atorvastatin-loaded lipoic acid nanovesicles (AVT@cLAVs), respectively. All formulations were administered intravenously twice within one week. After reaching the treatment time, ApoE... - / - Mice were euthanized. The aorta was excised and fixed with paraformaldehyde, then longitudinally dissected and stained with 0.3% Oil Red dye. The plaque area was then quantitatively analyzed. Results showed that the control group exhibited highly positive areas. Compared with the control group, there was no significant difference in the LA monomer group (P>0.05), while the positive area decreased by 49.8% in the AVT group (P<0.01) and by 52.3% in the cLAVs group (P<0.01), with no significant difference compared to the AVT group (P>0.05). These results indicate that the therapeutic effect of 20 mg / kg cLAVs is comparable to that of 30 mg / kg AVT. Furthermore, the positive area of ​​AVT@cLAVs decreased by 74.3%, which is 1.4 times and 1.5 times that of cLAVs and AVT, respectively, indicating that the efficacy of cLAVs encapsulated with the positive-positive drug atorvastatin was further enhanced.

[0121] Example 14

[0122] In vivo results of lipoic acid nanovesicles in the treatment of systemic lupus erythematosus (SLE). Twenty-week-old female NZBWF1 (SLE) mice were randomly divided into three groups (n=10 per group): a model group (saline), a LA monomer group (50 mg / kg), and a lipoic acid nanovesicle group (50 mg / kg). NZW / LacJ (n=10) served as the control group (saline). Different doses of the drug were administered intraperitoneally at a volume of 10 mL / kg twice weekly for eight weeks. After treatment, mice were euthanized by isoflurane anesthesia, and samples were collected to measure plasma endotoxin levels, T-box transcription factor (T-bet), and GATA-binding protein 3 (GATA-3) expression. The results showed that compared with the control group, the model group exhibited a higher endotoxin level (P < 0.001); there was no significant difference between the LA monomer group and the model group (P > 0.05); the endotoxin level in the nanovesicle group decreased by 45.6% compared with the model group (P < 0.01); compared with the control group, the expression and mRNA levels of T-Bet protein in the model group were significantly decreased (P < 0.001), while the expression and mRNA levels of GATA-3 protein were significantly increased (P < 0.001); there was no significant difference between the LA monomer group and the model group (P > 0.05); compared with the model group, the expression and mRNA levels of T-Bet protein in the lipoic acid nanovesicle group increased by 45.8% and 42.5%, respectively (P < 0.05), while the expression and mRNA levels of GATA-3 protein decreased by 47.5% and 27.5%, respectively (P < 0.05). These results indicate that lipoic acid nanovesicles have excellent therapeutic effects on lupus erythematosus.

[0123] In vivo results of lipoic acid nanovesicles in the treatment of diabetic cataracts. SD rats were randomly divided into four groups of 10 rats each: control group (saline), model group (saline), LA monomer group (30 mg / kg), and lipoic acid nanovesicle group (30 mg / kg). Except for the normal control group, diabetic cataract models were established in the other groups by intravenous injection of STZ (45 mg / kg). Different doses of the drug were administered intraperitoneally at a volume of 10 mL / kg, three times a week for 8 consecutive weeks. After the experiment, the rats were sacrificed, and the levels of MDA and GSH in the lens were measured. The lens was stained with hematoxylin and eosin (HE). The results showed that compared with the control group, the MDA content in the model group was significantly increased (P < 0.001), and the glutathione level was significantly decreased (P < 0.001). The MDA content and glutathione level in the lipoic acid monomer group showed no significant changes compared with the model group (P > 0.05). The MDA content in the lipoic acid nanovesicles decreased by 48.5% (P < 0.01) and the glutathione level increased by 21.5% (P < 0.05) compared with the model group. HE results showed that the anterior lens capsule, including epithelial cells and lens fibers, in the lipoic acid nanovesicle group of diabetic rats was arranged in an orderly manner, similar to that in the control group, while the lens in the LA monomer group showed no significant changes compared with the model group. These results indicate the clinical potential of lipoic acid nanovesicles in the treatment of diabetic cataracts.

[0124] In vivo results of lipoic acid nanovesicles in the treatment of colitis. Male C57BL / 6 mice were randomly divided into four groups (n=10 per group): control group (saline), model group (saline), LA monomer group (30 mg / kg), and lipoic acid nanovesicle group (30 mg / kg). Except for the normal control group, all other groups were induced with colitis by oral administration of 3% dextran sulfate sodium (DSS) for 6 days, followed by continuous oral administration of the drug for 7 days. Mice were sacrificed at the end of the experiment to measure inflammatory factors and for histopathology. Results showed that compared with the control group, the model group showed high expression of TNF-α and IL-6 (P < 0.001). Compared with the model group, there were no significant changes in TNF-α and IL-6 in the lipoic acid monomer group (P > 0.05), while the levels of TNF-α and IL-6 in the lipoic acid nanovesicles decreased by 36.7% and 40.2%, respectively (P < 0.01). HE staining further confirmed that DSS causes severe damage to colonic tissue by disrupting colonic crypt structure and accumulating inflammatory cells; LA monomer treatment could not alleviate DSS-induced colonic damage, while thioctic acid nanovesicles treated mice resulted in colonic tissue structure approaching normal. These results indicate the clinical potential of oral thioctic acid nanovesicles in the treatment of colitis.

[0125] Example 15

[0126] Lipoic acid nanovesicles were prepared according to the methods described in Examples 1 and 2. Then, Arg-Gly-Asp (RGD) was grafted onto the lipoic acid nanovesicles via an amidation reaction using 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to obtain nanomedicines modified with targeting groups. The efficacy of RGD-grafted nanovesicles in treating atherosclerosis was verified according to the method described in Example 8. The results showed that compared to ungrafted RGD nanovesicles, RGD grafting enhanced the efficiency of the nanovesicles reaching the target site, achieving a therapeutic effect equivalent to that of vesicles at only half the dosage.

[0127] Example 16

[0128] Referring to the experimental methods described in Examples 4-15, but replacing the vesicles with micelles and aggregates respectively, the experimental results obtained are basically consistent with those in Examples 4-15.

[0129] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalent alterations can be made within the spirit and scope defined by the claims of the present invention, but all such changes will fall within the protection scope of the present invention.

Claims

1. The application of lipoic acid nanoparticles in the preparation of a drug for treating at least one of ischemic stroke, pulmonary fibrosis, non-alcoholic fatty liver disease, premature ovarian failure, diabetic cataract, and lupus erythematosus, wherein the lipoic acid nanoparticles contain lipoic acid cross-linked polymerized via disulfide bonds.

2. The application according to claim 1, characterized in that, In the lipoic acid nanoparticles, the disulfide bonds in the five-membered ring of lipoic acid are broken first, and the lipoic acid molecules cross-link with each other.

3. The application according to claim 1, characterized in that, The surface potential of the nanoparticles is negative.

4. The application according to claim 1, characterized in that, The lipoic acid nanoparticles are micelles, vesicles, or aggregates formed by the self-assembly of lipoic acid.

5. The application according to claim 1, characterized in that, Using lipoic acid nanoparticles as a carrier, other active pharmaceutical ingredients are encapsulated. The lipoic acid nanoparticles serve both as active pharmaceutical ingredients and as nanocarriers.

6. The application according to claim 5, characterized in that, The other active pharmaceutical ingredients are at least one of vitamin C, vitamin E, coenzyme Q10, and atorvastatin.

7. The application according to claim 1, characterized in that, The nanoparticles have a particle size of 10-200 nm.

8. The application of lipoic acid nanoparticles in the preparation of anti-inflammatory drugs, characterized in that, The lipoic acid nanoparticles are loaded with vitamin C, and the lipoic acid in the lipoic acid nanoparticles is cross-linked and polymerized through disulfide bonds.

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