U.S. patent application number 15/301853 was filed with the patent office on 2017-07-06 for graphene nanostructure-based pharmaceutical composition for preventing or treating neurodegenerative diseases.
The applicant listed for this patent is THE JOHNS HOPKINS UNIVERSITY, SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION. Invention is credited to Byung Hee Hong, Donghoon Kim, Hanseok Ko, Je Min Yoo.
Application Number | 20170189359 15/301853 |
Document ID | / |
Family ID | 54240900 |
Filed Date | 2017-07-06 |
United States Patent
Application |
20170189359 |
Kind Code |
A1 |
Hong; Byung Hee ; et
al. |
July 6, 2017 |
Graphene Nanostructure-Based Pharmaceutical Composition for
Preventing or Treating Neurodegenerative Diseases
Abstract
The present disclosure relates to a pharmaceutical composition
for preventing or treating neurodegenerative diseases, the
pharmaceutical composition including a graphene nanostructure as an
active ingredient.
Inventors: |
Hong; Byung Hee; (Seoul,
KR) ; Yoo; Je Min; (Seoul, KR) ; Ko;
Hanseok; (Luthervile, MD) ; Kim; Donghoon;
(Baltimore, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION
THE JOHNS HOPKINS UNIVERSITY |
Seoul
Baltimore |
MD |
KR
US |
|
|
Family ID: |
54240900 |
Appl. No.: |
15/301853 |
Filed: |
April 3, 2015 |
PCT Filed: |
April 3, 2015 |
PCT NO: |
PCT/KR2015/003385 |
371 Date: |
March 23, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C01B 32/182 20170801;
A61P 3/00 20180101; A61K 41/0052 20130101; A61P 3/10 20180101; A61N
2005/066 20130101; A61P 25/28 20180101; A61P 21/00 20180101; A61K
49/0052 20130101; A61N 5/062 20130101; A61P 25/16 20180101; A61K
33/44 20130101; A61P 9/10 20180101; A61P 25/00 20180101; A61P 25/14
20180101; A61N 2005/067 20130101; A61K 31/194 20130101; A61P 21/02
20180101; A61K 49/0021 20130101; C01B 2204/32 20130101 |
International
Class: |
A61K 31/194 20060101
A61K031/194; A61K 49/00 20060101 A61K049/00; A61N 5/06 20060101
A61N005/06; A61K 41/00 20060101 A61K041/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 4, 2014 |
KR |
10-2014-0040400 |
Claims
1. A pharmaceutical composition for preventing or treating
neurodegenerative diseases, comprising a graphene nanostructure as
an active ingredient.
2. The pharmaceutical composition for preventing or treating
neurodegenerative diseases of claim 1, further comprising a
pharmaceutically acceptable carrier or excipient.
3. The pharmaceutical composition for preventing or treating
neurodegenerative diseases of claim 1, wherein the
neurodegenerative diseases include a member selected from the group
consisting of Alzheimer's disease, Parkinson's disease,
Huntington's chorea, HIV dementia, stroke, senile systemic
amyloidosis, primary systemic amyloidosis, secondary systemic
amyloidosis, type II diabetes, amyotrophic amyloidosis,
hemodialysis-related amyloidosis, transmissible spongiform
encephalopathy, and multiple sclerosis.
4. The pharmaceutical composition for preventing or treating
neurodegenerative diseases of claim 2, wherein the pharmaceutically
acceptable carrier or excipient includes a member selected from the
group consisting of vaseline, lanolin, polyethylene glycol,
alcohol, and combinations thereof.
5. The pharmaceutical composition for preventing or treating
neurodegenerative diseases of claim 1, wherein the graphene
nanostructure includes graphite, graphene, or graphene quantum
dots.
6. The pharmaceutical composition for preventing or treating
neurodegenerative diseases of claim 1, wherein the graphene
nanostructure inhibits fibril formation caused by protein
misfolding.
7. The pharmaceutical composition for preventing or treating
neurodegenerative diseases of claim 1, wherein the graphene
nanostructure inhibits transition of misfolded proteins.
8. The pharmaceutical composition for preventing or treating
neurodegenerative diseases of claim 1, wherein the graphene
nanostructure is not accumulated in the body.
9. The pharmaceutical composition for preventing or treating
neurodegenerative diseases of claim 1, wherein a terminal
functional group of the graphene nanostructure is bonded to a
material targeting a neuronal protein.
10. The pharmaceutical composition for preventing or treating
neurodegenerative diseases of claim 1, wherein the graphene
nanostructure shows a photothermal effect upon irradiation of a
far-infrared laser.
Description
TECHNICAL FIELD
[0001] The present invention relates to a pharmaceutical
composition for preventing or treating neurodegenerative diseases,
the pharmaceutical composition including a graphene nanostructure
as an active ingredient.
BACKGROUND
[0002] It has been known that protein misfolding causes normal
proteins to lose their function and abnormal proteins to be
accumulated in cells and produce toxicity and thus causes various
diseases such as Alzheimer's disease, Parkinson's disease,
Huntington's chorea, Lou Gehrig's disease, cancer, cystic fibrosis,
and type II diabetes. That is, malfunction of proteostasis causes
protein misfolding and its intracellular abnormal accumulation.
[0003] All the causes of neurodegenerative diseases have not yet
been found, but it has been well known that aggregation of neuronal
proteins is a main cause. Fibrillated proteins are gradually
transmitted to adjacent neurons and finally necrose all the neurons
in a specific part of the brain, so that the specific part cannot
perform its functions. As for Parkinson's disease, the disease
progresses while neurons that produce a neurotransmitter called
dopamine are gradually necrosed. Sinemet is now the most common
drug prescribed to Parkinson's disease patients, and Sinemet and
other Parkinson's disease drugs do not function to fundamentally
treat or delay the disease but supply Levodopa (L-DOPA) which is
converted into dopamine in neurons to temporally reduce the
symptoms. In the end, as the disease continues to progress, the
effect of the drugs decreases, which causes death.
[0004] As one of anti-amyloid compounds researched relating to
protein misfolding, Congo Red has an effect of inhibiting fibril
formation caused by protein misfolding but is highly toxic to the
body and cannot function to inhibit transition of misfolded
proteins and thus delay the progress of a disease. Further, a
conventional drug is formulated into a specific shape with a
uniform size and thus does not have any particular advantage in the
treatment of a disease in terms of entropy.
[0005] Accordingly, a lot of studies have been carried out to treat
protein misfolding (Korean Patent Laid-open Publication No.
10-2009-0019790), but there has been no great achievement about a
drug which is not toxic to the body but has an excellent inhibitory
activity.
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0006] Accordingly, the present disclosure provides a
pharmaceutical composition for preventing or treating
neurodegenerative diseases, the pharmaceutical composition
including a graphene nanostructure as an active ingredient.
[0007] However, problems to be solved by the present disclosure are
not limited to the above-described problems. Although not described
herein, other problems to be solved by the present disclosure can
be clearly understood by those skilled in the art from the
following descriptions.
Means for Solving the Problems
[0008] In accordance with a first aspect of the present disclosure,
there is provided a pharmaceutical composition for preventing or
treating neurodegenerative diseases, the pharmaceutical composition
including a graphene nanostructure as an active ingredient.
Effects of the Invention
[0009] According to the exemplary embodiments of the present
disclosure, the pharmaceutical composition for preventing or
treating neurodegenerative diseases, the pharmaceutical composition
including a graphene nanostructure as an active ingredient is the
first attempt to use a graphene nanostructure for preventing and
treating neurodegenerative diseases. The graphene nanostructure is
not toxic to the body and not accumulated in the body, but it has
an excellent effect such as inhibiting fibril formation caused by
protein misfolding to 80% and is effective in inhibiting transition
of misfolded proteins and thus delaying the progress of a disease.
Further, unlike the conventional drug, the graphene nanostructure
is not limited to a specific shape and the graphene nanostructures
are different from each other in molecular weight, molecular
formula, and shape, which inhibits formation of crystals in terms
of entropy, and, thus, it is possible to fundamentally treat a
disease. Furthermore, the graphene nanostructure exhibits
fluorescence in a UV-vis range, and it is possible to track
movement of the graphene nanostructure to act as a drug in the body
by appropriately regulating the intensity of fluorescence. Also, it
is possible to track a neuronal protein by bonding a material
targeting the neuronal protein to a terminal functional groups of
the graphene nanostructure. As such, thee graphene nanostructure
may be induced to a place near the neuronal protein and then a
far-infrared laser less damaging a cell may be irradiated, so that
fibrillation and aggregation can be inhibited by a photothermal
effect of the graphene nanostructure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows TEM and AFM images of graphene quantum dots in
accordance with an example of the present disclosure.
[0011] FIG. 2 is a graph showing the result of PL analysis on
graphene quantum dots in accordance with an example of the present
disclosure.
[0012] FIG. 3 is a graph showing the result of FT-IR analysis on
graphene quantum dots in accordance with an example of the present
disclosure.
[0013] FIG. 4 is a graph showing the result of Zeta potential
measurement on graphene quantum dots in accordance with an example
of the present disclosure.
[0014] FIG. 5 shows a fibrillation inhibiting effect of a graphene
nanostructure in accordance with an example of the present
disclosure.
[0015] FIG. 6A and FIG. 6B provide (A) images and (B) graphs
showing the result of analysis of a neuron survival rate affected
by a graphene nanostructure in accordance with an example of the
present disclosure.
[0016] FIG. 7A and FIG. 7B provide (A) images and (B) graphs
showing the result of analysis of 8-OHG staining which shows a
reactive oxygen species generation inhibitory activity of a
graphene nanostructure in accordance with an example of the present
disclosure.
[0017] FIG. 8A and FIG. 8B provide (A) schematic diagrams of a test
for confirming a-syn transition inhibition of a graphene
nanostructure in accordance with an example of the present
disclosure and (B) images showing the result thereof.
[0018] FIG. 9 shows the result of TEM analysis of fibrillation
inhibition of a graphene nanostructure in accordance with an
example of the present disclosure.
[0019] FIG. 10 shows the result of AFM analysis of fibrillation
inhibition of graphene quantum dots in accordance with an example
of the present disclosure.
[0020] FIG. 11A and FIG. 11B show the result of high-resolution TEM
analysis of fibrillation inhibition of a graphene nanostructure in
accordance with an example of the present disclosure.
[0021] FIG. 12 shows the result of BN-PAGE analysis on PFFs after
injection of a graphene nanostructure in accordance with an example
of the present disclosure.
[0022] FIG. 13 provides images showing luminescent characteristics
of a graphene nanostructure and Congo Red in accordance with an
example of the present disclosure.
[0023] FIG. 14 is a graph showing photothermal characteristics of a
graphene nanostructure in accordance with an example of the present
disclosure.
[0024] FIG. 15 shows FT-IR spectra of samples prepared in
accordance with an example of the present disclosure.
[0025] FIG. 16 shows the result of fluorescence measurement on
samples prepared in accordance with an example of the present
disclosure.
MODE FOR CARRYING OUT THE INVENTION
[0026] Hereinafter, embodiments and examples of the present
disclosure will be described in detail with reference to the
accompanying drawings so that the present disclosure may be readily
implemented by those skilled in the art.
[0027] However, it is to be noted that the present disclosure is
not limited to the embodiments and examples but can be embodied in
various other ways. In drawings, parts irrelevant to the
description are omitted for the simplicity of explanation, and like
reference numerals denote like parts through the whole
document.
[0028] Through the whole document, the term "on" that is used to
designate a position of one element with respect to another element
includes both a case that the one element is adjacent to the
another element and a case that any other element exists between
these two elements.
[0029] Further, through the whole document, the term "comprises or
includes" and/or "comprising or including" used in the document
means that one or more other components, steps, operation and/or
existence or addition of elements are not excluded in addition to
the described components, steps, operation and/or elements unless
context dictates otherwise. Through the whole document, the term
"about or approximately" or "substantially" are intended to have
meanings close to numerical values or ranges specified with an
allowable error and intended to prevent accurate or absolute
numerical values disclosed for understanding of the present
disclosure from being illegally or unfairly used by any
unconscionable third party. Through the whole document, the term
"step of" does not mean "step for".
[0030] Through the whole document, the term "combination of"
included in Markush type description means mixture or combination
of one or more components, steps, operations and/or elements
selected from a group consisting of components, steps, operation
and/or elements described in Markush type and thereby means that
the disclosure includes one or more components, steps, operations
and/or elements selected from the Markush group.
[0031] Through the whole document, the term "graphene quantum dots
(GQDs)" refers to nano-sized fragments of graphene oxides or
reduced graphene oxides.
[0032] Through the whole document, the term "graphene" refers to a
material forming a polycyclic aromatic molecule with multiple
carbon atoms covalently bonded to each other. The covalently bonded
carbon atoms form a six-member ring as a repeating unit, but can
further include a five-member ring and/or a seven-member ring.
[0033] Through the whole document, the term "graphene oxide" may be
abbreviated as "GO", and may include a structure in which a
functional group containing oxygen such as a carboxyl group, a
hydroxyl group, or an epoxy group is bonded onto graphene, but may
not be limited thereto.
[0034] Through the whole document, the term "reduced graphene
oxide" refers to graphene oxide decreased in a percentage of oxygen
through a reduction process and may be abbreviated as "rGO", but
may not be limited thereto.
[0035] Hereinafter, the exemplary embodiments of the present
disclosure will be described in detail, but the present disclosure
may not be limited thereto.
[0036] In accordance with a first aspect of the present disclosure,
there is provided a pharmaceutical composition for preventing or
treating neurodegenerative diseases, the pharmaceutical composition
including a graphene nanostructure as an active ingredient.
[0037] In accordance with an exemplary embodiment of the present
disclosure, the pharmaceutical composition may further include a
pharmaceutically acceptable carrier or excipient, but may not be
limited thereto. The pharmaceutically acceptable carrier or
excipient is not limited as long as it can be used in a
pharmaceutical composition, and may include a member selected from
the group consisting of, for example, vaseline, lanolin,
polyethylene glycol, alcohol, and combinations thereof, but may not
be limited thereto.
[0038] In accordance with an exemplary embodiment of the present
disclosure, the neurodegenerative diseases are relevant to protein
misfolding, and may include a member selected from the group
consisting of, for example, Alzheimer's disease, Parkinson's
disease, Huntington's chorea, HIV dementia, stroke, senile systemic
amyloidosis, primary systemic amyloidosis, secondary systemic
amyloidosis, type II diabetes, amyotrophic amyloidosis,
hemodialysis-related amyloidosis, transmissible spongiform
encephalopathy, and multiple sclerosis, but may not be limited
thereto.
[0039] In accordance with an exemplary embodiment of the present
disclosure, the graphene nanostructure may include graphite,
graphene, or graphene quantum dots, but may not be limited
thereto.
[0040] The graphene quantum dots may have a size in the range of,
for example, from about 1 nm to about 20 nm, from about 5 nm to
about 20 nm, from about 10 nm to about 20 nm, from about 15 nm to
about 20 nm, from about 1 nm to about 15 nm, from about 1 nm to
about 10 nm, or from about 1 nm to about 5 nm, but may not be
limited thereto.
[0041] In accordance with an exemplary embodiment of the present
disclosure, the graphene nanostructure may include graphene
nanostructures with various sizes in the range of from about 1 nm
to about 100 nm, for example, from about 10 nm to about 100 nm,
from about 30 nm to about 100 nm, from about 50 nm to about 100 nm,
from about 70 nm to about 100 nm, from about 90 nm to about 100 nm,
from about 1 nm to about 90 nm, from about 1 nm to about 70 nm,
from about 1 nm to about 50 nm, from about 1 nm to about 30 nm, or
from about 1 nm to about 10 nm, but may not be limited thereto.
[0042] In accordance with an exemplary embodiment of the present
disclosure, the graphene nanostructure may inhibit fibril formation
caused by protein misfolding, and also inhibit transition of
misfolded proteins, but may not be limited thereto.
[0043] In accordance with an exemplary embodiment of the present
disclosure, the graphene nanostructure is not accumulated in the
body and not toxic to the body.
[0044] In accordance with an exemplary embodiment of the present
disclosure, the graphene nanostructure may inhibit generation of
reactive oxygen species in neurons through a mechanism that
inhibits mitochondrial dysfunction caused by fibrillated proteins,
but may not be limited thereto.
[0045] In accordance with an exemplary embodiment of the present
disclosure, the pharmaceutical composition may include a material
targeting a neuronal protein, for example, Congo Red as an
anti-amyloid material or thioflavin T or S as an amyloid detecting
dye, which is bonded to a terminal functional group of the graphene
nanostructure, but may not be limited thereto.
[0046] The functional group according to the present disclosure may
include oxygen atoms and may be --OH, --COON, or --C.dbd.O, but may
not be limited thereto.
[0047] In accordance with an exemplary embodiment of the present
disclosure, the graphene nanostructure may show a photothermal
effect upon irradiation of a far-infrared laser, but may not be
limited thereto.
[0048] In accordance with an exemplary embodiment of the present
disclosure, the pharmaceutical composition of the present
disclosure has an advantage of being able to track a neuronal
protein by bonding a material targeting the neuronal protein to a
terminal functional groups of the graphene nanostructure. As such,
the graphene nanostructure may be induced to a place near the
neuronal protein and then a far-infrared laser less damaging a cell
may be irradiated, so that fibrillation and aggregation can be
inhibited by a photothermal effect of the graphene
nanostructure.
MODE FOR CARRYING OUT THE INVENTION
[0049] Hereinafter, examples of the present disclosure will be
described in more detail, but the scope of the present disclosure
is not limited thereto.
EXAMPLES
Preparation Example 1
[0050] GQDs were prepared with reference to the article disclosed
in Nano Letters 2012 [Nano Lett, 12, 844-849 (2012)]. A carbon
fiber was put into a solution including a mixture of sulfuric acid
and nitric acid at a ratio of 3:1 and then heated at 80.degree. C.
for 24 hours (thermo-oxidation process). After completion of the
reaction, the product was purified through dialysis and vacuum
filtration and GQDs in the form of powder were finally obtained
using a Rotovap. The prepared GQDs were particles with structurally
various sizes (about 5 nm to about 20 nm) (FIG. 1). The prepared
GQDs had other characteristics such as exhibiting fluorescence
under a UV lamp (Emission: 490 nm, and 550 nm) (JASCO FP-8300
Fluorescence Spectrometer) (FIG. 2) and showing a photothermal
effect upon irradiation of 808 nm NIR laser. According to the FT-IR
spectrum (Thermo Scientific Nicolet iS 10 FT-IR Spectrometer), a
carboxyl group (--COOH) at a terminal of GQDs was observed at 1724
cm.sup.-1 and an aromatic C.dbd.C peak was observed at 1614
cm.sup.-1 (FIG. 3). A surface charge analyzed from Zeta potential
(Malvern Zetasizer Nano ZS) was shown as about -20 mV (FIG. 4).
Preparation Example 2
[0051] A material in which Congo Red as a material targeting a
neuronal protein was bonded to a terminal functional group of the
prepared graphene nanostructure (GQDs) was prepared by a reaction
as shown in the following Reaction Formula. Samples 1, 2, and 3
were prepared by applying different amounts of the Congo Red (100
.mu.g/ml, 250 .mu.g/ml, and 500 .mu.g/ml), respectively:
##STR00001##
Test Example 1
[0052] A fibrillation inhibition effect of a graphene quantum dot
was determined using PFFs (pre-formed fibrils) as an
alpha-synuclein test model. Specifically, fibrils were formed about
a week after injection of the PFFs into neurons and finally caused
necrosis of the neurons. Fibrillation causes phosphorylation of
alpha-synuclein and can be recognized by staining as shown in FIG.
5. It was observed that p-a-syn (phosphorylated alpha-synuclei)
were dense in response to injection of PFFs (1 .mu.g/mL) only, but
upon injection of GODS (1 .mu.g/mL), the p-a-syn (phosphorylated
alpha-synuclei) almost disappeared to the level that nothing was
injected (FIG. 5). As can be seen from FIG. 5, the p-a-syn was
reduced to about 80%, which can be practically considered that
fibrillation is almost entirely inhibited. Further, a neuron
survival rate increases by about 20% (FIG. 6B). A neuron survival
rate (analyzed by TUNEL screening) in case of injecting GQDs only
was higher than a case where only a PBS medium was injected, and,
thus, it was confirmed that the GQDs were not toxic to neurons
(FIG. 6A and FIG. 6B). The left images of FIG. 6A show TUNNEL
screening in which a damaged cell is stained red, the middle images
show DAPI staining in which DNA in a cell nucleus is stained blue,
and the right images show quantification thereof.
[0053] In a test of whether the graphene nanostructure used herein
inhibits generation of reactive oxygen species in neurons through a
mechanism that inhibits mitochondrial dysfunction caused by
fibrillated proteins, primarily cultured neurons were stained with
8-OHG (8-oxo-2'-deoxyguanosine as a main product of DNA oxidation)
and then analyzed (FIG. 7A). As can be seen from FIG. 7A, it was
observed that the amount of 8-OHG generated by the influence of
reactive oxygen species remarkably decreased by addition of the
graphene nanostructure. A test of mitochondrial dysfunction was
confirmed through a basal respiratory rate and a maximal
respiratory rate of a cell, and mitochondrial Complex I activity
assay (FIG. 7B). As shown in the graph of FIG. 7B, it was observed
that when only PFFs were injected into neurons, a respiratory rate
of mitochondria remarkably decreased and Complex I activity also
decreased, and when GQDs and PFFs were injected into the neurons,
the respiratory rate recovered to a normal level. The result of
analysis of mitochondrial dysfunction through this test is
considered important since it corresponds to the neuron survival
rate shown in FIG. 6B.
Test Example 2
[0054] It is important to simply inhibit fibrillation and increase
a neuron survival rate, but it is very important to inhibit
transition to adjacent neurons in order to treat neurodegenerative
diseases and slow the progress of the neurodegenerative disease. In
order to confirm this fact, a microfluidic device was set up and it
was checked which chamber GQDs should be put into in order to
inhibit transition. FIG. 8A provides schematic diagrams of the test
and FIG. 8B shows the result of the test. In case of C1 (Chamber
1), GQDs were injected into neurons in a first chamber and in case
of C2 (Chamber 2), GQDs were injected into neurons in a second
chamber, and then, transition of fibrillation of alpha-synuclein to
adjacent neurons was observed. In a positive control group
including only PFFs without GQDs, it was observed that a
fibrillated alpha-synuclein was transmitted to Chambers 1 to 3.
Secondly, in a device in which GQDs were put into a first neuron,
fibrillation was not well developed from the first, and, thus,
fibrillation of alpha-synuclein was rarely observed from second and
third neurons. Finally, in a device in which GQDs were put into an
intermediate neuron, fibrillation was developed to some degree in
the first chamber, and the amount of fibrillation was noticeably
decreased in the second neuron where the GQDs were included, and in
the last neuron, it was observed that fibrillation was hardly
developed. It is deemed that a difference in the degree of initial
fibrillation between this device and a device which is the positive
control group including only PFFs without GQDs is an error inherent
in the device and caused by incomplete separation of GQDs from each
neuron in the device.
Test Example 3
[0055] A sampling was performed in the same manner as an
intracellular experiment (1 .mu.g/ml GQDs and 1 .mu.g/ml PFFs were
incubated at 37.degree. C. and then sampled on a silica substrate)
and fibrillation inhibition of GQDs was analyzed using an OM
(optical microscope), and as a result thereof, when only PFFs were
incubated, a large lump expected as an aggregate of fibrils was
observed (FIG. 9A and FIG. 9B), whereas when PFFs and GQDs were
incubated, GQDs and alpha-synuclein oligomers were shown as being
tangled (FIG. 9C and FIG. 9D). In the present result, the total
amount of alpha-synuclein and GQDs was excessive and the images
were not sufficiently clear to distinctly show the structures
thereof, but it could be seen that the results of the two samples
were definitely different from each other.
[0056] A sampling was performed in the same manner as the sampling
for OM analysis and fibrillation inhibition of GQDs was analyzed
using an AFM (atomic force microscope), and as a result thereof,
when only PFFs were incubated, a lewy body formed by aggregation of
fibrils and PFFs was observed (FIG. 10B), whereas when PFFs and
GQDs were incubated, GQDs and alpha-synuclein oligomers were shown
as being tangled (FIG. 10D).
[0057] A sampling was performed on a TEM (transmission electron
microscopy) grid in the same manner as the sampling for OM and AFM
analyses, and an analysis was conducted using a high-resolution
transmission electron microscope. FIG. 11A shows data obtained by
sampling PFFs only on the TEM grid and conducting an analysis. As
can be seen from the images, a very thick and long fibril bundle
was observed. Meanwhile, in a sample in which GQDs were also
included, as shown in the AFM image, GQDs and alpha-synuclein
oligomers were shown as being tangled (FIG. 11B).
[0058] The images shown in FIG. 9 to FIG. 11 showed a marked
difference between injection or non-injection of GQDs with PFFs,
but these images were obtained in a dried state and thus could not
show the exact state of PFFs after injection of GQDs. For more
detailed analysis, BN-PAGE (blue native polyacrylamide gel
electrophoresis) was performed, and a result thereof was surprising
(FIG. 12). In FIG. 12, Column 1 is about a negative control group
in which only GQDs are included, Columns 2 and 4 are about samples
in which only PFFs are included, and Columns 3 and 5 are about
samples in which both PFFs and GQDs are included, and as a result
of gel insertion, it could be seen that when GQDs were injected, a
strong band was detected from an area corresponding to
alpha-synuclein monomers (14.46 kDa). This is very important data
showing that when GQDs are added to PFFs, fibrillation of PFFs can
be inhibited and PFFs are returned to monomers.
Test Example 4
[0059] The GQDs prepared in Preparation Example 1 and a Congo Red
solution (control group) were irradiated with infrared rays and
fluorescence was photographed in the dark, and a result thereof was
as shown in FIG. 13.
[0060] Further, FIG. 14 shows that when a NIR laser was irradiated,
the GQDs showed a higher photothermal effect than Congo Red or
distilled water and was increased in temperature depending on the
concentration of GQDs (500 .mu.g/m L, 250 .mu.g/mL, 100 .mu.g/mL)
as the concentration of the GQDs was increased.
Test Example 5
[0061] A FT-IR analysis and a fluorescence measurement were
performed using various samples (GQDs, Sample 1, Sample 2, and
Sample 3 prepared in Preparation Examples 1 and 2, and a Congo Red
solution as a comparative example).
[0062] Referring to FIG. 15 showing FT-IR spectra, at a --NH.sub.2
peak 3419 cm.sup.-1 of Congo Red, the peak decreased as the
concentration of Congo Red increased, and even at a carboxyl group
(--COOH) peak 1710 cm.sup.-1 of a terminal of GQDs, the peak
decreased as the concentration of Congo Red increased, and a
peptide bond (--CONH) peak 1660 cm.sup.-1 was gradually formed by
conjugation of GQDs and Congo Red, and an aromatic C.dbd.C peak
1614 cm.sup.-1 was maintained in all of the samples as
expected.
[0063] Meanwhile, fluorescences of the samples depending on a
wavelength were measured as shown in FIG. 16. Herein, Congo Red was
used as a control group, and Sample 1 showed the highest
fluorescence peak.
[0064] The above description of the present disclosure is provided
for the purpose of illustration, and it would be understood by
those skilled in the art that various changes and modifications may
be made without changing technical conception and essential
features of the present disclosure. Thus, it is clear that the
above-described examples are illustrative in all aspects and do not
limit the present disclosure. For example, each component described
to be of a single type can be implemented in a distributed manner.
Likewise, components described to be distributed can be implemented
in a combined manner.
[0065] The scope of the present disclosure is defined by the
following claims rather than by the detailed description of the
embodiment. It shall be understood that all modifications and
embodiments conceived from the meaning and scope of the claims and
their equivalents are included in the scope of the present
disclosure.
* * * * *