TWI413524B - Use of gold nanoclusters in ameliorating oxidate stress and/or aging - Google Patents

Abstract

The invention discloses a method, a composition and a use of using au nanocluster to relieve the oxygen partial pressure and/or aging of culture cell, or relieve the oxygen partial pressure and/or aging caused by a vessel factor or a disease. The grain size of the disclosed au nanocluster is between 0.1 nanometers and 20 nanometers, and the au nanocluster coated with a dihydrolipoic acid is preferable.

Description

Method, composition and use for relieving oxidative stress and/or aging with a cluster of gold nanoparticles

The present disclosure is directed to the use of a cluster of gold nanoparticles; and in particular to novel uses for slowing oxidative stress and/or aging with red light gold nanoclusters.

According to previous research results, the degradation process caused by the aging process and related diseases is partly due to the oxidative transfer of free radical oxidation pressure and/or thio/disulfate redox reaction (Beckeamen and Ames, Physiol. Rev. 1998 78:547-581). It has also been postulated that oxidative stress is due to related vascular diseases or vascular factors. Therefore, there is an urgent need in the art to find a formulation that can alleviate oxidative stress against aging and various vascular disease symptoms. Thus, formulations which can slow the oxidative stress and even the aging process have the potential to be developed into agents or pharmaceutical compositions useful for the treatment of vascular diseases.

The following is a brief description of the contents of the present invention, which is intended to provide a basic understanding of the present disclosure. This Summary is not an overview of the disclosure, and does not define the main/critical elements of the disclosure or the scope of the invention. The present invention is intended to be illustrative of the present disclosure, and further details are described in the embodiments.

The present invention is based on the unexpected discovery that red light gold nanoclusters can effectively slow down the oxidative stress and/or aging of a cultured cell, and thus can be used as a preparation to effectively resist or inhibit cell aging, and can be further developed into a An agent that treats oxidative stress and/or aging induced by vascular factors (eg, inflammatory or vascular disease).

The present disclosure is a method of slowing the oxidative stress and/or aging of cultured cells. The method comprises contacting an effective amount of a nano-nano cluster with the cultured cell, wherein the golden nano-cluster has a particle size of about 0.1 to 20 nm.

In one example, the gold nanoclusters are dihydrolipoic acid (DHLA) coated gold nanoclusters. The cultured cells are selected from human arotic endothelial cells (HAEC), human epidermal cells, human endothelial progenitor cells (HEPC), human embryonic stem cells, and humans. A group consisting of human mesenchymal stem cells. The total amount of the golden nano-clusters in contact with the cultured cells is about 1-1,000 nM.

Additionally, the present disclosure is the use of the aforementioned gold nanoclusters to produce an agent that slows the oxidative stress and/or aging of cultured cells.

Further, the present disclosure is a pharmaceutical composition for treating oxidative stress and/or aging symptoms of a subject. The pharmaceutical composition comprises an effective amount of the aforementioned gold nanoclusters and a pharmaceutically acceptable excipient. The individual is a human or an animal, preferably a mammal.

On the other hand, a method of treating oxidative stress and/or aging symptoms of a body with a cluster of gold nanoparticles is also within the scope of the present invention. The method comprises administering to the individual a pharmaceutical composition as described above to slow the oxidative stress and/or aging of the individual. This oxidative stress and/or aging is caused by a vascular factor, such as by inflammation or vascular disease. The vascular disease may be atherosclerosis, coronary artery disease (CAD), myocardial infarction (MI), ischemia (ischemia), stroke, peripheral vascular disease, or Pulmonary vascular disease. The aforementioned individual may be a human or an animal, preferably a mammal.

Embodiments of the present disclosure will be described in detail within the scope of the embodiments and claims. The features, objectives, and advantages of the present disclosure will be more fully understood from the embodiments and appended claims.

It will be appreciated by those skilled in the art that many changes and modifications may be made to the present invention without departing from the scope of the invention. The examples herein are merely illustrative of the invention, and the scope of the invention is not limited thereto.

The specific nomenclature described in the present disclosure is as follows. The technical and scientific terms used in this specification have the same meaning as those of ordinary skill in the art, unless otherwise specified.

The singular forms "a", "the" and "said" are used in the <RTI ID=0.0> </ RTI> </ RTI> <RTIgt;

The term "about" as used in this disclosure is generally a specific value or range of 10%, 5%, 1%, or 0.5%. In addition, the term "about" refers to an acceptable standard error of an average value recognized by a skilled artisan.

The term "pharmaceutically acceptable" as used in the present disclosure is a human//or animal compatible with the benefit/risk ratio and has no adverse side effects (eg, toxicity, Ingredients for irritating and/or allergic reactions).

The term "an effective amount" as used in the present disclosure refers to an amount of a nano-nano cluster sufficient to produce an intended reaction. For in vitro assay applications, the effective amount that slows the oxidative stress and/or aging of the cultured cells will vary with different factors, such as the type of cell contacted with the cluster of gold nanoparticles or the passage of cells. The number of times of cultivation. The effective amount can be expressed by the total concentration of the gold nanoclusters (nM), the total mass of the gold nanoclusters (for example, grams, milligrams or micrograms), and the total mass of the gold nanoclusters. The ratio of the total volume of the medium (eg, mg/ml). Preferably, the concentration of the gold nanoclusters applied is between about 1 and 1,000 nM; preferably between about 10 and 30,000 nM; more preferably between about 30 and 100 nM. For in vivo testing applications, for example, in vivo applications that slow the oxidative stress and/or aging symptoms of a body, the effective amount will also vary with different factors, such as the particular therapeutic condition, the physiological condition of the subject ( For example, weight, age or gender), type of mammal or animal, type of other treatment (if any) administered simultaneously, and specific formulation used. The effective amount can be expressed as the total mass of the gold nanoclusters (eg, grams, milligrams, or micrograms), or the ratio of the total mass of the golden nanoclusters to the individual's body weight (eg, mg/ kg).

The term "excipient" as used in the present disclosure refers to any inert substance (for example, powder or liquid) that can be a medium/carrier for a group of gold nanoparticles. The excipient is generally safe, non-toxic, and broadly encompasses any of the known materials used in the pharmaceutical industry for the preparation of pharmaceutical compositions, for example, fillers, diluents (diluents) , agglutinants, binders, lubricating agents, glidants, stabilizers, colorants, wetting agents, decomposers Disintegrants) and so on.

The term "vascular factor" or "vascular disease" as used in the present disclosure refers to any disease or disease that affects the vascular system, including the heart and blood vessels. In the present disclosure, a vascular factor or disease includes any factor or disease that causes loss of vascular function, such as inflammation, stenosis of blood vessels, or vascular occlusion. Examples of vascular diseases include, but are not limited to, atherosclerosis, coronary artery disease (CAD), myocardial infarction (MI), ischemia (ischemia), stroke, peripheral vascular disease (peripheral vascular disease). ), or pulmonary vascular disease.

Embodiments of the present disclosure are directed to preparing pharmaceutical compositions or agents with a cluster of gold nanoparticles to slow oxidative stress and/or aging of cultured cells, or oxidative stress and/or oxidative stress caused by vascular factors or diseases. Symptoms of aging.

The Chennai clusters of the present disclosure are red-lighted gold nanoclusters; in particular, gold nanoclusters coated with dihydrolipoic acid. The gold nanoclusters coated with dihydrolipoic acid used in the present invention are well known in the art, and the process thereof (Lin et al., 2009 ACS Nano 3: 395-401) is also in the technical field. It is well known and, therefore, no further explanation of its preparation process is required. The gold nanoparticle cluster coated with dihydrolipoic acid emits 650 nm of polarized light at an excitation wavelength of about 420 nm, and thus its emission wavelength ranges from red light to near infrared. Each of the Jinnai clusters has a particle size of between about 0.1 and 20 nanometers, preferably between about 1 and 15 nanometers, more preferably between about 2 and 13 nanometers. The particle size of the Jinnai cluster disclosed above is a Chennai cluster in a dry state, however, if the Chennai cluster used in the present disclosure is water soluble or at least dispersible in a liquid medium and/or Or in water, it would be better; due to coupling with peripheral solvent molecules (eg, water), the hydrodynamic size of the Jinnai clusters is significantly greater than its dry size. In one embodiment, the hydrodynamic size of the Jinnai cluster is approximately equivalent to 0.1 to 30 kilocalories (kDa) of polyethylene glycol.

According to an embodiment, the Chennai cluster of the present disclosure has antioxidant and/or aging activity when contacted with cultured cells. In this embodiment, about 1 to 1,000 nM of the gold nanoclusters are added to the culture medium for incubation with the cultured cells for a period of time, for example, 1 to 10 hours, preferably about 3 to 7 hours, more preferably about 4 to 6 Hours, and fresh medium was replaced during the reaction. The cultured cells treated with the Jinnai cluster can maintain at least 7 days, preferably 14 days, more preferably 21 days, and particularly preferably 28 days. Cells suitable for treatment with the nanogold pellets of the invention include, but are not limited to, human aortic endothelial cells, human epidermal cells, human endothelial precursor cells, human embryonic stem cells, and human mesenchymal stem cells.

In another aspect, the Chennai clusters of the present disclosure can be used to prepare a pharmaceutical composition or a medicament for treating oxidative stress and/or aging symptoms of a subject. The pharmaceutical composition or the agent of the present disclosure has an effective amount of a gold nanocluster; and a pharmaceutically acceptable excipient. A suitable individual receiving the pharmaceutical composition or the agent is oxidative stress and/or aging caused by vascular disease, for example, by atherosclerosis, coronary artery disease (CAD), myocardial infarction (myocardial) Infraction, MI), ischemia, stroke, peripheral vascular disease, or oxidative stress and/or aging caused by pulmonary vascular disease. The individual is a human or mammal, such as a monkey and a chimpanzee.

The pharmaceutical composition or the administration method of the medicament may be systemic or local. The Cannell clusters disclosed in this application can be administered using any of the commonly accepted modes of administration. Administration can be by, for example, oral, lingual, sublingual, buccal, rectal or parenteral (ie, bypassing the intestinal tract, such as intravenously, intraarterially, heart) Intracardially, intracutaneously, subcutaneously, transdermally, intraperitoneally or intramuscularly, oral or intravenous administration is preferred.

For the purposes of this disclosure, the Chennai clusters of the present disclosure can be made into a general formulation, for example, lozenges, dragees, pills, granules, aerosols, syrups, emulsions, suspensions, solutions, ointments. A cream or gel or any form, especially using an inert, substantially non-toxic, and pharmaceutically acceptable carrier or solvent. To this end, the therapeutically effective amount of the Chennai cluster of the present disclosure in any case, i.e., an amount sufficient to achieve a specified or desired dosage range, is from about 0.001 to about 99% by weight of the total mixture ( Between the percentage concentrations), preferably from about 0.01 to about 95% (percentage concentration). Although, depending on the type of body weight and/or route of administration, the individual response of the agent, the type of formulation, and/or the time or interval of administration, it is necessary to suitably deviate from the above dosage range. Therefore, in some cases, a dose smaller than the above-mentioned dose range may be administered or, in other cases, a dose exceeding the above upper limit value. In the case of high dose administration, it is recommended to divide the high dose into several single doses for administration within a prescribed interval, for example, within one day.

The gold nanoclusters of the present disclosure may be diluted in a solvent and/or carrier (and emulsifiers and/or dispersing agents, as appropriate) under appropriate conditions to formulate them. For example, in the case of using water as a diluent, an organic solvent may be used as an auxiliary solvent if circumstances permit.

The dosage of the nano-nano clusters of the present disclosure ranges from about 0.001 to about 500 mg/kg body weight, preferably from about 0.001 to about 100 mg/kg body weight, more preferably from about 0.001 to about 100 mg/kg body weight, depending on the route of administration. 0.01 to about 50 mg / kg body weight. Although, depending on the weight and/or route of administration, the individual response of the agent, the type of formulation, and/or the time or interval of administration, it may be necessary to properly deviate from the above dosage range. Therefore, in some cases, the dose may be less than the above-mentioned dose range or exceed the upper limit value, and the desired dose may be achieved. When a high dose is administered, it is recommended that the high dose be divided into several single doses for administration over a specified period of time, for example, within one day; the administration mode is administered in several single doses or continuously (ie, continuous intravenous injection). . This mode of administration is more likely to be applied to chronic treatment (ie, the form of a lozenge).

Example 1 Preparation of a gold nano cluster

The fluorescent gold nanoclusters of the present invention were prepared by the method previously described (see Lin et al., ACS Nano 2009 3:395-401). Briefly, 6-nano and dodecyldimethylammonium bromide were synthesized via a single phase reaction (see Jana and Peng, J Am Chem Soc 2003 125: 14280-14281). Didodecyldimethylammonium) Stablely bound gold nanoclusters (AuNP@DDAB), the composition of which is shown in Figure 1. The gold precursor solution (gold chloride dissolved in the dodecyldimethylammonium bromide-toluene solution) was gradually added dropwise to gradually absorb the plasma until the solution turned yellow and transparent. Next, the previously prepared nano-clusters are added to the reduced state lipoic acid for ligand replacement; the reduced lipoic acid needs to be prepared freshly, which is based on the molar ratio of lipoic acid to tetrabutylammonium bromide (TBAB). Mix 4:1 ratio. The above procedure produced a mixture of dark brown nano-cluster clots, which were agglomerated by ultraviolet light (365 nm, 30 minutes) of nano-cluster clots. The supernatant was removed, methanol was added to the nanoclusters clot to resuspend and disperse the nanocluster clots, followed by precipitation with chloroform to remove free surfactant. The dried nano-cluster precipitate can again be suspended in borate buffer (pH 9). This was followed by ultracentrifugation (110,000 rpm) three times to remove excess lipoic acid. In order to collect the Jinnai cluster, PBS buffer was added to a 30 kilo-dollar molecular weight cut-off (MWCO) concentrated centrifuge tube until the nano-cluster transparent solution colloid was stable and had no plasma absorption wave front. The concentration of the Jinnai cluster is about 450,000 M ^-1 cm ^-1 at a wavelength of 420 nm.

Example 2 Reducing Active Oxygen Species (ROS) in Cultured Cells 2.1 culturing the cells and transporting the gold nanoclusters of Example 1.

Human arterial endothelial cells were cultured in endothelial cell growth medium MV (this medium was named "complete culture medium" purchased from PromoCell, Heidelberg, Germany). The cells were seeded at a density of 10,000 cells per square centimeter onto a 1% gelatin coated plastic dish or 2% gelatin coated coverslip and cultured in a 37 ° C incubator containing 95% air and 5% CO ₂ . The Chennai cluster of Example 1 was delivered to human arterial endothelial cells in serum-free complete medium with and without the addition of a cationic lipid reagent (LipofectAMINE 2000, Invitrogen, Carlsbad, California, USA). Briefly, the Chennai cluster of Example 1 and a lipid carrier were diluted in 250 microliters of serum-free or antibody-free medium. After 5 minutes of incubation, the diluted gold nanotusules of Example 1 and the lipid carrier were thoroughly mixed and cultured for 10 minutes. The gold nanocapsule/lipid carrier complex is added to a plastic dish containing cells and a conditioned medium (i.e., medium cultured for 3 to 5 days in advance via feeder cells). After culturing for 4 hours, the medium containing the gold nanoparticles cluster of Example 1 was replaced with fresh medium, and then the cell type change of the human arterial endothelial cells was observed under a microscope and the reactive oxygen species were analyzed by flow cytometry. . At room temperature, all samples were recorded with a microscope (DM IRBE, Leica, Wetzlar, Germany) objective lens 20 times/aperture 0.4 and a CCD camera (DC 300F, Leica). The results are shown in Fig. 2.

Fig. 1 is a photograph of human arterial cells treated with a gold nanocapsule of Example 1 and photographed by a phase contrast microscope or a fluorescence microscope on days 1, 7, 21 or 28 after the treatment, respectively. These photographs confirm that the Chennai cluster of Example 1 is capable of maintaining the cell type of human arterial epithelial cells for at least 28 days.

2.2 Decreasing reactive oxygen species with the gold nanoclusters of Example 1

Using a fluorescent dye, 2,7-dichlorodihydrofluorescein diacetate dye (DCFDA) (purchased from Invitrogen, Grand Island, NY, USA) The oxygen content of human arterial endothelial cells was evaluated to estimate the oxidative stress in the cells. The final concentration of the fluorescent dye reacted with the cells was 10 μM. After 1 hour of incubation, the cells were washed with HBSS buffer, followed by suspension of the cells with trypsin and immediately analyzed by FACScan flow cytometry. The FL1 detector detects the dichlorofluorescent yellow (DCF) signal, which is the oxidized form of the DCFDA fluorescent substance, thereby detecting the median value of the fluorescence intensity, thereby relatively assessing the degree of oxidation in the cell. The results are shown in Table 1.

Table 1 Reduction of reactive oxygen species in human arterial epithelial cells by human clusters of gold nanoparticles

It can be confirmed from the above table data that the gold nanoclusters of Example 1 can effectively reduce the amount of active oxygen species or slow down the oxidation pressure, and the active oxygen species decreases by 12%, 26% and 31 on days 14, 21 and 28, respectively. %.

Figure 1 is a schematic diagram showing the chemical structure of the Jinnai cluster;

Figure 2 is a photograph of human arterial cells taken with or without the gold nanoclusters of Example 1 and photographed by phase contrast microscopy or fluorescence microscopy on days 1, 7, 21 or 28 after treatment, respectively.

Claims (13)

A method for slowing oxidative stress and/or aging of cells cultured in vitro, comprising adding an effective dose of one of a group of gold nanoparticles to a cultured cell, wherein the particle size of the golden nanoclusters is from 1 to 20 Nano. The method of claim 1, wherein the gold nanoclusters are gold nanoclusters coated with dihydrolipoic acid on the outer layer. The method of claim 1, wherein the cultured cells are selected from the group consisting of human aortic endothelial cells, human epidermal cells, human endothelial precursor cells, human embryonic stem cells, and human mesenchymal stem cells. The method of claim 1, wherein the effective dose of the Chennai cluster is between about 1 and 1,000 nM. A use of a gold nanoclusters to slow the oxidative stress and/or aging of cells cultured in vitro, wherein the Jinnai clusters have a particle size of from 1 to 20 nm. The use of claim 5, wherein the gold nanoclusters are gold nanocapsules coated with dihydrolipoic acid on the outer layer. The use according to claim 5, wherein the cultured cells are selected from the group consisting of human aortic endothelial cells, human epidermal cells, human endothelial precursor cells, human embryonic stem cells, and human mesenchymal stem cells. A use of a golden nano-cluster for the manufacture of a body for treatment A pharmaceutical composition of oxidative stress and/or aging symptoms, wherein the Chennai cluster has a particle size of between about 1 and 20 nanometers. The use of claim 8, wherein the gold nanoclusters are gold nanoclusters coated with dihydrolipoic acid on the outer layer. The use of claim 8, wherein the oxidative stress and/or aging is caused by a vascular factor. The use of claim 10, wherein the vascular factor is an inflammatory or vascular disease. The use according to claim 11, wherein the vascular disease may be any one of the following: atherosclerosis, coronary artery disease (CAD), myocardial infarction (MI), ischemia (ischemia), Stroke, peripheral vascular disease, or pulmonary vascular disease. The use of claim 8, wherein the individual is a human.