WO2020009551A1 - Graphene quantum dot as therapeutic agent for disease associated with abnormal fibrillation or aggregation of neuroprotein - Google Patents

Abstract

The present invention provides a graphene quantum dot as a therapeutic agent for diseases associated with abnormal fibrillation or aggregation of neuroproteins. The graphene quantum dot according to the present invention suppresses α-syn fibrillation or disaggregates already formed α-syn fibrils, and shows the working effect of passing through the blood brain barrier (BBB). Therefore, the graphene quantum dot according to the present invention can be advantageously used as a therapeutic agent for diseases associated with abnormal fibrillation or aggregation of neuroproteins, such as neurodegenerative diseases, inflammatory diseases, and metabolic diseases.

Description

Graphene quantum dots as therapeutic agents for diseases related to abnormal fibrosis or aggregation of neuroproteins

FIELD OF THE INVENTION The present invention relates to graphene quantum dots and their use, and more particularly, to graphene quantum dots and their use as therapeutic agents for degenerative neurological, inflammatory or metabolic diseases associated with abnormal fibrosis or aggregation of neuroproteins.

Alpha-synuclein (α-syn) is a protein abundant in the human brain, found mainly at the ends of nerve cells (neurons), and is known to interact with phospholipids and proteins within this structure. When α-syn becomes abnormally aggregated, it forms α-syn fibrils (α-syn PFFs), and by this α-syn fibrils synucleinopathy, that is, degenerative neuropathy Metabolic diseases are known to occur. Therefore, the development of therapeutic agents that can treat degenerative neurological diseases, metabolic diseases, etc. by actively inhibiting the fibrillation of α-syn or by decomposing the already formed α-syn fibrils (Korean Patent Publication 10-A) 2018-0081465). However, there are no practically available therapeutic agents that have a significant effect on synuclein disease, and therefore, there is an urgent need for development of therapeutic agents.

Graphene quantum dots, on the other hand, have been used only for drug carriers. However, the present inventors have found the effect of inhibiting α-syn fibrillation or decomposing α-syn fibrils already formed, and completed the present invention.

It is an object of the present invention to provide graphene quantum dots which exhibit therapeutic activity in diseases associated with abnormal fibrosis or aggregation of neuroproteins.

Another object of the present invention is to provide a use of the graphene quantum dots according to the present invention.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, and other tasks not mentioned above may be clearly understood by those skilled in the art from the following description. will be.

In the present invention, graphene quantum dots having a negative charge on the surface, having an average diameter of 0.5 to 10 nm, an average height of 0.1 to 3 nm, and a wt% ratio of carbon and oxygen in a ratio of 4.0 to 6.5: 3.0 to 6.0 To provide. The average diameter may be more preferably 1 to 5 nm, the average height may be 0.5 to 2.5 nm, but is not limited thereto.

In one embodiment of the invention, the graphene quantum dot may comprise a carboxyl group preferably with a terminal functional group, the terminal functional group is preferably -C = O peak and aromatic -C of the carboxyl group in the FT-IR spectrum The absorbance ratio of the = C- peak may be 1: 1 or more, and more preferably, the absorbance ratio may be 1: 1 to 2: 1.

In another embodiment of the present invention, the -C═O peak may appear at 1700-1750 cm ⁻¹ and the aromatic —C═C- peak may appear at 1600-1650 cm ⁻¹ .

In another embodiment of the present invention, the graphene quantum dots can inhibit α-syn fibrillation or break down the already formed α-syn fibrils and may cross the blood brain barrier.

In another embodiment of the present invention, the graphene quantum dot is not toxic in the body, it can be stably discharged in vitro through the urine.

The present invention also provides a pharmaceutical composition for the prevention or treatment of diseases associated with abnormal fibrosis or aggregation of neuronal proteins comprising the graphene quantum dots as an active ingredient.

In one embodiment of the present invention, the diseases associated with abnormal fibrosis or aggregation of neuronal proteins are degenerative neurological diseases, inflammatory diseases or metabolic diseases, the neurodegenerative diseases Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, dementia, Stroke, amyloidosis, fibrosis, encephalopathy, multiple sclerosis, etc., the inflammatory diseases include erythema, atopic, rheumatoid arthritis, Hashimoto's thyroiditis, pernicious anemia, Edison's disease, type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, thyroid Hypofunction, hyperthyroidism, scleroderma, Behcet's disease, inflammatory bowel disease, myasthenia gravis, Meniere's syndrome, Guilian-Barre syndrome, Sjogren's syndrome, endometriosis, psoriasis, Vitiligo, systemic scleroderma, ulcerative colitis, etc., wherein the metabolic disease is diabetes, Blood pressure, hyperlipemia, or the like, but dyslipidemia, nonalcoholic fatty liver, if the disease that can be caused by abnormal fibrosis or aggregation of α-syn is not limited thereto.

The present invention also provides a method for preventing or treating diseases related to abnormal fibrosis or aggregation of neuroproteins, comprising administering to a subject a composition comprising the graphene quantum dots as an active ingredient.

The present invention also provides a prophylactic or therapeutic use of diseases associated with abnormal fibrosis or aggregation of neuronal proteins of the composition comprising the graphene quantum dots as an active ingredient.

The graphene quantum dots according to the present invention can inhibit the formation of α-syn fibrils or decompose the already formed α-syn fibrils, exhibit not only cytotoxic effects through the blood brain barrier, but also no cytotoxicity. Therefore, graphene quantum dots according to the present invention can be effectively used as a therapeutic agent for degenerative neurological diseases, inflammatory diseases, metabolic diseases and the like associated with abnormal fibrosis or aggregation of neuronal proteins.

1A-1K show the results of analysis of the effect of graphene quantum dots on the inhibition of α-syn fibrillation and degradation of α-syn fibrils already formed.

2A to 2H show detailed analysis of the interaction between graphene quantum dots and α-syn fibrils already formed during the decomposition of fibrils.

3a to 3j show the effect of graphene quantum dots on neuronal cell death and propagation of α-syn fibrils between neurons by α-syn PFFs in vitro.

4A to 4O show the effect of graphene quantum dots on pathologies induced by α-syn PFFs in vivo.

5 is a schematic diagram showing an overview of the therapeutic effect of graphene quantum dots on Parkinson's disease etiology.

6A to 6G show the results of analyzing the synthesis and biotinylation of graphene quantum dots and binding of biotin-graphene quantum dots to α-syn fibrils.

7 shows the results of graphene quantum dots on the degradation of α-syn fibrils.

8 shows the effect of graphene quantum dots on the degradation of α-syn PFFs.

9 is a result of ¹ H- ¹⁵ N HSQC spectrum analysis of synthesized graphene quantum dots.

10A to 10G show the effects of graphene quantum dots on neuronal cell death induced by α-syn PFFs and limited neurites growth.

11a to 11j show the effect of graphene quantum dots on mitochondrial dysfunction induced by α-syn PFFs and free radical induction.

12A to 12H show results of graphene quantum dots and cell live imaging-related results on primary neuronal toxicity and pathological induction at various treatment time points induced by α-syn PFFs.

13A to 13N show the blood brain barrier permeability of graphene quantum dots.

14 shows the effect of graphene quantum dots on glial cell activation induced by α-syn PFFs in the substantia nigra.

15A to 15C show results of graphene quantum dots on glial cell activation in brain stem of hA53T α-syn transgenic mice.

16A-16E show the results of prolonged in vitro and in vivo toxic effects of graphene quantum dots.

17A to 17H show comparison results of nano-GOs and rGQDs with respect to α-syn PFFs.

The inventors have completed the present invention by finding graphene quantum dots that can exhibit therapeutic activity in diseases associated with abnormal fibrosis or aggregation of neuronal proteins. The present inventors have a graphene quantum dot has an average diameter of 0.5 to 10 nm, the average height is 0.1 to 3 nm, the wt% ratio of carbon and oxygen has a structure consisting of 4.0 to 6.5: 3.0 to 6.0 ratio, the surface is It was confirmed that the negatively charged graphene quantum dots. In addition, it was confirmed that the graphene quantum dots are not toxic to cells and tissues at all, and can effectively prevent and treat abnormal fibrosis or aggregation of neuroproteins, that is, α-syn. In addition, since the graphene quantum dot can penetrate the blood brain barrier, it was confirmed that it exhibits a remarkable therapeutic effect in various degenerative neurological diseases, inflammatory diseases, metabolic diseases, etc. due to abnormal fibrosis or aggregation of α-syn.

Throughout this specification, the term "graphene" means that a plurality of carbon atoms are covalently linked to each other to form a polycyclic aromatic molecule, wherein the carbon atoms connected by the covalent bonds are 6-membered rings as basic repeating units. It is also possible to form, but further comprise a 5-membered ring and / or a 7-membered ring.

Throughout this specification, the term “graphene quantum dots (GQDs)” means graphene with nano-sized fragments.

Throughout this specification, the term "reduced graphene quantum dot" refers to graphene oxide having a reduced oxygen atom ratio through a reduction process, which may be abbreviated as "rGQDs".

Throughout this specification, the term "graphene oxide" is also called graphene oxides and may be abbreviated as "GOs". The graphene may include a structure in which a functional group containing an oxygen atom such as a carboxyl group, a hydroxy group, or an epoxy group is bonded to the graphene, but may not be limited thereto.

Throughout this specification, the term "nano-graphene oxide" may be abbreviated as "nano-GOs" as nano-scale graphene oxide having an average diameter of 15 nm or more and an average height of 5 nm or more.

Hereinafter, a graphene quantum dot and its use according to the present invention will be described in detail.

Graphene quantum dots

The present invention provides graphene quantum dots that can exhibit therapeutic activity in diseases associated with abnormal fibrosis or aggregation of neuroproteins.

Specifically, the graphene quantum dots according to the present invention have a negative charge on the surface, with an average diameter of about 0.5 to about 10 nm and an average height of about 0.1 to about 3 nm. Moreover, the wt% ratio of carbon and oxygen is a structure comprised by the ratio of 4.0-6.5: 3.0-6.0.

Since the graphene quantum dots according to the present invention exhibit negative charges on the surface, the graphene quantum dots may bind to the ends of α-syn fibrils, thereby inhibiting the formation of α-syn fibrils or converting the already formed α-syn fibrils into monomers Can be disassembled. Through this mechanism, the graphene quantum dots according to the present invention can exhibit therapeutic activity in diseases associated with abnormal fibrosis or aggregation of neuroproteins, for example, diseases such as Parkinson's disease and Lewy body dementia.

According to the present invention, the graphene quantum dots of the present invention may include oxygen atom-containing functional groups such as carboxyl groups, ketone groups, aldehyde groups, hydroxy groups, and epoxy groups as terminal functional groups. In particular, the carboxyl group exhibiting negative charge may be included as a main functional group.

According to an embodiment of the present invention, in the FT-IR spectrum of the graphene quantum dots of the present invention, the absorbance ratio of the -C═O peak and the aromatic —C═C- peak of the carboxyl group is about 1: 1 or more. In particular, the absorbance ratio is about 1: 1 to about 30: 1, about 1: 1 to about 20: 1, about 1: 1 to about 1:15, about 1: 1 to about 1:10, about 1: 1 To about 1: 7, about 1: 1 to about 1: 5, about 1: 1 to about 1: 3, or about 1: 1 to about 2: 1. However, the absorbance ratio is characterized in that the lower limit is about 1: 1, and the absorbance ratio may be about 1: 1 or more.

In the present invention, the -C = O peak means a peak appearing at about 1700 to about 1750 cm ^-1 , and the aromatic -C = C- peak means a peak appearing at about 1600 to about 1650 cm ^-1 . .

Graphene quantum dots according to the present invention have a mean lateral size of about 0.5 to about 10 nm and an average height of about 0.1 to about 3 nm, indicating a small size of nanoscale.

Specifically, the average diameter of the graphene quantum dots according to the present invention may be about 0.5 to about 10 nm, about 0.7 to about 7 nm, about 0.8 to about 6 nm or about 1 to about 5 nm.

In addition, the average height of the graphene quantum dots according to the present invention may be about 0.1 to about 3 nm, about 0.2 to about 3 nm, about 0.3 to about 3 nm, about 0.5 to about 3 nm or about 0.5 to about 2.5 nm.

The graphene quantum dots of the present invention mentioned above can suppress the fibrillation of the α-syn monomer or decompose the previously formed α-syn fibrils using negative charges generated on the surface of the quantum dots and have no toxicity in the body. In addition, the graphene quantum dots of the present invention exhibit a small size of the nano-scale can easily pass through the blood brain barrier (BBB). In addition, the graphene derivative of the present invention has not been found to accumulate in the body.

Therefore, the graphene quantum dots according to the present invention can be usefully used as a therapeutic agent for diseases related to abnormal fibrosis or aggregation of neuronal proteins.

Uses of Graphene Quantum Dots

The present invention provides the use of graphene quantum dots according to the present invention.

As described above, the graphene quantum dots of the present invention can cross the blood brain barrier to inhibit α-syn fibrillation or decompose the already formed α-syn fibrils into monomers. Therefore, the graphene quantum dots according to the present invention can be usefully used as a therapeutic agent for diseases related to abnormal fibrosis or aggregation of neuronal proteins.

According to one embodiment of the invention, the present invention provides a pharmaceutical composition for the prevention or treatment of diseases associated with abnormal fibrosis or aggregation of neuronal protein comprising a graphene quantum dot according to the present invention as an active ingredient.

In the present invention, the diseases associated with abnormal fibrosis or aggregation of neuronal proteins are degenerative neurological diseases, inflammatory diseases or metabolic diseases.

Specifically, the degenerative neurological disease may be selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, dementia, stroke, amyloidosis, fibrosis, encephalopathy and multiple sclerosis. However, the present invention is not limited thereto.

In addition, the inflammatory diseases include erythema, atopy, rheumatoid arthritis, Hashimoto's thyroiditis, pernicious anemia, Edison's disease, type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, hypothyroidism, hyperthyroidism, scleroderma, Behcet's disease, From the group consisting of inflammatory bowel disease, myasthenia gravis, Meniere's syndrome, Guilian-Barre syndrome, Sjogren's syndrome, endometriosis, psoriasis, vitiligo, systemic scleroderma and ulcerative colitis It may be selected. However, the present invention is not limited thereto.

In addition, the metabolic disease may be selected from the group consisting of diabetes mellitus, hypertension, hyperlipidemia, dyslipidemia and nonalcoholic fatty liver. However, the present invention is not limited thereto.

The pharmaceutical composition of the present invention may further include one or more pharmaceutically acceptable carriers in addition to the graphene quantum dots according to the present invention for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components, as necessary. And other conventional additives such as bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Accordingly, the pharmaceutical compositions of the present invention may be patches, solutions, pills, capsules, granules, tablets, suppositories, and the like. These formulations can be prepared by conventional methods used in the art for formulation or by methods disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA, and formulated into various formulations depending on the individual disease or component. Can be.

The pharmaceutical compositions of the present invention may be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex, The range varies depending on the state of health, diet, time of administration, method of administration, rate of excretion and the severity of the disease. The daily dosage of the graphene quantum dots according to the present invention is about 1 to 1000 mg / kg, preferably 5 to 100 mg / kg, and may be administered once to several times a day.

The pharmaceutical composition of the present invention may further include one or more active ingredients exhibiting the same or similar medicaments in addition to the graphene quantum dots according to the present invention.

According to another embodiment of the present invention, the present invention provides a method for preventing or treating diseases associated with abnormal fibrosis or aggregation of neuroproteins comprising the administration of a therapeutically effective amount of graphene quantum dots according to the present invention.

As used herein, the term “therapeutically effective amount” refers to the amount of graphene quantum dots effective for the prevention or treatment of diseases associated with abnormal fibrosis or aggregation of neuroproteins.

The method of preventing or treating diseases associated with abnormal fibrosis or aggregation of neuroproteins of the present invention includes administering the graphene quantum dots, thereby not only treating the disease itself before the onset of the symptoms, but also inhibiting or avoiding the symptoms thereof. In the management of a disease, the prophylactic or therapeutic dose of a particular active ingredient will vary depending on the nature and severity of the disease or condition and the route by which the active ingredient is administered. Dosage and frequency of dose will vary depending on the age, weight and response of the individual patient. Appropriate dosage regimens can be readily selected by those of ordinary skill in the art that naturally consider such factors. In addition, the method of preventing or treating diseases associated with abnormal fibrosis or aggregation of neuronal proteins of the present invention may further include the administration of a therapeutically effective amount of an additional active agent to help treat the disease in conjunction with the graphene quantum dots. In addition, the additional active agent may exhibit a synergistic or auxiliary effect with the graphene quantum dots.

According to another embodiment of the invention, the invention provides the use of the graphene quantum dots according to the invention for the manufacture of a medicament for the treatment of diseases associated with abnormal fibrosis or aggregation of neuroproteins. Graphene quantum dots for the preparation of the medicament may be mixed with an acceptable adjuvant, diluent, carrier and the like, and may be prepared in a complex formulation with other active agents to have a synergistic action of the active ingredients.

The matters mentioned in the uses, compositions and methods of treatment of the invention apply equally unless they contradict each other.

As used herein, "prevention" means any action that inhibits or delays the onset of a disease associated with abnormal fibrosis or aggregation of neuroproteins by administration of a composition according to the present invention.

As used herein, "treatment" means any action in which the symptoms of a disease associated with abnormal fibrosis or aggregation of neuroproteins are improved or beneficially altered by administration of a composition according to the invention.

In the present specification, "individual" refers to a subject to which the composition of the present invention can be administered, and the subject is not limited.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Example : Graphene Quantum dots  ( GQDs Manufacturing

Carbon fiber (0.9 g) was added to a mixed solution of sulfuric acid (300 ml) and nitric acid (100 ml) and then heated at 80 ° C. for 24 hours (thermo-oxidation process, see FIG. 1). Dilution with deionized water was followed by dialysis with a regenerated nitrocellulose membrane (Cat #: 06-680-2G; MWCO 1,000 Daltons; Fisher Sicentific) to completely remove acid and excess carbon fragments. The GQD solution was then vacuum-filtered with a porous inorganic membrane filter (Cat #: 6809-5002; Whatman-Anodisc 47; GE Healthcare) to remove large particles. Thereafter, the solution was rotovap to obtain graphene quantum dots (GQDs) in powder form. The obtained graphene quantum dots contain both the graphitic domain and edge functional groups, which are the main elemental components of nitrogen (N), carbon (C), hydrogen (H), sulfur (S), and oxygen (O). Elemental analysis (EA) was performed. Elemental analysis was performed using an Elemental analyzer Flash2000 (Thermo Fisher Scientific). The results are shown in Table 1.

Graphene Quantum Store	element	N	C	H	S	O	Sum
	mol	0.10	2.80	1.29	0.07	1.62	-
	wt%	2.12	52.21	2.00	3.31	40.36	100

As shown in Table 1, the graphene quantum dot of the present invention was confirmed that the wt% ratio of carbon and oxygen, which is a core element, is composed of a ratio of 5.2 ± 1: 4.0 ± 1.

Comparative Example: Preparation of nano-GOs and rGQDs

To prepare nano-GOs, pristine graphene oxides (GOs) were synthesized by an improved Hummer method according to the literature. The obtained GO powder was added to demineralized water at a concentration of 10 mg / ml and tip-ultrasound treatment for 3 hours to obtain nano-GOs.

Reduction of GQDs was performed by autoclave based hydrothermal method at 200 ° C. for 2 hours to prepare rGQDs.

The products were analyzed by FT-IR spectroscopy and AFM analysis, and rGQDs and raw GQDs did not differ substantially in AFM results.

Experiment method

1. AFM Imaging

For AFM imaging, each sample (10 μg / ml) and α-syn fibrils (10 μg / ml) were dropped on a 1 cm ² silicon oxide substrate and dried at room temperature. Analysis was carried out in a non-contact mode (scan size: 25 μm ² , scan rate: 0.8 Hz) using XE-100 AFM (Park Systems). Images were acquired with the XE Data Collection Program (XEP 1.8.0).

2. FT-IR measurement

Samples were dried completely in a high vacuum desiccator and made by conventional KBr pellet method. And it was measured in Nicolet 6700 FT-IR spectrometer (Thermo Scientific) 32 times, the wave range of 4000 to 40 cm ^-1 conditions.

3. Ththiofluorotin T Fluorescence and Turbidity Analysis

For ThT analysis, 50 μl of each sample was centrifuged at 16,000 × g for 30 minutes. The pellet was then resuspended in 200 μl of 25 μM ThT (Cat #: T3516; Sigma-Aldrich) dissolved using 10 mM glycine buffer (pH 9.0). ThT fluorescence was measured at 482 nm (excitation at 440 nm) using a fluorescence spectrophotometer.

For turbidity analysis, α-syn fibrils were diluted at a rate of 1/10 with phosphate buffered saline (PBS). The diluted α-syn fibrils were then transferred to Corning's 96-well plates, and the absorbance intensity at 360 nm was measured using a microplate multiple reader to evaluate the turbidity of each sample.

4. TEM Imaging

The sample was adsorbed onto a 400 mesh carbon-coated copper grid (manufactured by EMS) which had been glow discharged for 2 minutes. Then, three drops of 50 mM Tris-HCl (pH 7.4) solution were rapidly dropped into the grid, followed by two successive drops of 0.75% uranyl formate for 30 seconds each. The stain was removed using # 1 Whatman filter paper. Samples were digitized images using an electron microscope Phillips CM 120 TEM operating at 80 kV and an ER-80 CCD (8 megapixels) from AMT, dried sufficiently before measurement. For neurons, primary cultured cerebral cortical derived neurons were cultured at a density of 100,000 cells / well on a 35 mm dish coated with poly-D-lysine. Neurons were treated with 1 μg / ml PFFs with or without 1 μg / ml graphene quantum dots in DIV 10. And after 7 days, neurons were washed with PBS containing 1% sodium nitrite (pH 7.4), 3% (v / v) paraformaldehyde (PFA), 1.5% (v / v) glutalaldehyde, 100 mM caco Fixed using fixed solution consisting of dilate and 2.5% (v / v) sucrose (pH 7.4) and post-fixed for 1 hour. Images were collected on a Phillips EM 410 TEM equipped with a Soft Imaging System Megaview III digital camera.

5. Dot-blot Analysis

Samples were loaded onto a pre-wet nitrocellulose membrane (pore size: 0.45 μm) using a Bio-Dot microfiltration device (Cat #: 170645; Bio-Rad) and adhered to the membrane using negative pressure. After washing each membrane with Tris-buffered saline, the membranes were blocked with Tween-20 containing Tris buffered saline containing 5% skim milk powder. Samples were bound overnight at 4 ° C. with a form-specific anti-α-syn filament antibody (Cat #: ab209538; 1: 1, abcam) and then at room temperature with HRP-conjugated secondary antibody (GE Healthcare) extracted from rabbits. Incubated for hours. The membrane was then washed several times with Tris buffered saline, imaged using ECL solution, and analyzed using ImageJ software.

6. BN-PAGE and SDS-PAGE

For BN-PAGE, prepare α-syn fibrils and α-syn PFF using NativePAGETM sample preparation kit (Cat #: BN2008; Life technologies) and use NativePAGETM Novex 4-16% Bis- for 90 minutes at 200 V Electrophoresis was performed using Tris gel (Cat #: BN1002Box; Life technologies). Cathode buffer solution contains 50 mM trisine, 15 mM bis-tris and 0.02% brilliant blue G (pH 7.0), and the anode buffer solution is 50 mM bis-tris (pH 7.0) It consists of. Gels were stained according to the manufacturer's instructions using the SilverQuestTM Silver Staining Kit (Cat #: LC6070; Life technologies).

For SDS-PAGE, 10 DIV cortical derived neurons were treated with α-syn PFF (5 μg / ml) in the presence or absence of 5 μg / ml graphene quantum dots for 7 days. Soluble protein of neurons was prepared using a cocktail of 1% TX-100 and protease and phosphatase inhibitors (Cat #: PPC1010; Sigma-Aldrich) in PBS solution at 4 ° C. . The lysate was then sonicated at 12,000 x g for 30 minutes at 4 ° C. The pellet was washed several times and suspended in PBS containing 2% SDS for insoluble protein preparation. The lysates were diluted using 2 × Laemmli Sample Buffer (Cat #: 1610737, Bio-rad). 20 μg of protein was then loaded into NovexTM 8-16% Tris-Glycine Gel (Cat #: XP08160BOX, Life technologies) and electrophoresis was performed at 130 V for 85 minutes. The protein is then transferred to a nitrocellulose membrane, blocked for 1 hour with 0.1% Tween-20 containing Tris containing 5% skim milk powder, anti-pS129-α-syn (Cat #: ab59264, 1: 1,000). , abcam), SNRP25 (Cat #: 111-002, 1: 2,000, Synaptic Systems) or VAMP2 (Cat #: ab3347, 1: 1,000, abcam) antibody was attached overnight at 4 ° C., and then HRP isolated from rabbit or rat -Incubated with conjugated secondary antibody (GE Healthcare) for 1 hour at room temperature. Blots were visualized with ECL solution and analyzed with ImageJ software.

7. Preparation of Ultrasonicated α-syn PFF

α-syn PFF was prepared according to the conventional method reported by Volpicelli-Daley et al (Nat Protoc 9, 2135-2146, doi: 10.1038 / nprot. 2014.143 (2014)). Mouse recombinant full length α-syn was cloned into ampicillin-resistant bacterial expression vector pRK172. The plasmid was then introduced and used in BL21 (DE3) RIL-competent E. coli (Cat #: 23045, Life technologies). After bacterial incubation, the α-syn monomers were isolated according to the above mentioned documents and purified purely through several purification steps, including anion exchange, dialysis and size exclusion chromatography. In vitro α-syn fibrils were aggregated with stirring at 37 ° C., 1,000 rpm for 7 days using an Eppendorf orbital mixer (Cat #: 538400020). Fragments of α-syn fibrils were made by treating a total of 60 pulses (˜0.5 s each) at 20% intensity using an ultrasound device with a 1/8 ”probe-sonypicizer. After 7 days of incubation at 37 ° C. with neurons, the fragments were naturally converted into mature fibrils in neurons and showed toxicity to the cells.

8. Biotinylation and Binding Analysis of Graphene Quantum Dots

To introduce the carboxyl group into the graphene quantum dots for functionalization, 50 mg of graphene quantum dots were dissolved in conjugation buffer (pH 4.7). Subsequently, 12.5 mg of EDC reagent (N-3 (Dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride, Cat #: 03449, Sigma-Aldrich) was added to introduce a carboxyl group into the graphene quantum dot. After stirring for 1 hour, 25 mg of EZ-Link Amine-PEG3-Biotin (Cat #: 21347, Thermo Scientific) was added to EDC-activated graphene quantum dots for 12 hours to bond the amide between graphene quantum dots and biotin. Was formed. The solution was dialyzed with a regenerated nitrocellulose membrane (Cat #: 06-680-2G, MWCO 1,000 Daltons, Fisher Scientific) to remove unreacted biotin and EDC reagents. The solution was then used in a rotary thickener to obtain the final product in powder form. For binding analysis between graphene quantum dots and α-syn fibrils, 5 mg / ml α-syn fibrils were replaced with 5 mg / ml biotinylated graphene quantum dots and streptavidin-coupled 0.8 nm gold nanoparticles (Cat #: 800.099, Aurion) for 1 hour. Thereafter, streptabadine-bound ultrafine gold particles bound to biotinylated-graphene quantum dots with high affinity were enhanced with GoldEnhanceTM EM Plus solution (Cat #: 2114, Nanoprobes) for 5 minutes. Unreacted solution was removed with a 100 kDa MWCO spin column (Cat #: UFC510024, Millipore-Sigma) followed by TEM analysis.

9. Purification of ¹⁵ N-labeled α-syn

The α-syn gene cloned into the pRK172 vector was introduced into E. coli BL21 (DE3) for α-syn overexpression. For the preparation of isotopically-labeled α-syn, 100 μg / ml in M9 medium containing 0.5 g of ¹⁵ NH ₄ Cl and 1 g of ¹³ C glucose (Cambridge Isotope Laboratory Inc., Andover, MA) per liter Cells were grown at 37 ° C. with ampicillin. After induction with isopropyl ß-D-thiogalactopyranoside (IPTG), the heat treated cell lysates were successively subjected to DEAE-Sephacel anion exchange, Sephacryl S-200 size exclusion and S-Sepharose cation chromatography Purified. The purified α-syn was dialyzed three times with 12 L of fresh 20 mM 2- (N-morpholino) ethanesulfonic acid (MES) buffer (pH 6.5) three times and dispensed at a concentration of 1 mg / ml at -80 ° C. Was stored. Samples were concentrated to 5 mg / ml using Nanosep 10 K membrane (Pall Gelman, Germany) at 4 ° C. immediately before the experiment.

10. NMR spectral analysis

Detailed NMR studies of the interaction between α-syn and graphene quantum dots were performed using a 950 MHz spectrometer (Bruker, Germany) equipped with a cryo-genic probe. Homogeneously classified ¹⁵ N-labeled α-syn (5 mg / ml, 100 μl) was reacted with GQD (5 mg / ml, 100 μl) with stirring at 1,000 rpm for 3 days at 37 ° C. ^{For 1} H- ¹⁵ N HSQC measurements, ¹⁵ N-labeled α-syn samples were prepared using 10 mM containing 20 mM MES buffer (pH 6.5). ¹ H- ¹⁵ N HSQC spectra of α-syn reacted with α-syn and graphene quantum dots were obtained at 37 ° C. The data obtained were treated with NMRPipe24 and analyzed with Sparky25.

11. Simulation Details

To investigate the interaction between graphene quantum dots and α-syn fibrils at the molecular level, 200 ns molecular dynamics (MD) simulations were performed with Gromacs 5.1. The initial structure of the hydrophobic NAC domain (residues 71-82) of α-syn was applied from the ssNMR structure (PDB ID: 2N0A) with the CHARMM forcefield, and the structure of the graphene quantum dots was obtained from https://cgenff.paramchem.org. Designed by CGenFF as protocol.

12. CD measurement

α-syn fibrils (5 mg / ml, 100 μl) were mixed with an aqueous solution of graphene quantum dots (5 mg / ml, 100 μl) and depolymerized under shaking culture at 37 ° C., 1,000 rpm for 7 days. For far-UV CD measurements, samples were diluted (1/2) with distilled water. CD spectra from 190 nm to 260 nm were measured at 0.5 nm intervals using a J-815 spectropolarimeter (Jasco, Japan) and a 0.2 mm long quartz cuvette. The spectrum of the buffer solution was excluded from the sample spectrum. CD signals were normalized in units of deg cm ² / dmol relative to the mean residual ellipticity [θ]. The fractional secondary structure content of α-syn fibrils and depolymerized α-syn fibrils was calculated using the CONTIN / LL algorithm of the DichroWeb online server. When calculated using CONTIN / LL, DichroWeb's Reference Set 7 was used and optimized for the wavelength range from 190 to 240 nm.

13. Primary Neuron Culture

Primary cortical derived primary neurons were prepared using 15 day old C57BL / 6 mouse embryos. Isolated neurons were prepared in culture medium consisting of Neurobasal Media (Cat #: 21103049, Life technologies) containing B27 supplement (Cat #: 17504044, Life technologies) and L-glutamine (Cat #: 25030149, Life technologies) Were plated in a culture dish coated with 50 μg / ml poly-D-lysine (Cat #: P6407, Sigma-Aldrich). Cultures were exchanged twice a week and maintained in a 37 ° C., 7% CO ₂ incubator. After 5 days of culture, 30 μM of 5-fluoro-2′-deoxyuridine (Cat #: F0503, Sigma-Aldrich) was treated to inhibit the growth of glial cells. All procedures for experiments using mice were approved and followed according to the guidelines of the Johns Hopkins University Animal Care and Use Committee.

14. Cell Survival and Cytotoxicity Analysis

For cell survival and cytotoxicity assays, primary cortical neurons were cultured at a density of 10,000 cells / cm ² in poly-D-lysine coated glass coverslips and medium exchanged twice a week in a 7% CO ₂ incubator. And incubated. Cytotoxicity of primary cultured neurons was determined after treatment of α-syn PFFs (1 μg / ml) on 10 DIV mouse cortical neurons for 7 days in the absence or presence of graphene quantum dots (1 μg / ml), LDH cytotoxicity analysis kit (Cat #: 88954, Pierce) was measured. Cell death was also determined using the TUNEL Assay Kit (Cat #: 12156792910, Roche). Viability of primary cultured neurons was measured using the alamarBlue cell viability assay kit (Cat #: DAL1025, Molecular Probes ™) and the neurite outgrowth staining kit (Cat #: A15001, Molecular Probes ™). The neurite outgrowth staining kit includes an orange dye that stains the cell membrane surface as a marker of neurite outgrowth, and cell-permeable staining that is converted from a non-fluorescent substrate to a green fluorescent product by intracellular esterases.

15. In vitro immunofluorescence

Mouse primary cerebral cortical neurons were plated at a density of 20,000 cells / cm ² on coverslips coated with poly-D-lysine. After fixing neurons using 4% PFA, 5% normal donkey serum (Cat #: 017-000-121, Jackson ImmunoResearch), 2% bovine serum albumin (Cat #: A7030, Sigma-Aldrich) and 0.1% Trion Blocking with X-100 (Cat #: T8787, Sigma-Aldrich) for 1 hour at room temperature. anti-8-OHG (Cat #: ab62623, 1: 1,000, abcam), anti-pS129-α-syn (Cat #: ab59264, 1: 1,000, abcam) and anti-MAP2 (Cat #: MAB3418, 1: 1,000 , Millipore) were incubated overnight at 4 ° C with antibodies. Samples were washed with PBS containing 0.1% Trion X-100, followed by FITC-conjugated secondary antibody (donkey anti-mouse FITC; Cat #: 715-095-151, donkey anti-rabbit FITC; Cat #: 711 -095-152, Jackson ImmunoResearch) and Cy3-conjugated secondary antibody (donkey anti-mouse FITC; Cat #: 715-165-151, donkey anti-rabbit CY3; Cat #: 711-165-1527, Jackson ImmunoResearch) The mixture was incubated with coverslips at room temperature for 1 hour. Fluorescence images were obtained through Zeiss confocal microscopy (LSM 710, Zeiss Confocla).

16. Complex I Activity Assay

Mitochondrial complex I enzyme activity was measured using the complex I enzyme activity microplate assay kit (Cat #: ab109721, abcam) according to the manufacturer's instructions. Briefly, primary cortical neurons were plated at a density of 1,000,000 cells / plate on 6 cm culture dishes coated with poly-D-lysine. Neurons were treated with 1 μg / ml of α-syn PFFs in the presence or absence of 1 μg / ml graphene quantum dots on day 10 in vitro. After 7 days of treatment, proteins were extracted from primary neurons using PBS containing 1/10 volume of detergent and incubated for 30 minutes on ice. The final protein concentration of the sample was adjusted to 5.5 mg / ml, centrifuged at 12,000 x g for 20 minutes, and the supernatant was placed in a microplate well and incubated for 3 hours at room temperature. After 3 hours, the plates were washed twice with buffer and 200 μl of assay solution was added. Mitochondrial complex I enzyme activity was measured at OD450 for 30 minutes at about 1 minute intervals.

17. Mitochondrial morphology evaluation

Cortical primary neurons were plated at a concentration of 10,000 cells / cm ² on glass coverslips coated with poly-D-lysine. Primary cultured cerebral cortical neurons were treated with α-syn PFF 1 μg / ml in the presence or absence of 1 μg / ml graphene quantum dots at DIV 10. After 7 days of treatment, neurons were stained with MitoTracker ^® Orange CMTMRos probe (Cat #: M7510, Life technologies) according to the manufacturer's instructions. And washed with: (A1429DJ, Life technologies Cat # ) briefly, and incubated for 30 min with primary MitoTracker ^® Orange CMTMRos probe of the neurons 100 nM, cell imaging solution. Stained mitochondria were imaged using Zeiss confocal microscopy (LSM710), and mitochondrial morphological features such as length or aspect ratio (AP, ratio of long axis and short axis of ellipse equivalent to mitochondria) were analyzed using ImageJ software.

18. Determination of oxygen consumption rate

Oxygen Consumption Rate (OCR) was measured using the Seahorse XF cell mito stress test kit (Cat #: 103015, Agilent) by modifying the manufacturer's instructions as follows. Briefly, primary cultured cerebral cortical neurons were plated at a concentration of 500,000 cells / well on hippocampal XF24 cell culture plates. Neurons were treated with α-syn PFF 1 μg / ml in the presence or absence of 1 μg / ml graphene quantum dots at DIV 10. After 7 days of treatment, neurons were washed with warm PBS and incubated for 1 hour at 37 ° C. in hippocampus assay medium. Thereafter, the plate was placed in an XF24 Extracellular Flux Analyzer (Seahorse Biosicince) and oxygen consumption was measured. Oxygen consumption was measured at 37 ° C. with a 1 min mixing, 1 min atmospheric and 2 min measurement protocol, followed by 45 min incubation in a CO ₂ free incubator and analyzed by XF24 analyzer. Sequential injections of oligomycin, carbonyl canide m-chlorophenylhydrazone (CCCP) and rotenone were used to assess basal respiration, coupling of the respiratory chain and mitochondrial respiratory dose. The measured oxygen consumption rate was normalized using the protein concentration of each well. The data expressed the change in percentage compared to the control.

19. Live Imaging

For slow confocal live imaging, primary cortical neurons plate 10,000 cells on a glass bottom dish (Cat #: 150682, NuncTM) coated with poly-D-lysine, 37 ° C., 7% CO ₂ incubator. Incubated at. In DIV 7, primary cultured neurons were treated with 100 nM LysoTrackerTM Blue DND-22 containing FITC labeled α-syn PFFs, 1 μg / ml GQDs-biotin and streptavidin Qdot complex and cell imaging solution (Cat #: L7525, Life technologies) for 1 hour. The petri dish was mounted on a Zeiss confocal microscope (LSM710) equipped with a temperature controlled CO ₂ culture system and then low speed images were captured at specified intervals at 488 nm and 561 nm laser excitation.

20. Microfluidic chamber

As a triple-compartment microfluidic device, Xona Microfluidic (Cat #: TCND1000) was used. Glass cover slips were coated with poly-D-lysine to attach to the microfluidic device. Plate about 100,000 neurons per chamber, pretreat 0.5 μg of GQDs in chamber 1 (C1) or chamber 2 (C2) in DIV 7 and then process 0.5 μg of α-syn PFF in chamber 1 (C1) It was. Treatment of α-syn PFF in the first chamber was performed in all test groups to create the appropriate transfer conditions for the next chamber. A 50 μl-difference of medium volume was maintained between the three compartments to control the direction of the fluid flow. Neurons were fixed using PBS with 4% PFA 14 days after α-syn PFFs treatment. Fixed neurons in the chamber were incubated for 1 hour at room temperature using a blocking solution containing 5% normal donkey serum, 2% bovine serum albumin and 0.1% Triton X-100. Neurons were incubated with anti-pS129-α-syn (Cat #: ab59264, 1: 1,000, abcam) and anti-MAP2 (Cat #: MAB3418, 1: 1,000, Millipore) antibodies overnight at 4 ° C., and the chamber was 0.1 After washing with PBS containing% Triton X-100, the mixture was incubated for 1 hour at room temperature with a mixture of FITC-conjugated secondary antibody (Jackson ImmunoResearch) and Cy3-conjugated secondary antibody (Jackson ImmunoResearch). Fluorescence images were obtained through a Zeiss confocal microscope.

21. Animals

All experimental procedures were performed according to the Animal Care and Use Guide Guidelines of the National Institute of Animal Health, approved by the Johns Hopkins Medical Institute Animal Care and Use Committee. Human α-syn-A53T transgenic mice (B6.Cg-Tg, Prnp-SNCA * A53T; 23 Mkle / J, stock #: 006823) were purchased from Jackson Lab.

22. In Vitro Blood Brain Barrier Permeability of Graphene Quantum Dots

Primary cultured mouse astrocytes for in vitro blood-brain-barrier (BBB) permeation experiments were prepared using 10 C58BL / 6 mice. Briefly, primary cultured mixed glial cells (neuroglia) were prepared using 1-d-old C57BL / 6 mice. Meninges were removed from the isolated cerebral cortex, and the cerebral cortex was disrupted with a 30 ml syringe with a 19-gauge, 0.5-inch needle attached. Cells were then plated in 75 cm ² T-flasks with DMEM supplemented with 10% FBS, 1 mM HEPES, 2 mM glutamine and antibiotic / antibacterial (Life technologies). Mixed glial cells were maintained in a 5% CO ₂ incubator at 37 ° C. and the culture was changed twice a week. Two weeks later, pure astrocytes were isolated using an astrocyte isolation kit (Cat #: 130-096-053, Miltenyl Biotec). Primary brain microvascular endothelial cells (BMEC) of C57BL / 6 mice were purchased from Cell Biologics. High purity (> 95%) astrocytes and BMECs were monitored by staining cell specific markers GFAP (for astrocytes) and CD31 (for BMEC, Cat #: ab28364, 1: 500, abcam). For in vitro blood brain barrier fabrication, 10 ⁶ cells / ml of astrocytes were added to the underside of collagen-coated 0.4 μm transwell inserts (Cat #: CLS3491, Sigma-Aldrich), under 37 ° C., 5% CO ₂ conditions. Incubate for 48 hours. The chamber was then carefully placed on a 6-well plate with 10 ⁷ cells / ml BMEC plated onto the insert. To confirm the structural integrity of in vitro BBB, epithelial electrical resistance using epidermal volt / ohm (TEER) instruments (Cat #: 300523, EVOM2, world precision instruments) at 0, 2, 4, 6 and 8 days after BMEC seeding. Was measured. To verify completeness, the blood brain barrier permeable 3 kDa dextran-fluorescein (Cat #: D3306, Life technologies) was loaded inside the transwell (blood side) and placed on a fluorescence spectrophotometer (dextran-fluorescein). Ex = 494 nm / Em = 521 nm, the ratio of blood brain barrier impermeable dextran rhodamine Ex = 555 nm / Em = 580 nm) was measured. Graphene quantum dots, biotin-graphene quantum dots, nano-GO remaining at 520 nm (Ex = 310 nm) using FluoroBriteTM DMEM medium (Cat #: A1896701, Life technologies) in the inner (blood side) and outer (cell membranes) And the concentration of rGQDs.

23. Exosome Isolation

To measure the concentration of graphene quantum dot-biotin in the released exosomes, BMEC or astrocytes were placed in a 6 cm culture dish and treated with 5 μg of graphene quantum dot-biotin for 12 hours. After 12 hours, exosomes were separated using exosome separation reagent (Cat #: 4478359, ThermoFisher) after 24 and 48 hours after the culture medium was changed.

24. In Vivo Blood Brain Barrier Permeability of Graphene Quantum Dots

For in vivo immunostaining of graphene quantum dot-biotin, 8-week-old C57BL / 6 mice were intraperitoneally injected with 2 mg / kg graphene quantum dot-biotin. The brain was isolated, fixed for 6 hours with 4% PFA, then dehydrated in 30% sucrose for 48 hours and subjected to immunohistochemistry analysis. Graphene quantum dot-biotin signals from olfactory bulb, neocortex, midbrain and cerebellum were visualized using the DAB kit. Graphene quantum dot-biotin positive signal in the cells of the above-mentioned region was confirmed by immuno EM staining after 20 minutes of incubation using GoldEnhanceTMEM Plus solution according to the manufacturer's instructions. In in vivo blood brain barrier experiments, 8 week old C57BL / 6 mice were intraperitoneally or intravenously injected with biotin (2 mg / kg) or vehicle. On days 7 and 14, brain and blood were obtained and the brain was homogenized with PBS containing 1% TX-100. The whole blood was coagulated by standing at room temperature for 30 minutes, and the coagulum was removed by centrifugation at 2,000 x g for 10 minutes. The concentration of graphene quantum dot-biotin was measured using QuantTag Biotin Quantification Kit (Cat #: BDK-2000, Vector Laboratories). The brain / plasma concentration ratio of graphene quantum dot-biotin was calculated for the brain / plasma ratio.

25. Stereoscopic Evaluation of Dopamine Neuronal Cell Death

All experimental procedures were performed according to the Animal Care and Use Guide Guidelines of the National Institute of Animal Health Care, which was approved by the Johns Hopkins Medical Institute Animal Care and Use Committee. 8-10 week old male C57BL6 mice were purchased from Jackson laboratory. Mouse was anesthetized with pentobarbital (60 mg / kg) and 2 μl of PBS or PFFs (5 μg / 2 μl) was used on one side of the striatum using a stereotaxic instrument (Cat #: Model 900; David KOPF instruments). Injection into hemispheres. The experimental group was intraperitoneally injected with 50 μl of GQD (50 μg per mouse) every 6 months for 6 months. Six months after injection, mice were perfused with PBS followed by 4% PFA. After fixing with 4% PFA for 12 hours, brains were dehydrated with 30% sucrose and analyzed by immunohistochemistry. The entire brain, including the midbrain nigra (SN, substantia nigra), was cut into 50 μm coronal sections and every fourth section was used for killing cell number analysis. Rabbit polyclonal anti-TH (Cat #: NB300-19; 1: 1,000; Novus Biologicals), rabbit polyclonal anti-pS129-α-syn (Cat #: ab59264; 1: 1,000; abcam) with blocking solution Incubated. After visualization using the DAB kit (Cat #: SK-4100; Vector Laboratories), biotinylated secondary antibody and streptavidin-conjugated horseradish peroxidase (HRP) (Cat #: PK-6101; Vector Laboratories Cultured). Stained tissue sections were placed on slides and then stained with Nissl bodies using thionine. The total number of TH and Nissl positive neurons in SN was measured using the Optical Factionator probe program of Stereo Investigator software (MBF Bioscience).

26. In vivo Glial Cell Immunohistochemistry

Animal brains were perfused with PBS and 4% PFA. After fixation after freezing, sections were made and cultured with anti-Iba-1 antibody (Cat #: 019-19741; 1: 1,000; Wako) or with anti-GFAP antibody (Cat #: Z0334; 1: 2,000; Dak). . The cultured tissue was again incubated with biotin-conjugated anti-rabbit antibody and ABC reagent (Cat #: PK-6101; Vector Laboratories) and then using DAB peroxidase substrate (Cat #: SK-4100; Vector Laboratories) Each section was stained. The number and density of glial cells in the SN was measured by ImageJ software (http://rsb.info.nih.gov/ij/, NIH). For major organ pathology, male C57BL / 6 mice 8-10 weeks old were intraperitoneally injected with 50 μg of graphene quantum dots every other week for 6 weeks. After 6 months of injection, animals were perfused and fixed using PBS and 4% PFA. Liver, kidney and spleen were separated and stained with H & E staining kit (Cat #: H-3502; Vector Laboratories).

27. Analysis of α-syn Aggregation Formation

HEK293T cells were plated on glass slides and transfected with pCMV5 vectors with myc-labeled α-syn variants (pCMV5-myc-SNCA-A53T, kindly gifted by Dr. Thomas C. Sudof) with A53T mutations Next, PBS (pH 7.4) or graphene quantum dots (0.1 μg / ml) were treated. After 48 hours of treatment, HEK293T cells were washed three times with PBS and fixed at room temperature for 20 minutes using PBS containing 4% PFA. After washing three times with PBS, the fixed cells were washed for 4 minutes using PBS containing 0.1% Triton X-100 (Sigma Aldrich). Thereafter, the cells were washed three more times with PBS and blocked for 20 minutes with PBS containing 5% donkey serum. α-syn expression was monitored using α-syn antibody (Cat #: 610787; 1: 1,000; BD Biosciences). Signals appearing at 550 nm and 570 nm in a laser scanning confocal microscope were imaged. The number of immune positive aggregates per field was measured and then quantified using ImageJ software (http://rsb.info.nih.gov/ij/, NIH) and normalized using the number of cells stained with DAPI.

28. Behavior Analysis

(1) cylinder test

The use of asymmetric forelimbs was used for the test. The animals were placed in a 20 cm clear plastic cylinder and the number of paw contact with the cylinder wall was measured. Wall contact was measured about 20-30 times per animal (when fully performed in the forward / rear and opposite to the contact). The number of forelimb contacts corresponding to the injured cerebrum was expressed as a percentage divided by the total number of forelimb contacts. All analyzes were performed by investigators blinded to different groups.

(2) pole test

All animals were acclimated for 30 minutes prior to behavioral experiments. The behavioral test rod was made using a 75 cm diameter metal rod wrapped with bandage gauze. The mouse was placed over the head at the top of the pole (7.5 cm from the top of the pole) and the total time taken to reach the pole pole was recorded. All mice were trained continuously for 2 days prior to the actual experiment. Each training session consisted of three individual training sessions, and on the test day all mice were evaluated in three sessions and the total time was recorded. The maximum blocking time to stop testing and recording was 60 seconds. Results for turn down, downhill and total time in seconds were recorded.

(3) clasping test

For hA53T α-syn mice, hind limbs were tested. The hind limb grip of the animal with the tail was observed for 10 seconds, and the score for each test was determined based on the following criteria.

1) Score 0: The hind legs spread outwards and away from the abdomen.

2) Score 1: One hind leg retracted for more than 5 seconds towards the abdomen.

3) Score 2: Two hind limbs partially retracted towards the abdomen for more than 5 seconds.

4) Score 3: Both hind legs fully retracted and touched the abdomen for more than 5 seconds.

29. Statistics

Data were measured as mean ± standard deviation from at least 3 independent experiments. To assess statistical significance, the Student t test or ANOVA test was performed with a Bonferroni post hoc analysis using Prism6 software (GraphPad). Assessments with p <0.05 were considered significant.

Experiment result

1. Effect of Graphene Quantum Dots on α-syn Fibrillation and Fibrillation

Figure 1a is a schematic diagram of the α-syn fibrillation and fibril decomposition effects with or without graphene quantum dots prepared according to the present invention.

Figure 1b is a graph quantifying the degree of α-syn fibrillation using the ThT fluorescence results. As shown in FIG. 1B, when only α-syn is present, α-syn fibrillation is rapidly increased to increase the fluorescence value. However, when the graphene quantum dots are reacted together, α-syn fibrils are not formed after 150 hours. There was no change in the fluorescence value.

1C shows turbidity assay results (n = 4, **** P <0.001, post-hoc Bonferroni tests at each reaction time point 0, 2, 4, 6, 8, 10, 12, 24, 72 and 168 hours). 2-way ANOVA, error bars are standard deviations). As shown in FIG. 1C, when only α-syn is present, α-syn fibrillation is rapidly increased to increase turbidity, but when the graphene quantum dots are reacted together, α-syn fibrils are not formed to increase turbidity. I could confirm that it did not.

1D is an image confirmed by TEM after fibrillation in the presence (right) and absence (left) of graphene quantum dots. As shown in FIG. 1D, α-syn fibrillation proceeded in the absence of graphene quantum dots, but it was confirmed that α-syn fibrils were not formed even after 7 days when the graphene quantum dots were reacted together.

FIG. 1E is a graph showing the degradation results of α-syn fibrils already formed after incubation with graphene quantum dots using samples of reactions monitored by ThT fluorescence. As shown in FIG. 1E, when the α-syn fibrils and the graphene quantum dots were reacted together, the α-syn fibrils were decomposed to reduce the ThT fluorescence.

Figure 1f shows several response time points (0, 1, 3, 12, 24, 72 and 168 hours, n = 4 at each time point, **** P <0.001, two-way ANOVA with post-hoc Bonferroni test, error Bars are graphs showing the results of turbidity analysis according to the standard deviation. As shown in FIG. 1F, when the α-syn fibrils and the graphene quantum dots were reacted together, it was confirmed that the α-syn fibrils were decomposed to decrease turbidity.

FIG. 1G is a graph showing the end-to-end length (n = 50 fibrils at each time point) and the number of fibrils (n = 4 at each time point) for the α-syn fibril decomposition effect of graphene quantum dots to be. As shown in Figure 1g, after the 6-hour reaction of the graphene quantum dots and α-syn fibrils significantly reduced the length of the α-syn fibrils, and after 24 hours the number of α-syn fibrils also decreased I could confirm it.

Figure 1h shows the amount of α-syn fibrils remaining at the same time point (0, 6, 12, 24 and 72 hours) in the presence of graphene quantum dots of the length of α-syn fibrils and the number of α-syn fibrils It is a graph showing the result of product analysis. As shown in Figure 1h, it was confirmed that the amount of α-syn fibrils significantly reduced by the graphene quantum dots.

1I is a TEM image of α-syn fibrils after several reaction time points (6 hours, 12 hours, 1 day, 3 days and 7 days) in the absence (top) or presence (bottom) of graphene quantum dots. As shown in FIG. 1I, the length of α-syn fibrils is reduced in the presence of graphene quantum dots, and it was directly confirmed that the formed α-syn fibrils were effectively decomposed.

Figure 1j shows the results of the measurement of α-syn fibrils by dot-blot analysis at various reaction time points (0 hours, 12 hours, 1 day, 3 days and 7 days) using an α-syn filament specific antibody. . As shown in FIG. 1J, as the reaction time was increased, the amount of α-syn fibrils was significantly decreased, and it was confirmed that α-syn fibrils were not detected at day 7.

FIG. 1K shows the results of BN-PAGE analysis of α-syn prepared as a sample of the reaction after various reaction time points (0 hours, 3 hours, 6 hours, 12 hours, 1 day, 3 days and 7 days). As shown in FIG. 1K, the 7-day reaction with graphene quantum dots showed that α-syn fibrils were decomposed to exist in the monomer (monomer) state.

Through the above results, it was confirmed that the graphene quantum dots of the present invention can not only inhibit the fibrillation of α-syn, but also effectively decompose the α-syn fibrils already formed.

2. During the decomposition process Graphene Quantum dots  Already formed α- syn Fibril  Mutual Analysis of the action

Figure 2a is a result of confirming the binding between the biotinylated graphene quantum dots and α-syn fibrils at low and high magnification using TEM. Triangular arrows indicate biotinylated graphene quantum dots combined with tiny gold-streptavidin nanoparticles. As shown in Figure 2a, it was confirmed that the graphene quantum dots and α-syn fibrils are combined.

Figure 2b is a graph showing the change in the length of the average width of the α-syn fibrils in the digestion process after 1 hour incubation (n = 20, * P <0.05, Student's t test, error bars are standard deviation in each group). As shown in Figure 2b, it was confirmed that the graphene quantum dots are combined with the α-syn fibrils to increase the average width.

2C is a graph showing the NMR chemical shift differences obtained from the ¹ H- ¹⁵ N HSQC spectrum. The reduced intensity ratio of chemical shift of NMR for each residue after binding with graphene quantum dots can be confirmed. As shown in Figure 2c, it was confirmed that the graphene quantum dots are bonded to the NAC and C-terminal portion of the α-syn fibrils.

2D is a snapshot image up to 65 ns and interaction between graphene quantum dots and α-syn fibrils already formed using time-lapse simulation kinetics. As shown in FIG. 2D, it was predicted that the graphene quantum dots bind to α-syn fibrils and change the structure of α-syn fibrils.

FIG. 2E is a time-dependent plot of the atomic position RMSD, SASA for α-syn fibrils group (black) and α-syn fibrils and graphene quantum dot groups (red), total potential energy (ΔUtot), electrostatic Energy (ΔElec) and Van der Waals energy (ΔEvan) for the α-syn fibrils and graphene quantum dot groups.

2F shows time-dependent plots calculated by the DSSP algorithm. As shown in Figure 2f, it was confirmed that beta-sheet is reduced, alpha-helix is increased.

2G is a graph showing the CD spectrum of α-syn monomer and α-syn fibrils in the absence and presence of graphene quantum dots after 7 days of incubation.

FIG. 2H is a graph showing the content of partial secondary structures of α-syn fibrils and degraded α-syn fibrils by graphene quantum dots calculated using the algorithm of CONTIN / LL. As shown in Figure 2h, ß-sheet was reduced, it was confirmed that the α-helix is increased.

Through the above results, it can be seen that the graphene quantum dots of the present invention can promote the decomposition of α-syn fibrils by directly binding to α-syn fibrils and modifying the structure of α-syn fibrils.

3. α- in vitro syn To PFFs  Effect of Graphene Quantum Dots on Neuronal Cell Death, Pathology and Metastasis

3A-C show α-syn PFFs (1 μg / ml) in the absence and presence of graphene quantum dots (1 μg / ml) for 7 days in 10 DIV mouse-derived cortical neurons, It is a graph showing the results of neuronal death (FIG. 3A), alamar Blue (FIG. 3B) and LDH analysis (FIG. 3C) evaluated by TUNEL. As shown in Figures 3a-c, graphene quantum dots did not show toxicity to the cells, it was confirmed that significantly reduced the cytotoxicity represented by α-syn PFFs.

Figure 3d is an image showing the results of the immunoblot (immunoblot) using the p-α-syn antibody, Figure 3e is a graph quantifying the amount of p-α-syn of the SDS insoluble fraction normalized to ß-actin (N = 3). As shown in Figure 3d and 3e, when treated with graphene quantum dots it was confirmed that the α-syn PFFs are significantly reduced.

FIG. 3F shows p-α-syn and p-α-syn antibody immunomicroscopic images in neurons, and FIG. 3G quantifies the extent of p-α-syn immune fluorescence intensity normalized to a PBS control (n = 3). It is a graph. As shown in FIGS. 3F and 3G, the red fluorescence was significantly increased in the experimental group treated with α-syn PFFs, but when treated with graphene quantum dots, the α-syn PFFs were decomposed to significantly reduce the red fluorescence value. Could.

3H is a schematic representation of a microfluidic device consisting of three chambers for identifying pathological α-syn transition.

3I is a representative image of p-α-syn immunostained neurons observed on the microfluidic device 14 days after addition of α-syn PFFs. 3J is a graph showing the quantification of p-α-syn immunofluorescence intensity. The area occupied by p-α-syn was measured in each chamber (n = 3, * P <0.05, ** P <0.01, *** P <0.001, NS, two-through the post-hoc Bonferroni test). Wei ANOVA, error bars are standard deviation). As shown in FIGS. 3i and 3j, the control group treated with α-syn PFFs on the first column showed high red fluorescence (α-syn fibrils) in all columns, whereas graphene quantum dots and α-syn PFFs were observed. In the experimental group treated with the first column (C1) and the α-syn PFFs together, the amount of α-syn PFFs was significantly decreased in the first column and the experimental group treated with the graphene quantum dots in the second column (C2). I could confirm that it does not.

Through the above results, it was confirmed that the graphene quantum dots of the present invention can significantly inhibit α-syn fibrillation by α-syn PFFs in neurons. In addition, it was confirmed that as α-syn PFFs metastasize in neurons, graphene quantum dots may also inhibit fibrillation by α-syn PFFs that metastasize together. Through this, it was confirmed that the graphene quantum dots of the present invention can be effectively used to suppress and treat neuronal disease metastasis caused by α-syn fibrillation.

4. Effect of graphene quantum dots on α-syn induced pathology in vivo

4A is a schematic diagram showing coordinates in which α-syn PFFs (5 μg) were injected into the striatum of C57BL / 6 mice. 50 μg of graphene quantum dots or PBS were intraperitoneally injected for 6 months every other week.

4B is an image of representative TH immunohistochemical staining of hemisphere SNs injected with α-syn PFFs in the absence (top) or presence (bottom) of graphene quantum dots. As shown in FIG. 4B, in the experimental group injected with only α-syn PFFs, neurons were killed and reduced in shade, whereas in the experimental group administered with graphene quantum dots, neuronal cell death by α-syn PFFs was suppressed and thus shaded. Was confirmed to recover similar to the normal control.

FIG. 4C shows the stereoscopic analysis of the number of TH- and Nissl-positive neurons in SN through stereoanalysis after 6 months of α-syn PFF injection in and without graphene quantum dots (n = 6 per group). This graph shows the result of the calculation. As shown in Figure 4c, in the experimental group administered with graphene with α-syn PFFs it was confirmed that the neuronal cell death by α-syn PFFs is reduced.

Figure 4d is a representative TH-immunohistochemical staining image of the hemisphere striatum injected with α-syn PFFs, Figure 4e is a graph showing the result of quantifying the density of TH-immune positive fibers in the striatum (n = for each group) 5 to 6). As shown in Figures 4d and 4e, in the experimental group administered with α-syn PFFs and graphene quantum dots, it was confirmed that the decrease in density of TH-immune positive fibers by α-syn PFFs was recovered.

4F is a graph showing cylinder test results during behavioral deficit assessment as measured by the use of the forefoot of mice (n = 5 to 6 for each group). In addition, Figure 4g is a graph showing the results of the pole test, the ability to grasp the pole during the behavioral deficit evaluation of mice (n = 5 to 6 for each group). As shown in Figures 4f and 4g, the abnormal behavior of the mice by the administration of α-syn PFFs when the graphene quantum dot is administered together was found to show a similar behavior type to the normal mouse control.

Figure 4h is an image showing the immunostaining image of the representative p-α-syn in the hemisphere SN injected with striatum and α-syn PFFs, Figure 4i is the immunity of p-α-syn in striatum and SN (n = 5) It is a graph showing the result of quantification of reactive neurons. As shown in Figure 4h and 4i, in the experimental group administered with graphene quantum dot was confirmed that the number of α-syn PFFs positive neurons significantly reduced.

4J is a schematic diagram showing the distribution of LB / LN-like pathologies in the CNS of hemispheres injected with α-syn PFFs (p-α-syn positive neurons: red dot, p-α-syn positive neurons: red line) . As shown in Figure 4j, in the experimental group administered with graphene quantum dot was confirmed that the degree of pathological distribution was significantly reduced.

4K is a schematic diagram of the observation area of the hA53T α-syn Tg model. 50 μg of graphene quantum dots or PBS were intraperitoneally injected for 4 months.

4L is an image of p-α-syn and α-syn fibrils measured by dot-blot analysis. As shown in FIG. 4L, in the experimental group to which graphene quantum dots were administered, p-α-syn and α-syn fibrils were significantly reduced in the brain stem (brainstem).

4M is a representative α-syn immunostaining image (n = 5 for each group) in the cortex, ventral midbrain and brain stem of hA53T α-syn Tg. As shown in FIG. 4M, the aggregates of α-syn were observed in the cortex, ventral midbrain, and brain stem of hA53T α-syn Tg mice, whereas graphene quantum dots were administered. In the case of the α-syn aggregates was confirmed that hardly observed.

4N is a graph showing the results of quantification (five in each group) of p-α-syn immune reactive neurons in hA53T cortex (Ctx), ventral midbrain (VM), and brainstem (BS). As shown in Figure 4n, it was confirmed that the number of p-α-syn immunoreactive neurons significantly reduced in the experimental group administered with graphene quantum dots.

FIG. 4O is a graph (left) showing the results of the test withdrawal during the behavioral deficit evaluation of the mouse and the graph (right) showing the results of the pole experiment during the behavioral deficit evaluation of the mouse (n = 5 for each group). As shown in FIG. 4O, the hA53T α-syn Tg mouse had an increased deletion behavior. However, in the experimental group administered with graphene quantum dots, the deletion behavior was recovered.

Based on the above results, the graphene quantum dots of the present invention effectively inhibit abnormal fibrosis or aggregation of α-syn or effectively decompose the formed α-syn aggregates in vivo, and are exhibited by abnormal fibrosis or aggregation of α-syn. It was confirmed that the pathological symptoms could be treated.

5. Therapeutic Effect of Graphene Quantum Dots on the Pathogenesis of Synuclein Disease

5 is a schematic diagram of the anti-aggregation efficacy of graphene quantum dots for the etiology caused by the metastasis and proliferation of pathological α-syn aggregation in vitro and in vivo. In the absence of graphene quantum dots, pathological α-syn monomers undergo spontaneous fibrosis to form fibrous aggregates, eventually leading to dopaminergic neurons, LB / LN analog construction and behavioral abnormalities. Graphene quantum dots, on the other hand, inhibit fibrillation of α-syn and break down fibrils formed into monomers to prevent loss of dopaminergic neurons, LB / LN analogs and behavioral disorders caused by abnormal α-syn fibrils And treatment.

6. Graphene Quantum dots  Synthesis Biotinylation  And Graphene Quantum dots Biotin and α syn Fibrillated  Binding analysis

6A is a schematic diagram showing graphene quantum dots synthesized by thermal oxidation cutting in carbon fiber.

FIG. 6B is an AFM image of synthesized graphene quantum dots, and FIG. 6C is a graph showing thickness distribution of graphene quantum dots. As shown in Figure 6b and 6c, it was confirmed that the average height of the graphene quantum dot is 0.1 to 3 nm.

6D is a schematic diagram of a synthetic procedure for biotinylating graphene quantum dots.

6E is an FT-IR spectrum of graphene quantum dots (black lines) and biotinylated graphene quantum dots (red lines). As shown in Figure 6e, it was confirmed that there is a change in the functional group when biotinylated graphene quantum dots.

FIG. 6F is a schematic diagram showing the preparation steps for binding affinity between biotinylated graphene quantum dots and nanogold-streptavidin, and FIG. 6G is a graphene quantum dot-biotin-nanogold-streptavidin complex (left). TEM image of α-syn fibrils already formed after 7 days in the presence of nanogold-streptavidin complex (right). As shown in FIG. 6G, biotinylated graphene quantum dots also bind to α-syn fibrils, and it was confirmed that the α-syn fibrils were degraded.

7. Effect of Graphene Quantum Dots on the Degradation of α-syn Fibrils

7A is a graph showing the distribution of α-syn fibril lengths at various reaction time points (0, 6, 12, 24 and 72 hours, n = 50 fibrils at each time point). As shown in FIG. 7A, before the reaction, a long α-syn fibril of 600 nm or more was observed, but as the reaction time increased, it was confirmed that the α-syn in monomeric form was observed.

FIG. 7B shows AFM images of α-syn fibrils after various reaction time points (0, 24 and 48 hours) and shows representative line profiles (graphene quantum dots: blue lines, α-syn fibrils: red lines) of designated areas. will be. As shown in FIG. 7B, even after the α-syn fibrils and the graphene quantum dots were reacted, there was no significant difference in height.

8. Effect of Graphene Quantum Dots on the Degradation of α-syn PFFs

FIG. 8A is a representative TEM image of fibrillated α-syn PFFs (5 mg / ml) reacted for 1 hour in the absence (top) and presence (bottom) of graphene quantum dots (5 mg / ml), and FIG. 8B is 1 hour After reaction, the end-to-end length of α-syn PFFs (n = 286; PFFs, n = 249; PFFs + GQDs, *** P <0.001; Student's t-test, error bars are standard deviations) . As shown in Figure 8a and 8b, it was confirmed that the end-to-end length was reduced by decomposing the graphene quantum dot fibrillated α-syn PFFs.

FIG. 8C is an image showing the results of evaluation of the amount of α-syn PFFs and degraded α-syn PFFs remaining after various reaction times by BN-PAGE. As shown in Figure 8c, the longer the reaction time was confirmed that the α-syn PFFs are present in the monomer form to decompose.

9. ¹ H- ¹⁵ N HSQC Spectrum Analysis

FIG. 9A shows that the ¹ H- ¹⁵ N HSQC spectrum of α-syn (red) incubated with graphene quantum dots completely overlaps with only ¹⁵ N-labeled α-syn monomer (black). The assigned residues appeared only in the ¹⁵ N-labeled α-syn monomer group. 9B shows the complete HSQC spectrum of ¹⁵ N-labeled α-syn monomers incubated with graphene quantum dots. Based on the peak intensity of the ¹⁵ N-labeled α-syn monomer group, residues (red) with no decrease or minimal reduction in peak intensity, residues with significant decrease in peak intensity (blue) and disappeared residues (Black) is noticeable. Through this, it was confirmed that the graphene quantum dot binds to the N-terminal or positively charged residue of the amino acid constituting α-syn.

10. α- syn With PFFs  For induced neuronal death and limited neurogenesis Graphene  Quantum dots effect

10A is an image of representative TUNEL-positive neurons. DIV 10 primary cortical neurons were treated with α-syn PFFs (1 μg / ml) in the absence and presence of graphene quantum dots (1 μg / ml). TUNEL analysis was performed after incubation for 7 days. As shown in FIG. 10A, in the experimental group treated with α-syn PFFs only, many red fluorescences were observed, whereas in the experimental group treated with α-syn PFFs and graphene quantum dots, red fluorescence was reduced, and α- It was confirmed that cell death by syn PFFs was reduced.

FIG. 10B is a representative microscopic image of growth degree and cell viability assay of neurites stained by outer cell membrane (red) and cell-permeability viability indicator (green), and FIGS. 10C and 10D show neurite growth and neuronal viability It is a graph showing the result of quantification. As shown in Figure 10b to 10d, the experimental group treated with α-syn PFFs alone reduced the growth of neurites, cell survival was reduced, while the experimental group treated with α-syn PFFs and graphene quantum dots together It was confirmed that the neurite outgrowth similar to the control group. It was also confirmed that cell death by α-syn PFFs was suppressed.

10E is a graph showing the results of immunoblot analysis of SNAP25 and VAMP2 protein levels. 10 DIV primary cortical neurons were treated with α-syn PFFs (5 μg / ml) in the absence and presence of graphene quantum dots (5 μg / ml), incubated for 7 days, and subjected to immunoblot. FIG. 10F is a graph showing normalization of the expression level of SNAP25 to beta-actin expression level followed by quantification (n = 3), and FIG. 10G is quantification of normalization of VAMP2 expression level to beta-actin expression level (n = 3). ) A graph showing the results. As shown in Figure 10e to 10g, the expression of SNAP25 and VAMP2 protein significantly decreased in the experimental group treated with α-syn PFFs alone, but in the experimental group treated with α-syn PFFs and graphene quantum dots, It was confirmed that the expression at a similar level.

11.α- syn To PFFs  Effects of Graphene Quantum Dots on Mitochondrial Dysfunction and Oxidative Stress Induction

11A shows representative 8-OHG (8-hydroxyguanine) of major cortical neurons treated with α-syn PFFs (1 μg / ml) and cultured for 7 days in the absence and presence of graphene quantum dots (1 μg / ml). Immunostained image (n = 4 for each group), FIG. 11B is a graph quantifying the amount of 8-OHG by immunofluorescence level. As shown in FIGS. 11A and 11B, when the α-syn PFFs are treated alone, the oxidative stress is increased, but when the α-syn PFFs and the graphene quantum dots are treated together, the oxidative stress due to the α-syn PFFs is decreased. It could be confirmed.

11C is a graph showing the results of measuring the activity of mitochondrial complexes after treatment with α-syn PFFs (1 μg / ml) and graphene quantum dots (1 μg / ml) and incubated for 7 days. As shown in FIG. 11C, it was confirmed that the inhibition of mitochondrial complex activity by α-syn PFFs was restored by graphene quantum dots.

11D is a representative MitoTracker positive microscope image. Primary cortical neurons at 10 DIV were treated with α-syn PFFs (1 μg / ml) in the absence and presence of graphene quantum dots (1 μg / ml). After incubation for 7 days, mitochondria were labeled with MitoTracker ^® Orange CMTM Ros (red). FIG. 11E is a graph showing the results of quantifying the length of stained mitochondria (n = 50 for each group), and FIG. 11F is a graph showing the aspect ratio of stained mitochondria (n = 50 for each group). And FIG. 11G is a representative TEM image of low and high magnification of stained mitochondria. As shown in Figure 11d to 11g, it was confirmed that the reduction of the length and aspect ratio of mitochondria by α-syn PFFs is recovered by the graphene quantum dots.

11H is a graph showing micro-plate based respiratory counts for neurons. Oxygen consumption was measured with an XF 24 Seahorse analyzer using primary cortical neurons treated with α-syn PFFs (1 μg / ml) in the absence and presence of graphene quantum dots (1 μg / ml) for 24 hours (per group). n = 4). FIG. 11I is a graph quantifying the basal respiratory rate of the respiratory measurement results, and FIG. 11J is a graph quantifying the maximum respiratory rate of the respiratory measurement results. As shown in Figure 11h to 11j, it was confirmed that the respiratory rate reduction by α-syn PFFs is recovered by the graphene quantum dots.

Through the above results, it was confirmed that the graphene quantum dot of the present invention exhibits a mitigating or therapeutic effect on oxidative stress due to abnormal fibrosis or aggregation of α-syn, abnormality of mitochondria, and dysfunction.

12. Graphene Quantum dots  Α- at different treatment points syn To PFFs  Effect of Graphene Quantum Dots and Cell Live Imaging on the Toxicity and Pathological Phenomena of Primary Neurons Induced by

Cortical neurons in 10 DIV mice were cultured on day 3 (n = 4, After) after 3 days (n = 4, Before), concurrent (n = 4, Simul) and graphene quantum dot treatment (1 μg / ml) α-syn PFFs (1 μg / ml) were treated.

12A shows the results of alarmarBlue fluorescence analysis. As shown in FIG. 12A, it was confirmed that cell death by α-syn PFFs was inhibited by graphene quantum dots. Α-syn PFFs treated 3 days before graphene quantum dot treatment, α-syn PFFs and graphene quantum dots treated simultaneously, graphene quantum dots treated and α-syn PFFs treated 3 days after both cell death It was confirmed that this was effectively suppressed.

12B is a graph showing the LDH analysis result. As shown in FIG. 12B, it was confirmed that LDH secretion by α-syn PFFs was suppressed by graphene quantum dots, through which cell death by α-syn PFFs was effectively inhibited by treatment of graphene quantum dots. I could confirm it.

12C is a representative image of p-α-syn by dot-blot analysis, and FIG. 12D is a graph showing quantified results normalized to a PBS control (n = 4 for each group). As shown in FIGS. 12c and 12d, it was confirmed that α-syn PFFs were effectively decomposed by graphene quantum dots.

FIG. 12E is a representative p-α-syn immunostained fluorescence microscopic image with p-α-syn antibody after 7 days of incubation, FIG. 12F quantifies the immunofluorescence intensity of p-α-syn normalized with PBS control Graph showing (n = 4 for each group). As shown in FIGS. 12E and 12F, it was confirmed that α-syn PFFs were effectively decomposed by graphene quantum dots.

FIG. 12g confirms that graphene quantum dots and α-syn PFFs are co-located on lysosomes of neurons in a live image. LysoTracker (blue), graphene quantum dot-biotin-streptavidin Qdot complex (red) and FITC-labeled α-syn PFFs (green) were used.

12h shows that the degradation process of α-syn PFFs by graphene quantum dots was monitored by a slow fluorescence signal using live imaging.

Through the above results, it was confirmed that the graphene quantum dots of the present invention effectively decomposed α-syn PFFs when treated before, simultaneously with, or after formation of α-syn PFFs. Through this, it was confirmed that the graphene quantum dots of the present invention can be used both for the prevention and treatment of diseases related to abnormal fibrosis or aggregation of neuronal proteins.

13. Blood Brain Barrier Permeability of Graphene Quantum Dots

13A is a schematic diagram of blood brain barrier permeability in vitro experiment.

13B shows the results of confluent monolayer formation of cerebral microvascular endothelial cells (BMEC) and astrocytes by immunofluorescence staining with CD31 (endothelial marker) or GFAP (astrocytic marker) antibody, respectively.

Figure 13C shows the epithelial electrical resistance (TEER) at various time points (0, 2, 4, 6 and 8 days, n = 4, *** P <0.001 per group, one-way ANOVA in post-hoc Bonferroni tests, error bars are standard) Measurement results).

Figure 13D shows the results observed by measuring the permeability of endothelial monolayers and astrocytes in the blood and brain sides using dextranfluorescein (3 kDa) and dextran-rhodamine (2,000 kDa). .

13E is a graph quantifying the permeability of graphene quantum dots and graphene quantum dots-biotin over time. As shown in FIG. 13E, it was confirmed that both graphene quantum dots and biotinylated graphene quantum dots exhibit high cell permeability.

FIG. 13F is an image showing the results of observing the graphene quantum dot and the graphene quantum dot-biotin α-syn fibril decomposition ability by dot-blot. As shown in FIG. 13F, it was confirmed that both graphene quantum dots and biotinylated graphene quantum dots penetrate both the endothelial cell monolayer and the astrocytic layer to effectively degrade α-syn fibrils.

FIG. 13G is a graph showing the results of ThT and turbidity analysis (n = 4 for each group, *** P <0.001, one-way ANOVA in post-hoc Bonferroni test, error bars with standard deviation). As shown in FIG. 13G, both graphene quantum dots and biotinylated graphene quantum dots penetrated both endothelial monolayers and astrocytic layers to effectively decompose α-syn fibrils, and thus, turbidity was effectively reduced.

FIG. 13H is an image of a confocal laser scanning microscope of BMEC and astrocytes first cultured after 1 hour incubation with graphene quantum dot-biotin. Graphene Quantum Dot-Biotin is labeled Qdotstreptavidin and lysosomes are labeled LysoTracker Orange. As shown in FIG. 13h, it was confirmed that biotinylated graphene quantum dots exist in lysosomes of endothelial cells and astrocytes.

FIG. 13I is a graph quantifying graphene quantum dot-biotin in exosomes extracted from BMEC or astrocytes. The amount of graphene quantum dot-biotin was measured using exosomes separated 24 and 48 hours after cell death (n = 3 for each group). As shown in FIG. 13I, as the reaction time was increased, the amount of biotinylated graphene quantum dots contained in endothelial cells and astrocytes increased.

FIG. 13J shows avidin-biotin biotinylated graphene quantum dots present in the cerebellum, midbrain, olfactory bulb, and neocortex after intraperitoneal injection of graphene quantum dot-biotin (2 mg / kg) to mice It is an image showing the results confirmed by immunohistochemistry by staining with a complex detection method or immunogold (immunogold) method. DAB positive staining signals were detected higher in mice injected with graphene quantum dot-biotin compared to vehicle injection control. Immunogold positive signals were observed both inside neurons (red triangles) or outside neurons (blue triangles). As shown in FIG. 13J, it was confirmed that the biotinylated graphene quantum dots pass all the BBB layers even in vivo.

FIG. 13K is a graph showing the results of comparing the difference between intraperitoneal injection (i.p .; red bar) and intravenous injection (i.v .; blue bar) of graphene quantum dot-biotin. It was confirmed that the results of both injection methods are substantially no difference, through this, it was confirmed that there is no difference in effect according to the injection method of graphene quantum dots.

13L shows graphene quantum dot-biotin concentrations of 1 to 8 weeks, 3 months and 6 months (n = 4 for each group) using a biotin quantitative kit following biweekly injection of graphene quantum dot-biotin. As shown in FIG. 13L, as the number of injections of biotinylated graphene quantum dots increases, it was confirmed that the graphene quantum dots remaining in the body also increased.

FIG. 13M shows concentrations in the brain (green bar) and plasma (blue bar) 1 hour, 7 days, and 14 days (n = 4) after one intraperitoneal injection of 2 mg / kg graphene quantum dot-biotin It is a graph showing the results of the measurement. 13N is a graph showing the results of measuring concentrations in the brain (green bar) and plasma (blue bar) after 3 and 6 months after multiple injections every other week. As shown in Figure 13m and 13n, it was confirmed that the graphene quantum dots remain in the body for 14 days even if a single injection, it was confirmed that the graphene quantum dots remain even up to 6 months when inoculated multiple times.

Through the above results, it was confirmed that the graphene quantum dots of the present invention not only effectively penetrate the blood brain barrier both in vitro and in vivo, but also remain in the body for a long time. Through this, it was confirmed that the graphene quantum dots of the present invention can exhibit a high therapeutic effect for diseases related to abnormal fibrosis or aggregation of neuronal proteins.

14. Blackness SN ) At syn PFFs -Induced glial cell activation Graphene Quantum dots  effect

14A is an image of representative immunohistochemical staining for Iba-1 at low and high magnification SNs. Microglia belonging to SN hemispheres injected with α-syn PFFs were stained with a specific marker, Iba-1 (specific marker of ionized calcium binding protein, microglia / macrophage). And FIG. 14B is a graph quantifying stained Iba-1-positive microglia (n = 5). As shown in FIGS. 14A and 14B, it was confirmed that Iba-1-positive microglia increased by α-syn PFFs was inhibited by graphene quantum dots.

14C is an image of representative immunohistochemistry for GFAP at low and high magnification SNs. Astrocytes were stained with GFAP (glial fibrillary acidic protein) antibodies in hemisphere SNs injected with α-syn PFFs. And FIG. 14D is a graph quantifying the stained GFAP intensity. GFAP intensity was normalized to PBS injection control (n = 5 for each group) (* P <0.05; NS, not significant; 2-way ANOVA using post-hoc Bonferroni test, error bars standard deviation). As shown in Figure 14c and 14d, it was confirmed that the astrocytic cells increased by α-syn PFFs are inhibited by graphene quantum dots.

Through the above results, it was confirmed that the neuronal inflammatory cells increased by α-syn PFFs are inhibited by graphene quantum dots.

15. hA53T  α- syn  In the brainstem of transgenic mice Glial cell  For activation Graphene Quantum dots  effect

In order to confirm the effect of the graphene quantum dot of the present invention on the activation of glial cells in the brain stem, experiments were carried out using locus coeruleus (LC) of hA53T α-syn Tg mice.

FIG. 15A is a graph of quantified images of microglia and stained Iba-1-positive microglia at low and high magnification brain stem LCs (each group is n = 5, * P <). 0.05; ** P <0.01; NS not significant, 2-way ANOVA using post-hoc Bonferroni test, standard error for error bars). Microglia in the brain stem of nTg or hA53T α-syn Tg were stained with Iba-1. As shown in Figure 15a, it was confirmed that the increased Iba-1-positive microglia in hA53T α-syn Tg mice is reduced by administration of graphene quantum dots.

FIG. 15B is a graph of GFAP stained images and stained GFAP in low and high magnification brain stems. Relative GFAP intensities were normalized to PBS injection control (n = 5, each group). As shown in Figure 15b, it was confirmed that the increased GFAP in hA53T α-syn Tg mice is reduced by administration of graphene quantum dots.

FIG. 15C quantifies and normalizes the number of immune positive aggregates per field and images confirming the effect of GQDs on α-syn aggregate formation in HEK293 cells with hA53T α-syn overexpression (n = 7 ** P <0.01, error Bars are graphs of standard deviations. PCMV5-myc-A53T α-syn was introduced into HEK293T cells and treated with graphene quantum dots (0.1 μg / ml). 48 hours after treatment with graphene quantum dots, cells were immunostained with α-syn antibody. White arrows indicate α-syn aggregates. As shown in FIG. 15C, it was confirmed that the formation of α-syn aggregates was effectively suppressed by graphene quantum dots.

16. Graphene Quantum dots  Long-term in vitro and in vivo vivo A) toxicity test

Figure 16a is a graph measuring the cytotoxicity of graphene quantum dots in DIV 10 cortical neurons cultured for 7 days by alamarBlue and LDH assay (n = 4, each time point, NS is not significant, post-hoc Bonferroni test 1-way ANOVA used, error bars are standard deviation). As shown in Figure 16a, it was confirmed that the graphene quantum dot does not exhibit cytotoxicity even in long-term culture.

16b is a graph showing survival curves of mice injected with graphene quantum dots. 50 μg of graphene quantum dots or vehicle were intraperitoneally injected into C57BL / 6 mice for 6 months and the in vivo toxicity of graphene quantum dots was monitored (n = 20 for each group). Statistically, after 8 months, there was no significant difference between the graphene quantum dots and the vehicle injection control group.

16C is an image stained with haematoxylin and eosin (H & E) of major organs. As shown in Figure 16c, it was confirmed that the graphene quantum dots do not show toxicity to the major organs.

16D is a schematic diagram of a method of injection and sampling for in vivo tracking of graphene quantum dot-biotin, and FIG. 16E is a graph measuring the concentration of graphene quantum dot-biotin over time in the brain (red) or urine (blue). to be. 50 μg of graphene quantum dot-biotin was intraperitoneally injected into C57BL / 6 mice and measured at various time points (1, 3, 7 and 14 days). As shown in Figure 16e, the biotinylated graphene quantum dots were discharged through the urine, it was confirmed that the concentration in the body is reduced.

Through the above results, it was confirmed that the graphene quantum dots of the present invention do not show toxicity in the body, and stably discharged through urine as time passes after the injection.

17. Comparison of nano-GOs and rGQDs for α-syn PFFs

FIG. 17A is a graph showing FT-IR spectra of raw graphene quantum dots (black), nano-GOs (nano graphene oxide, green) and reduced GQDs (rGQDs, purple) with designated peaks for functional groups. And FIG. 17B is an AFM image of nano-GOs and graphene quantum dots with representative line profiles. As shown in FIG. 17B, the nano-GOs exhibited an average height of about 5 nm, but in the case of graphene quantum dots, the height of 0.1 to 3 nm was confirmed.

FIG. 17C is a graph of quantification of cytotoxicity after treatment of cells with 20 μg / ml of each substance and incubation for 72 hours (n = 3 for each group, ** P <0.01, Student's t-test, error Bar is the standard deviation). As shown in FIG. 17c, the reduced graphene quantum dots and the nano-GOs exhibited cytotoxicity, but the graphene quantum dots did not exhibit cytotoxicity.

17D is a graph of monitoring cytotoxicity in SH-SY5Y cells using alarmarBlue assay at 12, 24 and 72 hours after each material (1, 10 and 20 μg / ml) was treated at various concentrations. As shown in FIG. 17D, the reduced graphene quantum dots and the nano-GOs showed cytotoxicity, but the graphene quantum dots did not show cytotoxicity.

17E is an image comparing the degradation effects of α-syn PFFs of graphene quantum dots, nano-GOs and rGQDs evaluated by dot blot. As shown in FIG. 17E, it was confirmed that the reduced graphene quantum dots and nano-GOs had no effect of decomposing α-syn PFFs.

And FIG. 17F is a graph showing ThT and turbidity analysis (n = 4 for each group, *** P <0.001; NS, not significant, 1-way ANOVA in post-hoc Bonferroni test, error bars with standard deviation). As shown in FIG. 17F, in the case of graphene quantum dots, turbidity and fluorescence values are decreased by decomposing α-syn PFFs effectively, but in the case of reduced graphene quantum dots and nano-GOs, it is confirmed that the α-syn PFFs cannot be decomposed Could.

17G is a TEM image obtained after 5 mg / ml of each material was treated with α-syn PFFs and reacted for 7 days. As shown in FIG. 17g, in the case of reduced graphene quantum dots and nano-GOs, it was confirmed that the α-syn PFFs could not be decomposed.

Figure 17h is a graph measuring the in vitro blood brain barrier permeability of graphene quantum dots, nano-GOs and rGQDs through blood brain barrier permeability in vitro experiment. As shown in FIG. 17H, the graphene quantum dots and the reduced graphene quantum dots passed through the blood brain barrier, but in the case of nano-GOs, it was confirmed that the passage rate was low.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

The graphene quantum dots of the present invention are not cytotoxic and can inhibit the formation of α-syn fibrils or decompose the already formed α-syn fibrils and exhibit an effect of crossing the blood brain barrier, thereby causing abnormal fibrosis or It is expected that it can be widely used as a therapeutic agent for various diseases associated with aggregation.

Claims (17)

1. The surface shows a negative charge,

   The average diameter is 0.5 to 10 nm and the average height is 0.1 to 3 nm,

   Graphene quantum dot, the wt% ratio of carbon and oxygen is composed of 4.0 to 6.5: 3.0 to 6.0 ratio.
2. The method of claim 1,

   The graphene quantum dot is a terminal functional group containing a carboxyl group, graphene quantum dot.
3. The method of claim 2,

   The terminal functional group is a graphene quantum dot, the absorbance ratio of the -C = O peak of the carboxyl group and the aromatic -C = C- peak in the FT-IR spectrum of 1: 1 or more.
4. The method of claim 3, wherein

   Graphene quantum dots, wherein the absorbance ratio is 1: 1 to 2: 1.
5. The method of claim 3, wherein

   Wherein the -C = O peak appears at 1700 to 1750 cm ^-1 and the aromatic -C = C- peak appears at 1600 to 1650 cm ^-1 .
6. The method of claim 1,

   Graphene quantum dots, wherein the average diameter is 1 to 5 nm.
7. The method of claim 1,

   Graphene quantum dots, wherein the average height is 0.5 to 2.5 nm.
8. The method of claim 1,

   The graphene quantum dot is to inhibit the α-syn fibrillation or to degrade the α-syn fibrils, graphene quantum dots.
9. The method of claim 1,

   The graphene quantum dot is to pass through the blood brain barrier (BBB), graphene quantum dot.
10. The method of claim 1,

    The graphene quantum dots are not toxic in the body, graphene quantum dots.
11. A pharmaceutical composition for preventing or treating a disease associated with abnormal fibrosis or aggregation of neuroprotein, comprising the graphene quantum dot according to any one of claims 1 to 10 as an active ingredient.
12. The method of claim 11,

    The disease associated with abnormal fibrosis or aggregation of the neuroprotein is a neurodegenerative disease, inflammatory disease or metabolic disease.
13. The method of claim 12,

    The degenerative neurological disease is one or more selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, dementia, stroke, amyloidosis, fibrosis, encephalopathy and multiple sclerosis.
14. The method of claim 12,

    The inflammatory diseases include erythema, atopy, rheumatoid arthritis, Hashimoto's thyroiditis, pernicious anemia, Edison's disease, type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, hypothyroidism, hyperthyroidism, scleroderma, Behcet's disease, inflammatory bowel One selected from the group consisting of disease, myasthenia gravis, Meniere's syndrome, Guilian-Barre syndrome, Sjogren's syndrome, endometriosis, psoriasis, vitiligo, systemic scleroderma and ulcerative colitis What is more, the pharmaceutical composition.
15. The method of claim 12,

    The metabolic disease is one or more selected from the group consisting of diabetes, hypertension, hyperlipidemia, dyslipidemia and non-alcoholic fatty liver, pharmaceutical composition.
16. A method for preventing or treating diseases related to abnormal fibrosis or aggregation of neuroproteins, comprising administering to a subject a composition comprising a graphene quantum dot according to any one of claims 1 to 10 as an active ingredient.
17. Use of the prophylaxis or treatment of diseases associated with abnormal fibrosis or aggregation of neuroproteins of a composition comprising the graphene quantum dots according to any one of claims 1 to 10 as an active ingredient.

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