Use of gold nanoclusters in the treatment of hypercholesterolemia or diseases associated with hypercholesterolemia

Abstract

The present invention relates to the use of dihydrolipoic acid (DHLA)-coated gold nanoclusters in the preparation of a medicament for treating hypercholesterolemia or a disease associated with hypercholesterolemia in an individual, wherein the gold nanoclusters have a particle size of about 1 to 10 nanometers.

Description

Technical Field

[0001] The present invention relates to the field of treating diseases. More specifically, the present invention relates to the use of gold nanoclusters coated with dihydrolipoic acid (DHLA) in the treatment of hypercholesterolemia or diseases associated with hypercholesterolemia (for example, atherosclerosis). Background Art

[0002] Hypercholesterolemia (also known as high cholesterol) is a disease caused by excessive blood cholesterol levels. Cholesterol is an essential substance for the formation of cell membranes, hormones, and compounds related to fat metabolism. However, excessive cholesterol abnormally accumulates in the blood vessel walls and forms plaques, which can lead to the development of atherosclerosis; atherosclerosis is a disease characterized by thickening of the blood vessel walls and loss of elasticity. Atherosclerosis is associated with the development and progression of different cardiovascular diseases (CVD), including coronary heart disease, angina pectoris, heart attack, stroke, transient ischaemic attack (TIA), peripheral arterial disease (PAD; such as limb ischemia), and restenosis.

[0003] Statins and surgery are two major current treatments for atherosclerosis. Statins are a class of lipid-lowering drugs that block the cholesterol synthesis pathway in the liver by inhibiting the action of HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase). As a treatment prescription for atherosclerosis, statins are usually accompanied by adverse side effects, such as dizziness, nosebleeds, sore throat, headache, constipation, diarrhea, indigestion, muscle or joint pain, hyperglycemia, and inflammation. In addition, it has also been reported that statins interact with other drugs, increasing the harmful risks such as muscle damage. When the condition of atherosclerosis is relatively severe and leads to irreversible ischemic conditions, surgery (for example, vascular bypass surgery, or angioplasty with or without stent implantation) provides another option for treating atherosclerosis. However, surgery is known to cause blood loss, wound hematoma, infection, or more seriously, nerve damage.

[0004] In view of this, there is an urgent need in the relevant field for a novel method that can treat atherosclerosis more safely and effectively. Summary of the Invention

[0005] The Summary of the Invention aims to provide a simplified abstract of the present invention to enable readers to have a basic understanding of the present invention. This Summary of the Invention is not a complete overview of the present invention, and its intention is not to point out the important / critical components of the embodiments of the present invention or to limit the scope of the present invention.

[0006] A first aspect of the present invention relates to a method for treating hypercholesterolemia or a disease associated with hypercholesterolemia in an individual. The method comprises administering to the individual an effective amount of gold nanoclusters coated with dihydrolipoic acid (DHLA), which is composed of a gold nanocluster and a plurality of DHLA, wherein the gold nanocluster is formed by a plurality of gold nanoparticles, and the plurality of DHLA are coated on the gold nanocluster. According to an embodiment of the present invention, the particle size of the gold nanoclusters coated with DHLA is about 1 to 10 nanometers; preferably, about 1 to 5 nanometers. In a specific embodiment of the present invention, the particle size of the gold nanoclusters coated with DHLA is about 2 nanometers.

[0007] According to certain embodiments of the present invention, the effective amount is about 0.001 - 10 milligrams per kilogram of the individual's body weight per day; preferably, about 0.01 - 1 milligram per kilogram of the individual's body weight per day; more preferably, about 0.01 - 0.1 milligram per kilogram of the individual's body weight per day. According to a specific embodiment of the present invention, the gold nanoclusters coated with DHLA are administered to the individual daily for 56 consecutive days.

[0008] The present invention also provides the use of gold nanoclusters coated with DHLA in the preparation of a drug or a pharmaceutical composition, wherein the drug or the pharmaceutical composition can be used to treat hypercholesterolemia or a disease associated with hypercholesterolemia in an individual. The drug or the pharmaceutical composition comprises the gold nanoclusters coated with DHLA of the present invention, and optionally, a pharmaceutically acceptable excipient.

[0009] Generally, diseases associated with hypercholesterolemia that can be treated by the DHLA-coated gold nanoclusters, drugs or pharmaceutical compositions of the present invention are atherosclerosis, hyperlipidemia, pancreatitis, gallstone, biliary tract cancer, or venous thromboembolism (VTE).

[0010] The individual is a mammal. Preferably, the individual is a human.

[0011] After referring to the following embodiments, those skilled in the art should be able to easily understand the basic spirit and other inventive purposes of the present invention, as well as the technical means and embodiments adopted by the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] To make the above and other purposes, features, advantages and embodiments of the present invention more obvious and understandable, the description of the accompanying drawings is as follows:

[0013] Figure 1 FIG. is a schematic diagram drawn according to an embodiment of the present invention, which is about the DHLA-coated gold nanoclusters of the present invention;

[0014] Figure 2 FIG. is a bar chart drawn according to Example 1 of the present invention, which is about the area ratio of atherosclerotic regions; n = 6 for each group; *, p < 0.05; **, P < 0.001;

[0015] Figure 3 FIG. is a bar chart drawn according to Example 2 of the present invention, which is about the content of total cholesterol in the serum of mice administered with a specific treatment; n = 6 for each group; *, p < 0.05; **, P < 0.001;

[0016] Figure 4A and Figure 4B FIG. is a bar chart drawn according to Example 2 of the present invention, which is about the expression levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) in mice administered with a specific treatment; n = 6 for each group; *, p < 0.05; **, P < 0.001; and

[0017] Figures 5A to 5C FIG. is a bar chart drawn according to Example 3 of the present invention, which respectively illustrates the number of macrophages attached to human aortic endothelial cells (HAEC) ( Figure 5A ), and the expression levels of adhesion molecules ICAM-1 ( Figure 5B ) and VCAM-1 ( Figure 5C ) on HAEC; n = 3 for each group; *, p < 0.05; **, P < 0.001. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0018] In order to make the description of the present invention more detailed and complete, the following provides an illustrative description of the embodiments and specific examples of the present invention; however, this is not the only form for implementing or applying the specific examples of the present invention. The embodiments cover the features of multiple specific examples and the method steps and their sequences for constructing and operating these specific examples. However, other specific examples can also be used to achieve the same or equivalent functions and step sequences.

[0019] I. Definitions

[0020] Unless otherwise defined in this specification, the meanings of scientific and technical terms used herein are the same as those understood and commonly used by those skilled in the art. In addition, in the case of no conflict with the context, the singular nouns used in this specification cover the plural forms of the nouns; and the plural nouns used also cover the singular forms of the nouns.

[0021] Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate values, the relevant values in the specific embodiments have been presented as precisely as possible herein. However, any numerical value inherently and inevitably contains standard deviations due to individual testing methods. Herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range. Or, the term "about" represents that the actual value falls within the acceptable standard error of the mean value, depending on the consideration of those skilled in the art. Except for experimental examples, or unless otherwise clearly stated, it should be understood that all ranges, amounts, numerical values and percentages (such as those used to describe material amounts, time lengths, temperatures, operating conditions, quantity ratios and others similar) used herein are modified by "about". Therefore, unless otherwise stated to the contrary, the numerical parameters in this specification and the claims are approximate values and can be changed as needed. At least these numerical parameters should be understood as the values obtained by indicating the significant digits and applying the general rounding method. Herein, a numerical range is expressed as from one endpoint to the other endpoint or between two endpoints; unless otherwise stated, the numerical ranges described herein include the endpoints.

[0022] "Administer" or "administration" in the present invention refers to a delivery mode that includes, but is not limited to, intravenous, intramuscular, intraperitoneal, intraarterial, intracranial or subcutaneous delivery of the medicament of the present invention (for example, gold nanoclusters coated with DHLA).

[0023] In the present invention, the term "treat" encompasses partially or fully preventing, ameliorating, alleviating, and / or managing a related symptom, secondary disorder, or condition associated with or caused by hypercholesterolemia, where reducing the levels of hypercholesterolemia can be helpful to an individual suffering from or suspected of suffering from these symptoms, conditions, or diseases. The term "treat" as used in this specification also refers to the application or administration of one or more of the DHLA-coated gold nanoclusters of the present invention to an individual suffering from a related symptom, secondary disorder, or condition associated with or caused by hypercholesterolemia, to partially or fully alleviate, slow down, cure the disease, delay the onset, inhibit the progression of the disease, reduce the severity of the disease, and / or reduce the occurrence of one or more related symptoms, secondary disorders, or conditions associated with or caused by hypercholesterolemia. Related symptoms, secondary disorders, or conditions associated with or caused by hypercholesterolemia include, but are not limited to, coronary heart disease, angina pectoris, heart disease, stroke, TIA, and PAD. Here, "treat" can also refer to the administration to an individual with early onset of these symptoms or conditions to reduce the risk of the individual developing related symptoms, secondary disorders, or conditions associated with or caused by hypercholesterolemia. A treatment is "effective" when it can reduce one or more conditions or clinical markers. Alternatively, a treatment is "effective" when it can reduce, slow down, or terminate the progression of a disease, symptom, or condition.

[0024] "Effective amount" as used herein refers to an amount of a drug sufficient to produce a desired therapeutic response. An effective amount also refers to a compound or composition whose therapeutic benefits outweigh its toxic or detrimental effects. The specific effective amount depends on a variety of factors such as the particular condition to be treated, the physiological condition of the patient (e.g., patient weight, age or gender), the type of mammal or animal being treated, the duration of treatment, the nature of the current therapy (if any), and the specific formulation used and the structure of the compound or its derivatives. For example, the effective amount can be expressed as the total weight of the drug (e.g., in grams, milligrams or micrograms) or as the ratio of the drug weight to body weight (in units of milligrams per kilogram (mg / kg)). Alternatively, the effective amount can be expressed as the concentration of the active ingredient (e.g., the DHLA-coated gold nanoclusters of the present invention), such as molar concentration, weight concentration, volume concentration, molality, mole fraction, weight fraction and mixing ratio. A person skilled in the art can calculate the human equivalent dose (HED) of a drug (such as the DHLA-coated gold nanoclusters of the present invention) based on the dose in an animal model. For example, a person skilled in the art can estimate the maximum safe dose for human use based on the "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers" announced by the US Food and Drug Administration (FDA).

[0025] In the present invention, "hypercholesterolemia" refers to any disease caused by a cholesterol level in the blood that is higher than the clinically recommended level. For example, when measuring blood cholesterol levels using low density lipoprotein (LDL), an individual may have hypercholesterolemia if the measured LDL level is higher than about 75 milligrams per deciliter (dl). Alternatively, when measuring blood cholesterol levels using plasma-free cholesterol, an individual may have hypercholesterolemia if the measured plasma-free cholesterol is higher than about 200 - 220 milligrams per deciliter.

[0026] "Hypercholesterolemia-associated disease" in the present invention refers to a disease, condition or symptom that originates from or is exacerbated by abnormally elevated blood cholesterol; for example, atherosclerosis (including various atherosclerotic cardiovascular diseases (ASCVD)), hyperlipidemia, pancreatitis, gallstones, cholangiocarcinoma, or VTE.

[0027] In the present invention, a "pharmaceutically acceptable" ingredient refers to an ingredient that can be used in the human body and / or animal body without producing adverse side effects (such as toxicity, irritation and / or allergic reactions), and has a reasonable benefit / risk ratio.

[0028] The term "excipient" in the present invention refers to any inert substance (such as a powder or liquid) that can form a carrier or vehicle for the DHLA-coated gold nanoclusters of the present invention. The excipient can be any commercially available excipient as long as it is generally safe and non-toxic to an individual.

[0029] The term "subject" refers to an animal including humans that can be treated with the DHLA-coated gold nanoclusters, drugs, pharmaceutical compositions or methods of the present invention. Unless specifically indicated, the term "subject" refers to both males and females.

[0030] II. Description of the Invention

[0031] The present invention is at least partially based on the inventors' first discovery that cholesterol, oxidative stress and vascular inflammation levels / degrees can be reduced by DHLA-coated gold nanoclusters, thereby treating atherosclerosis. Accordingly, the present invention relates to the use of DHLA-coated gold nanoclusters in the treatment of hypercholesterolemia or hypercholesterolemia-associated diseases.

[0032] The DHLA-coated gold nanoclusters of the present invention are known in the art, and their manufacturing process (Lin et al., ACS Nano (2009); 3: 395-401) is also known in the art. Therefore, there is no need to further explain the preparation process. The gold nanoclusters coated with dihydrolipoic acid can emit polarized light at 650 nm under the condition that the excitation wavelength is about 420 nm. Therefore, its emission wavelength range is from red light to near-infrared range. The particle size of each gold nanocluster is about 1 to 10 nm, and the preferred range is about 1 to 5 nm. In some operating examples, the particle size of the gold nanoclusters is about 2 nm. The particle size of the gold nanoclusters disclosed above is the gold nanoclusters in the dry state. However, it will be better if the gold nanoclusters used in the present invention are water-soluble or at least dispersible in a liquid medium and / or water; due to the coupling with surrounding solvent molecules (e.g., water), the hydrodynamic size of the gold nanoclusters is significantly larger than its dry size. In one embodiment, the hydrodynamic size of the gold nanoclusters is approximately equivalent to that of polyethylene glycol with a molecular weight of 0.1 to 30 kDa.

[0033] On the one hand, the present invention utilizes DHLA-coated gold nanoclusters to prepare a pharmaceutical composition or a drug for treating hypercholesterolemia or a disease related to hypercholesterolemia. The pharmaceutical composition or drug comprises DHLA-coated gold nanoclusters, and optionally, a pharmaceutically acceptable excipient.

[0034] The weight of the DHLA-coated gold nanoclusters accounts for about 0.01% to 99.9% of the total weight of the drug or the pharmaceutical composition. In some embodiments, the weight of the DHLA-coated gold nanoclusters accounts for at least 0.1% of the total weight of the drug or the pharmaceutical composition. In some embodiments, the weight of the DHLA-coated gold nanoclusters accounts for at least 5% of the total weight of the drug or the pharmaceutical composition. In some embodiments, the weight of the DHLA-coated gold nanoclusters accounts for at least 10% of the total weight of the drug or the pharmaceutical composition. In some embodiments, the weight of the DHLA-coated gold nanoclusters accounts for at least 25% of the total weight of the drug or the pharmaceutical composition.

[0035] In application, the DHLA-coated gold nanoclusters of the present invention can be formulated into different dosage forms, such as tablets, sugar-coated tablets, pills, granules, aerosols, syrups, suspensions, solutions, ointments, creams, gels, or any substantially non-toxic and pharmaceutically acceptable excipient or carrier, especially excipients or carriers prepared from inert substances.

[0036] The present invention also provides a method for treating hypercholesterolemia or a disease associated with hypercholesterolemia in an individual. The method comprises administering to the individual an effective amount of the DHLA-coated gold nanoclusters of the present invention, or a drug or pharmaceutical composition comprising the DHLA-coated gold nanoclusters.

[0037] The DHLA-coated gold nanoclusters, drug or pharmaceutical composition of the present invention can be administered systemically or locally. The DHLA-coated gold nanoclusters of the present invention can be administered by any conventional route of administration. For example, administration can be oral, lingual, sublingual, buccal, rectal or parenteral (i.e., intravenous, intraarterial, intracardiac, intradermal, subcutaneous, transdermal, intraperitoneal or intramuscular).

[0038] According to certain embodiments of the present invention, the individual is a mouse. To produce a therapeutic effect in the mouse, the individual is administered about 0.01 to 150 mg of the DHLA-coated gold nanoclusters per kilogram of body weight of the individual per day; preferably, about 0.1 to 15 mg per kilogram of body weight of the individual per day; more preferably, about 0.1 to 1.5 mg per kilogram of body weight of the individual per day. According to one working example, the individual is administered 0.57 mg of the DHLA-coated gold nanoclusters per kilogram of body weight of the individual per day.

[0039] Those skilled in the art can calculate the human equivalent dose of the DHLA-coated gold nanoclusters of the present invention based on the dosage used in the animal model. Accordingly, the human equivalent dose of the DHLA-coated gold nanoclusters of the present invention is about 0.001 to 10 mg per kilogram of body weight of the individual per day; for example, about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg per kilogram of body weight of the individual per day. Preferably, a human individual is treated with about 0.01 to 1 mg of the DHLA-coated gold nanoclusters of the present invention per kilogram of body weight of the individual per day. More preferably, a human individual is treated with about 0.01 to 0.1 mg of the DHLA-coated gold nanoclusters of the present invention per kilogram of body weight of the individual per day.

[0040] Alternatively, the effective amount of the DHLA-coated gold nanoclusters of the present invention can be adjusted according to clinical factors, such as the disease to be treated, the severity of the disease, the physiological parameters of the patient (including age, physical condition, body type, gender and weight), the duration of treatment, the treatments administered simultaneously (if any), the route of administration, and other considerations in the knowledge or professional judgment of those skilled in the art or clinical operators.

[0041] Those skilled in the art or clinical operators can adjust the administration course of the DHLA-coated gold nanoclusters according to different factors, such as age, gender, weight or other treatments (if any). For example, the DHLA-coated gold nanoclusters of the present invention can be administered to an individual 1-7 times a week (e.g., 1, 2, 3, 4, 5, 6 or 7 times a week) for 1, 2, 3, 4 or more weeks continuously. Alternatively, the DHLA-coated gold nanoclusters of the present invention can be administered to an individual 1-10 times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times) every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks or every 10 weeks; or once a month, once every 2 months, once every 3 months or less frequently. Preferably, the DHLA-coated gold nanoclusters of the present invention are administered to an individual daily for at least 14 days (i.e., 2 weeks); for example, administered for 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more days. More preferably, the DHLA-coated gold nanoclusters of the present invention are administered to an individual daily for at least 28 days (i.e., 4 weeks). According to an operating example of the present invention, the DHLA-coated gold nanoclusters of the present invention are administered to an individual daily for 56 days (i.e., 8 weeks) to achieve a therapeutic effect.

[0042] Diseases associated with hypercholesterolemia can be atherosclerosis, hyperlipidemia, pancreatitis, gallstones (including cholesterol gallstones and combined gallstones), cholangiocarcinoma (or biliary tract cancer (cholangiocarcinoma), including gallbladder carcinoma (GBC), intrahepatic cholangiocarcinoma (iCC), and extrahepatic cholangiocarcinoma (eCC)), or VTE (including deep vein thrombosis (DVT) and pulmonary embolism (PE)). According to certain method embodiments, the disease associated with hypercholesterolemia is atherosclerosis, wherein administering gold nanoclusters coated with DHLA can effectively reduce the formation of atherosclerotic plaques, contributing to the treatment and / or prevention of different ASCVDs, including coronary heart disease, angina pectoris, heart disease, stroke, TIA, peripheral artery disease, and vascular stenosis.

[0043] Basically, the individual is a mammal; for example, a human, mouse, rat, hamster, guinea pig, rabbit, dog, cat, cow, goat, sheep, monkey, and horse. Preferably, the individual is a human.

[0044] As can be imagined, the method of the present invention can be administered alone or in combination with other treatments to an individual, wherein the other treatment is helpful for treating hypercholesterolemia or atherosclerosis, for example, statins. Depending on different usage requirements, the method of the present invention can be administered to the individual before, simultaneously with, or after administering other treatments.

[0045] The following presents several experimental examples to illustrate certain aspects of the present invention to facilitate those skilled in the art to implement the present invention, and these experimental examples should not be regarded as limiting the scope of the present invention. It is believed that those skilled in the art can fully utilize and practice the present invention without excessive interpretation after reading the descriptions presented herein. All the publicly cited documents herein are regarded as part of this specification in their entirety.

[0046] Examples

[0047] Materials and Methods

[0048] Preparation of Gold Nanoclusters Coated with DHLA (FANC)

[0049] The fluorescent gold nanoclusters of the present invention are prepared by the method described previously (see Lin et al., ACS Nano (2009); 3: 395-401). Briefly, 6-nanometer gold nanoclusters stably bound to didodecyldimethylammonium (AuNP@DDAB) are synthesized via a single phase reaction (see Jana and Peng, J Am Chem Soc (2003); 125: 14280-14281), and their composition structure is as shown in Figure 1 . Then, a gold precursor solution (gold chloride dissolved in a didodecyldimethylammonium-toluene solution) is gradually added dropwise to gradually deplete the plasma absorption until the solution turns yellow and transparent. Next, the previously prepared nanoclusters are added to reduced lipoic acid for ligand replacement; the reduced lipoic acid needs to be freshly prepared, which is a mixture of lipoic acid and tetrabutylammonium bromide (TBAB) in a molar ratio of 4:1. The above steps generate a dark brown nanocluster clot mixture, and the nanocluster clots are irradiated with an ultraviolet lamp (365 nm, 30 minutes) to agglomerate the clots. The supernatant is removed, methanol is added to the nanocluster clots to resuspend and disperse the nanocluster clots, and then chloroform is added to precipitate and remove the free surfactant. This dried nanocluster precipitate can be resuspended again in a boric acid buffer (pH 9). Then, ultra-high speed centrifugation (110,000 rpm) is performed three times to remove the excess lipoic acid. To collect the gold nanoclusters, PBS buffer is added to a 30 kDa molecular weight cut-off (MWCO) concentrator centrifuge tube until the nanocluster transparent solution colloid is stable and has no plasma absorption peak. The concentration of the gold nanoclusters has an extinction coefficient of approximately 450,000 M -1 cm -1 at a wavelength of 420 nm.

[0050] Adhesion test

[0051] Human aortic endothelial cells (HAEC) are cultured in endothelial growth medium. At a density of 10,000 cells per square centimeter, the cells are seeded on plasticware coated with 1% gelatin or glass coverslips coated with 2% gelatin, and cultured in a humid environment at 37 °C with 95% air and 5% CO2 gas.

[0052] HAECs were mixed with DHLA-coated gold nanoclusters at specific concentrations (i.e., 0 nM, 50 nM, or 100 nM) for 3 days, followed by stimulation with 100 ng / mL lipopolysaccharide (LPS) for 24 hours. The activated HAECs were co-cultured with calcein AM-treated monocytes for an adhesion assay. After 2 hours of culture, unbound monocytes were removed, and cell images were recorded using a fluorescence microscope.

[0053] Animal experiments

[0054] Four-week-old ApoE - / - mice and wild-type C57BL / 6 mice were fed a normal diet or a granular Western diet (containing 0.21% cholesterol) and administered FANC (0.57 mg / kg body weight per day for 56 days) or placebo (PBS). The mice were divided into three groups: Group 1 was wild-type mice fed a normal diet; Group 2 was ApoE - / - mice fed a normal diet and treated with PBS; Group 3 was ApoE - / - mice fed a Western diet and treated with FANC.

[0055] Detection and quantitative analysis of atherosclerotic regions

[0056] According to the above animal experiments, after specific treatments were administered to ApoE-deficient and wild-type mice respectively, their aortas were isolated and analyzed by Sudan IV staining, which is a red β-naphthol diazo dye used to analyze lipid-containing substances (e.g., triglycerides, lipids, and lipoproteins) in cells and tissues. The stained aortic tissue and pathological morphology were observed under a microscope, and software was used to determine the area of atherosclerotic regions.

[0057] Analysis of cholesterol, MDA, and 4-HNE

[0058] To evaluate whether the DHLA-coated gold nanoclusters of the present invention would affect the levels of cholesterol and oxidative stress, the sera of ApoE-deficient mice after 56 days of treatment were isolated respectively. A cholesterol detection kit was used to analyze the total cholesterol level in the sera, and enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of MDA and 4-HNE (two oxidative stress indicators).

[0059] Example 1 Anti-atherosclerotic efficacy of DHLA-coated gold nanoclusters

[0060] This example will analyze the effect of DHLA-coated gold nanoclusters on atherosclerosis, in which ApoE-deficient mice were fed with normal food (as a control group) or Western-style food (i.e., high-fat food), and treated with PBS or DHLA-coated gold nanoclusters.

[0061] Compared with ApoE-deficient mice fed with normal food, the atherosclerotic plaque accounted for about 2.57 ± 0.5% of the lumen area. Feeding high-fat food significantly increased the area of atherosclerotic plaques in ApoE-deficient mice, and the area of atherosclerotic plaques increased about 6-fold, accounting for 15.35 ± 2.6% of the lumen area ( Figure 2 ). It is worth noting that administering DHLA-coated gold nanoclusters (labeled as "FANC" in Figure 2 ) significantly reduced the area of atherosclerotic plaques ( Figure 2 ). There was no significant effect on ApoE-deficient mice fed with normal food and treated with DHLA-coated gold nanoclusters. Similar results were presented in ApoE-deficient mice fed with high-fat food. Compared with the PBS control group, administering DHLA-coated gold nanoclusters reduced the atherosclerotic plaque by about 43.3%, and the area of atherosclerotic plaques decreased to 8.70 ± 2.5% of the lumen area ( Figure 2 ).

[0062] These results show that the DHLA-coated gold nanoclusters of the present invention have a therapeutic effect on atherosclerosis and can be used to treat different atherosclerotic cardiovascular diseases.

[0063] Example 2 Efficacy of DHLA-coated gold nanoclusters in inhibiting serum cholesterol and oxidative stress

[0064] This example will analyze whether the DHLA-coated gold nanoclusters of the present invention affect cholesterol content and oxidative stress. Figure 3 And Figure 4 analyzes and elaborates on the analysis results.

[0065] Figure 3 The data indicate that compared with feeding normal food, feeding high-fat food increased the total serum cholesterol in ApoE-deficient mice (normal food: 567.7 ± 32.5 mg / dL; high-fat food: 1020.0 ± 55.0 mg / dL). Administering DHLA-coated gold nanoclusters (in Figure 3Those labeled as "FANC" can significantly reduce cholesterol levels. Among ApoE-deficient mice fed a normal diet and treated with DHLA-coated gold nanoclusters, the total serum cholesterol decreased to 501.0 ± 46.7 mg / dL; while in ApoE-deficient mice fed a high-fat diet and treated with DHLA-coated gold nanoclusters, the total serum cholesterol decreased to 529.0 ± 300.0 mg / dL, a reduction of approximately 48.1%( Figure 3 ).

[0066] In addition to cholesterol, this experiment also detected the inhibitory effect of DHLA-coated gold nanoclusters on the expression of MDA and 4-HNE. Figure 4A and Figure 4B The results of the analysis pointed out that compared with wild-type mice (ApoE -+ / + mice) fed a normal diet, feeding either a normal diet or a high-fat diet would increase the levels of MDA and 4-HNE in the serum of ApoE-deficient mice. Compared with mice fed the same diet and treated with PBS, treatment with DHLA-coated gold nanoclusters (labeled as "FANC" in Figure 4A and Figure 4B ) could significantly reduce the levels of these oxidative stress indicators in the serum( Figure 4A and Figure 4B ).

[0067] These results confirm that the DHLA-coated gold nanoclusters of the present invention can effectively inhibit cholesterol levels and oxidative stress in an individual.

[0068] Example 3 Effect of DHLA-coated gold nanoclusters on adhesion molecules

[0069] Since the attachment of inflammatory cells (e.g., macrophages) to the blood vessel wall can cause vascular inflammatory reactions, which in turn lead to the generation and deterioration of atherosclerosis, this example will use LPS to stimulate HAEC to evaluate whether the DHLA-coated gold nanoclusters of the present invention (labeled as "FANC" in Figures 5A to 5C ) will affect vascular inflammatory reactions. Figures 5A to 5C The analysis results are described separately.

[0070] As shown in Figure 5A and Figure 5B , LPS can induce HAEC to express adhesion molecules (i.e., ICAM-1 and VCAM-1, two adhesion molecules that mediate vascular inflammatory reactions); treatment with DHLA-coated gold nanoclusters can significantly reduce the expression induced by LPS. In addition, the DHLA-coated gold nanoclusters of the present invention can also dose-dependently reduce the number of macrophages attaching to HAEC( Figure 5C ).

[0071] In summary, the present invention demonstrates that DHLA-coated gold nanoclusters can treat atherosclerosis by reducing cholesterol, oxidative stress, and the content or expression level of adhesion molecules (e.g., ICAM-1 and VCAM-1). Therefore, DHLA-coated gold nanoclusters can be used to prepare drugs for treating different ASCVDs.

[0072] Although specific embodiments of the present invention have been disclosed in the above embodiments, they are not intended to limit the present invention. Those skilled in the art should be able to make various changes and modifications without departing from the principles and spirit of the present invention. Therefore, the protection scope of the present invention shall be subject to that defined by the claims.

Claims

1. Use of a dihydrolipoic acid-coated gold nanocluster with a particle size of 1 to 10 nanometers for preparing a drug, wherein the drug can be used to treat hypercholesterolemia in an individual, wherein the dihydrolipoic acid-coated gold nanocluster is composed of a gold nanocluster and a plurality of dihydrolipoic acids, wherein the gold nanocluster is formed by a plurality of gold nanoparticles, and the plurality of dihydrolipoic acids are coated on the gold nanocluster.

2. The use according to claim 1, wherein the dihydrolipoic acid-coated gold nanocluster has a particle size of 1 to 5 nanometers.

3. The use according to claim 2, wherein the dihydrolipoic acid-coated gold nanocluster has a particle size of 2 nanometers.

4. The use according to claim 1, wherein the individual is a human.

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