A curcumin-based NIR fluorescence probe for detection of amyloid-beta (Aβ) plaques in Alzheimer's disease

Article abstract of DOI:10.1016/j.dyepig.2018.12.003

Amyloid-beta (Aβ) peptide as one of the main components of senile plaques is closely related to the onset and progression of incurable Alzheimer's disease (AD). In recent years, it is reported that curcumin derivatives have been used as the near-infrared

Full text of DOI:10.1016/j.dyepig.2018.12.003

Accepted Manuscript
A curcumin-based NIR fluorescence probe for detection of amyloid-beta (Aβ) plaques
in Alzheimer's disease
Guofu Si, Shujie Zhou, Guoyong Xu, Jiafeng Wang, Baoxing Wu, Shuangsheng Zhou
PII:
S0143-7208(18)31991-0
https://doi.org/10.1016/j.dyepig.2018.12.003
DYPI 7208
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 8 September 2018
Revised Date: 4 December 2018
Accepted Date: 5 December 2018
Please cite this article as: Si G, Zhou S, Xu G, Wang J, Wu B, Zhou S, A curcumin-based NIR
fluorescence probe for detection of amyloid-beta (Aβ) plaques in Alzheimer's disease, Dyes and
Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2018.12.003.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
A curcumin-based NIR fluorescence probe for detection of amyloid-beta (Aβ)
plaques in Alzheimer’s Disease
a
b
c
a
a, c
Guofu Si ，Shujie Zhou ，Guoyong Xu , Jiafeng Wang , Baoxing Wu
,
a, c
Shuangsheng Zhou
*
a
Department of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012,
P.R .China
b
Qlu school of Medicine , Shandong University, Jinan 502100, P.R. China
c
Institute of Physical Science and Information Technology, Anhui University, Hefei
2
30601, P R.China
Abstract：Amyloid-beta (Aβ) peptide as one of the main components of senile
plaques is closely related to the onset and progression of incurable Alzheimer's
disease (AD). In recent years, it is reported that curcumin derivatives have been used
as the near-infrared (NIR) fluorescence imaging probes of Aβ plaques for the early
diagnosis of AD. To further develop a curcumin-based NIR fluorescent probe for Aβ
plaques, in this work, we have synthesized three novel curcumin-based NIR Dyes.
Among them, the curcumin derivative Dye 2 showed a significant enhancement in its
fluorescence intensity (λem, 635 nm; 19.5-fold increase in quantum yield, Φ = 0.36;
K , 1.13 µM) after binding to Aβ plaques. Additionally, in vitro and in vivo
d
fluorescence imaging of Aβ plaques strained with Dye 2 confirmed that the
compound was a potential probe to detect Aβ plaques in AD. This work opens a
perspective to rationally design novel curcumin-based NIR compound for Aβ
detection.
Keywords: Curcumin, NIR fluorescence probe, Amyloid-beta deposits, Fluorescence
imaging, Amyloid-beta plaques, Alzheimer’s Disease
1
. Introduction
Alzheimer’s disease (AD) is the most common form of dementia in the elderly.
1

ACCEPTED MANUSCRIPT
Pathologically, AD is a neurodegenerative disease characterized by extracellular
senile plaques consisting of fibrillar amyloid-beta (Aβ) peptides. The formation of
neurofibrillary tangles (NFTs) eventually results in neurodegeneration and brain
atrophy. Furthermore, deposition of Aβ is considered to be the most important
pathophysiological hallmark of AD according to the amyloid-cascade hypothesis [1].
The diagnosis of AD has increasingly become the focus of attention in recent years.
Currently, memory and behavioral tests are widely used for late-stage AD diagnosis
[
2]. Up to now, computer image techniques, including computed tomography (CT)，
magnetic resonance imaging (MRI), positron emission tomography (PET) and so on,
have been employed for the early detection of AD pathology, and considerable
progress has been achieved [3–13]. However, these computer imaging techniques also
showed some limitations, such as high cost, lack of sensitivity, involvement of
radioactive compounds and sophisticated instrumentation as well as data analysis
variability. On the contrary, fluorescence imaging, particularly near-infrared (NIR)
probe has appeared to be an attractive alternative as a simple, inexpensive and rapid
technique that allows real-time monitoring of the Aβ aggregation in a sensitive and
high-resolution manner.
The NIR fluorescence imaging provides enormous potential as a noninvasive
method for in vivo imaging because biomolecules have low absorption and
autofluorescence in the NIR(﹥630 nm), which result in an optimal penetration depth
and high sensitivity， thus there has been an increasing demand for new chemical
materials that can be used as NIR fluorescence probes for the early detection of Aβ
plaques and interneuronal deposits of NFTs in AD. It is generally accepted that
good NIR probes for the detection of Aβ plaques should have the following
fundamental characteristics: 1) specificity to Aβ plaques; 2) emission wavelength >
6
30 nm and a large Stokes shift; 3) low cytotoxicity for in vivo imaging; 4) high
quantum yield and affinity binding; 5) reasonable stability in blood.
Curcumin is a natural yellow pigment that is derived from the rhizomes of
Curcuma longa. Besides its effective antioxidant, anti-inflammatory, antimicrobial
2

ACCEPTED MANUSCRIPT
and antiviral properties, the compound is also considered as a cancer chemopreventive
agent [14,15]. Additionally, curcumin and its derivatives have been reported to
possess good optical and electrical properties because of a highly π-electron
delocalized and symmetric structure [16,17]. Besides, curcumin has recently been
recognized as a fluorescence probe for the in vivo visualization of both Aβ plaques
and intraneuronal deposits of NFTs [18,19]. However, the practical use of curcumin
as a NIR probe is limited due to its fluorescence emission wavelength (﹤530 nm)
[
20] , which is outside the NIR range. To induce a red-shift of the fluorescence
emission wavelength, a compound with a push/pull electron architecture is generally
designed by introducing terminal electron-rich donor or electron-deficient acceptor
groups to a highly π-conjugated structure system according to principle of Marder [21]
and Perry [22]. For instance, curcumin derivatives (e.g. CRANAD-2, CRANAD-6,
CRANAD-17 and CRANAD-58) with push/pull structures have been devised and
prepared by Anna Moore [23] as NIR fluorescence probes for the in vivo imaging of
AD pathology. Those compounds show fluorescence emission in the NIR range, high
affinity to Aβ, and better fluorescence properties (considerable increase in
fluorescence intensity, large stokes shift and high quantum yield) upon binding to Aβ
aggregates.
We are interested in searching novel curcumin-based molecular probes that can be
used for the NIR fluorescence imaging of Aβ plaques. For this purpose, in the paper,
three
new
curcumin
derivatives,
1,7-bis[(2,5-dimethoxy-3-
(Dye 1),
,7-bis[(2-methoxy-3-diethylamino-5-methyl)phenyl]-1,6-heptadiene--3,5-dione (Dye
) and 1,7-bis[(2-methoxy-3-diethylamino-5-nitryl)phenyl]-1,6-heptadiene--3,5-dione
diethylamino)phenyl]-1,6-heptadiene--3,5-dione
1
2
(Dye 3), were synthetized. Simultaneously, the photophysical and cytotoxic properties
of these compounds were also investigated. Additionally, the application of the Dye 2
as an in vitro and in vivo NIR fluorescent probe for the imaging of Aβ plaques was
primarily studied. The synthetic route of Dyes 1-3 is presented in Scheme 1.
3

ACCEPTED MANUSCRIPT
Scheme 1 Synthetic route of as-prepared Dyes 1-3
2
2
. Materials and methods
.1. Materials and general instruments
All chemicals were available commercially and every solvent was purified by
conventional methods before use. Monomeric Aβ1-40 was purchased from r-Peptide
GA, USA). Double distilled water was used throughout all experiments. All
(
compounds were first dissolved in DMSO to 2 mM, and then diluted by phosphate
buffer solution (PBS, pH = 7.2, Gibco) to different concentration. Each solution used
for photophysical properties was freshly prepared and kept in the dark before
measurement. All aqueous solutions were prepared in distilled water. AD patients
brain frozen tissue (83 years old) and paraffin brain sections were obtained China
Pharmaceutical University (Nanjing, China) with the permission for the use of tissue
and clinical information for research purposes.
1
13
Melting points were determined on WRS-2 (Shanghai China). H NMR and
NMR spectra were performed on a Bruker 400 spectrometer with tetramethylsilane
Si(CH ) ) as the internal standard. The mass spectra were obtained on FINNIGAN
C
(
3
4
LCQ Advantage MAX LC/MS (Thermo Finnigan, American). Elemental analysis was
carried out with a Perkin-Elmer 240C analyzer.
4

ACCEPTED MANUSCRIPT
2
.2. Photophysical methods
Absorbance spectra were obtained on a Shimadzu UV-1650 PC spectrophotometer
Shimadzu, Sydney NSW) and emission spectra were obtained on a Varian CAREY
(
Eclipse fluorescence spectrophotometer (Varian, California USA). The quartz cuvettes
used with a 1 cm path length were used for all spectral collection. The fluorescence
quantum yields of target compounds in DMF were measured according to literature
[
14].
2
.3. Preparation of Aβ1-40 plaques and measurement of their binding constants
The preparation of Aβ1-40 plaques was carried out according to the method reported
previously [24]. In brief, Aβ1-40 monomer was diluted to 50 µM with PBS (pH = 7.2)
and incubated at 37 °C for 3 days. TEM formation of Aβ1-40 plaques..
The in vitro binding constants of Dyes 1-3 with Aβ1-40 plaques were determined as
below. A solution of Aβ plaques (a final concentration of 50 µM) was mixed with
different concentrations of each Dye (0, 20, 40, 60, 80 and 100 µM) and incubated at
room temperature for 5 min. Emission spectra of the resultant mixture were measured
by a fluorescence spectrometer. The apparent dissociation constants (K ) resulted
d
from the saturation experiments were calculated by software Prism 3.0 with nonlinear
one-site binding regression.
2
.4. Cytotoxicity and stability in mouse plasma experiments
The cytotoxic effects of the obtained compound were assessed using the MTT assay.
Human breast cancer (MCF-7) cell lines were seeded into a 96-well culture plate at
5
about 1.0 × 10 cell/well. 50 µL of the sample solution diluted with DMEM at
different concentrations was added to each well, respectively. The cells were
cultivated for 24 h at 37 °C, 5% CO and 95% air, followed by the addition of 50 L
2
MTT solution (5 mg/mL) to each well and incubated for an addition 2 h (37 °C, 5%
CO and 95% air). Then, DMEM was removed, the cells were dissolved in DMSO
2
(
150 µL/well), the absorbance was determined by UV spectrometer at 540 nm. The
percentage of cell viabilities was calculated by the following equation: cell viability
%) = (ODtreated/ODcontrol) × 100%, where ODtreated was obtained in the presence of
(
sample at various concentration, ODcontrol was obtained from the incubation medium.
5

ACCEPTED MANUSCRIPT
Three independent trials were conducted, and the average percentage of viable cells
was calculated relative to untreated cells[25].
Normal Kunming mice were purchased from Animal Laboratory of China
Pharmaceutical University (Nanjing, China). All animal experiments were performed
in compliance with the Animal Management Rules of the Ministry of Health of the
People's Republic of China. The in vitro stabilities of Dyes 1-3 in mouse plasma were
-1
determined as follows. After peritoneal injection of pentobarbital sodium (40 mg kg ),
plasma samples were drawn from cardiac puncture of the mouse and mixed with
heparin (China) to prevent clotting. Each dye was added to the mouse plasma (400 μ
L), and the plasma samples were incubated at 37 °C for 1 h. After incubation, plasma
samples were mixed with equal volumes of acetonitrile, followed by centrifugation
for 10 min to remove the denatured proteins. The supernatant was filtrated using a
0
.50 μm filter (Millipore; Billerica, MA, USA). Then, the filtrate was collected and
determined at different time points by fluorescence spectrometer.
2
.5. AD patients brain tissue sections staining
Dye 2 was selectively used for bioimaging studies because of low cytotoxicity,
strong fluorescence intensity, high quantum yield and binding to Aβ plaques. The
paraffin tissue of AD patients was sliced into 5 µm by a microtome (Leica RM2235,
Germany). The paraffin brain sections were washed with xylene, 80% ethanol
solutions and PBS (pH = 7.2), respectively. Auto fluorescence of the tissue was
quenched using potassium permanganate (0.20% in PBS) for 20 min, followed by
washing with PBS, then the sections were washed with potassium metbisulfite (1%)
and oxalic acid (1%) until the brown color was removed, and then washed with PBS.
The sections were stained with Dye 2 (20 µM) for 1 h, washed by PBS three four
times to remove unlabeled molecules, and sealed with 80% glycerin and coverslips.
The sections were imaged using a fluorescence microscope (Olympus IX81, Tokyo,
Japan) and a SPOT digital camera (Diagnostic Instruments, USA). Under the same
condition, the adjacent section was also stained with silver reagent, a pathological dye
commonly used for staining Aβ plaques in the brain [26].
2
.6. In vivo NIR imaging
6

ACCEPTED MANUSCRIPT
In vivo imaging studies were carried out with the IVIS Lumina system (Xenogen
Co., Alameda, CA, USA) fellowing the reported procedure [27-29]. The images were
acquired with a 600 nm excitation filter and a 680 nm emission filter. Data analyses
were proformed using the KodakTM1D Analysis software.
Mice (n = 4 for APP/PSI transgenic mice) were shaved before background
imaging, and each mouse was intravenously injected with solution of Dye 2 (3.0
mg/kg, 20% DMSO, 80% PBS). Fluorescence signals from the brain were recorded at
0
h, 1.0 h, 2.0 h, 3.0 h and 4.0 h after injection of the compound Dye 2.
2
.7. Preparation of compounds
2
,5-dimethoxy-N,N-diethylamino. 2,5-dimethoxyaniline (0.61 g, 4.0 mM) and
2
0 mL of acetone solution containing anhydrous potassium carbonate (1.2 g, 8.2 mM)
and bromoethane (0.5 mL) were added into a round-bottom flask equipped with a
magnetic stirrer. The resultant mixture was stirred for 15 h at 80 °C, cooled to RT, and
then filtered. The crude product was purified by column chromatography (ethyl
acetate : petroleum ether mixture = 3:2) to give a 2,5-dimethoxy-N,N-dimethylaniline
as colorless powder. Yield: 61.3%.
2
,5-dimethoxy-3-diethylaminobenzaldehyde. 5 mL of dry DMF was cooled to
0
0
°C and treated dropwise with POCl (0.9 mL, 10.0 mM). The solution was stirred at
3
°C for 1 h and at room temperature for another 1 h. Absolute ethanol (20 mL)
solution containing 2,5-dimethoxy-N,N-diethylamino (2.4 g, 10.0 mM) was added
dropwise into the mixture and refluxed at 70 °C for 12 h, then cooled to 5 °C. A
solution of 20 g of sodium acetate in 200 mL of cold water was added slowly with
stirring. The reaction mixture was then stirred for an additional hour and the resulting
solution was extracted with diethyl ether. The combined extract was washed with
saturated anhydrous NaHCO and then washed with water. The organic layer was
3
dried over anhydrous magnesium sulfate and the solvent was removed under reduced
pressure. The residue was purified by column chromatography (hexane : ethyl acetate
=
5:2) on silica gel. 1.08 g of colorless microcrystal was obtained. Yield: 51.7%.
7

ACCEPTED MANUSCRIPT
2
-methoxy-3-diethylamino-5-methylbenzaldehyde. 2-methoxy-3-diethylamino
(
0.35 g，20 mmol), a small quantity of KI and CH I (1.0 mL) were placed into a 20
3
mL. The mixture was stirred for 5 h at 60℃, filtered and the crude product. Absolute
ethanol (20 mL) solution containing crude product and treated dropwise with POCl3
(0.9 mL, 10.0 mM). was added dropwise into the mixture and refluxed at 80 °C for 8
h and treated dropwise with POCl (0.9 mL, 10.0 mM), then cooled to 5 °C, give a
3
2
-methoxy-3-diethylamino-5-methylbenzaldehyde as powder. Yield: 51.8%.
-methoxy-3-diethylamino-5-nitrylbenzaldehyde. 2-methoxy-3-diethylamino
0.35 g，20 mmol) and hydrogen nitrate were placed into 30 mL of dry DMF. The
mixture was stirred at 5 h at 70℃, filtered and the crude product. Absolute ethanol
20 mL) solution containing crude product and treated dropwise with POCl3 (1.5 mL,
5.0 mM), was added dropwise into the mixture and refluxed at 80 °C for 7 h, filtered
and give a 2-methoxy-3-diethylamino-5-nitrylbenzaldehyde as powder. Yield: 48.9%
,7-bis[(2,5-dimethoxy-3-diethylamino)phenyl]-1,6-heptadiene--3,5-dione (Dye
). Potassium tertbutoxide (0.10 g, 8.0 mM) was added to a solution of acetylacetone
0.3 mL, 3.5 mM) in DMF (5 mL) and the resultant mixture was stirred at room
temperature for under nitrogen atmosphere.
,5-dimethoxy-3-(diethylamino)benzaldehyde (1.74 g, 7.0 mM) was added and the
2
(
(
1
1
1
(
1
h
2
reaction mixture was stirred at 40 °C for 15 h. The reaction mixture was poured over
saturated aqueous ammonium chloride and then extracted with ethyl acetate. The
organic layer was washed with water and anhydrous MgSO , respectively. The
4
solvent was removed under reduced pressure, and the residue was purified by column
chromatography (hexane ：ethyl acetate = 4:3) to give Dye 1 (1.83 g) as pale yellow
1
microcrystal. Yield: 53.8%. m.p. 152-154 °C. H NMR (400 MHz, DMSO-d6) δ: 7.85
(d, J = 15.0 Hz, 2H), 7.46 (d, J = 12.8 Hz, 2H), 6.86-6.65 (m, 4H), 5.77 (s, 2H),
1
3
3
.68-3.45 (m, 12H), 3.20-3.15 (m, 8H), 2.90 (s, 12H); C NMR (75 MHz, DMSO-d6)
δ: 190.2, 154.4, 150..2, 145.1, 132.0, 131.7, 131.4, 128.2, 125.5, 122.7, 115.9, 115.3,
+
8
8.7, 44.4, 41.7, 24.5, 21.3; MS (ESI) m/z：539.65 ([M + H] , 100); Anal. calcd for
C H N O : C 69.12, H 7.86, N 5.20; found: C 68.89, H 7.63, N 5.06.
31
42
2
6
1
,7-bis[(2-methoxy-3-diethylamino-5-methyl)phenyl]-1,6-heptadiene-3,5-dione
8

ACCEPTED MANUSCRIPT
(Dye 2). The synthesis method of Dye 2 is similar to that of Dye 1. The compound
was obtained starting from 2-methoxy-3-diethylamino-5-methylbenzaldehyde. The
crude product was purified by column chromatography (ethyl acetate : petroleum
ether mixture = 9:5) to get pale yellow microcrystal. Yield: 51.6%. m.p. 145-147 °C.
1
H NMR (400 MHz, DMSO-d6) δ: 7.78 (d, J = 15.0 Hz, 2H), 7.42 (d, J = 12.8 Hz,
2
H), 6.82-6.64 (m, 4H), 5.68 (s, 2H), 3.63-3.42 (m, 12H), 3.18-3.13 (m, 8H), 2.88 (s,
1
3
1
2H); C NMR (75 MHz, DMSO-d6) δ: 190.0, 154.3, 150.1, 144.9, 131.7, 131.4,
31.1, 127.9, 125.2, 122.3, 115.5, 115.0, 88.4, 44.2, 41.5, 24.1, 21.1; MS (ESI) m/z：
1
+
5
07.63 ([M + H] , 100); Anal. calcd for C H N O : C 73.48, H 8.36, N 5.53; found:
3
1
42
2
4
C 73.26, H 8.21, N 5.34.
1
,7-bis[(2-methoxy-3-diethylamino-5-nitryl)phenyl]-1,6-heptadiene--3,5-dione
(Dye 3). The synthesis method of Dye 3 is similar to that of Dye 1. The compound
was obtained starting from 2-methoxy-3-diethylamino-5-nitrylbenzaldehyde. The
crude product was purified by column chromatography (ethyl acetate : petroleum
ether mixture = 7:6) to get pale yellow microcrystal. Yield: 43.7%. m.p. 125-127 °C.
1
H NMR (400 MHz, DMSO-d6) δ: 7.90 (d, J = 15.0 Hz, 2H), 7.51 (d, J = 12.8 Hz,
2
H), 6.92-6.71 (m, 4H), 5.83 (s, 2H), 3.71-3.50 (m, 6H), 3.23-3.15 (m, 8H), 2.94 (s,
1
3
1
2H); C NMR (75 MHz, DMSO-d6) δ: 190.5, 154.7, 150.5, 145.4, 132.3,131.9,
31.7, 128.5, 125.8, 122.9, 116.1, 115.6, 88.9, 44.7, 41.9, 24.8, 21.5; MS (ESI) m/z：
1
+
5
69.60 ([M + H] , 100); Anal. calcd for C H N O : C 61.25, H 6.38, N 9.86; found:
2
9
36
4
8
C 60.98, H 6.22, N 9.64. .
3
3
. Results and discussion
.1. Preparation and characterization of Dyes 1-3
Synthetic route of as-prepared Dyes 1-3 and their intermediates are shown in
Scheme 1. The high reactivity of acetyl acetone in alkaline condensation reactions
with aldehyde is well-known. In the light of the mechanism from the literature [30],
Dyes 1-3 were prepared by the condensation reaction between acetyl acetone and
2
,5-dimethoxy-3-(diethylamino)benzaldehyde, and 2-methoxy-3-diethylamino-5-
methylbenzaldehyde,
and
2-methoxy-3-diethylamino-5-nitrylbenzaldehyde,
9

ACCEPTED MANUSCRIPT
respectively. The structures of Dyes 1-3 were characterized by NMR, MS
spectroscopic methods and elementary analyses, and the satisfactory analysis data
were obtained (Supporting Information).
3
.2. The photophysical properties of Dyes 1-3
The photophysical data of Dyes 1-3 are showed in Table 1. Fig. 1a displays the
linear absorption spectra of Dyes 1-3 in DMF with a solution concentration of 1.0 ×
−
5
1
0
mol/L. From Fig. 1a, one can see that the absorption behaviors of the three
compounds are almost the same because of their similar structure. The three curves
show that absorption maximum at 328 nm for Dye 1, 331 nm for Dye 2, and 330 nm
for Dye 3, corresponding to the n-π* transition of the compound [31].
As expected, with introduction of terminal electron-rich donor or electron-deficient
acceptor groups, these compounds exhibited a large Stokes shift (more than 130 nm)
with their emission maximum (shown in Table 1), which fell in the range for NIR
probes. Particularly, the compound Dye 2 displayed a largest Stokes shift (λmax(ex) =
4
99 nm, λ max (em) = 635 nm). Furthermore, by comparing the fluorescence intensity,
the quantum yield of Dye 2 was significantly higher than that of curcumin and other
Dyes, which may be explained by better coplanarity and the stronger
electron-donating ability (methyl) of the Dye 2 molecule.
1
0

ACCEPTED MANUSCRIPT
Table 1 The optical properties of the compounds upon binding with and without Aβ
plaques
Unbouu
d
Bou
nd
c
g
(
free)
b
Compd
λ
ε ×
λex
Fold
a
4
d
e
1
0
λ
λ
λem
abs
ex
em
f
Ф1
(nm
Ф2
(Ф2/
-
1
(n
(M c (nm)
(nm)
(nm)
)
Ф1)
-
1
m) m )
32
Dye 1
Dye 2
Dye 3
Cur
3
3
3
2
.24
.86
.18
.87
479
499
497
421
609
635
633
522
0.018
0.019
479
499
497
423
609
635
633
0.32
0.37
0.29
0.22
17.8
19.5
18.1
15.7
8
33
1
33
0
0.016
0.014
31
5
521
a
b
absorption spectra of the compounds determined in DMF. Molar extinction
c
coefficient determined in DMF. Fluorescence properties of the compounds measured
d
in PBS in absence of Aβ plaques. Maximum excitation wavelength of the compound
e
f
measured in DMF. Maximum emission wavelength of the compound. Quantum
g
yield of the compound were measured in DMF. Fluorescence property of the
compound measured in PBS in presence of Aβ plaques.
3
.3. Fluorescence response of NIR probe toward Aβ plaques
Scanning electron microscopy (SEM) had been used here to analyze the
morphology of dyes (dye 1, dye 2 and dye 3). The SEM images of dyes were given in
Fig.S6. The shape of the particles is regular and the particles are of variable diameter
ranging from 25 to 37 nm. In phosphate buffered saline (PBS, PH = 7.2) at 37 °C,
dye(dye 1, dye 2 and dye 3) remained and maintained its original compound size for
up to 12 days, indicating the excellent stability of dyes probe in aqueous conditions.
Table 1 exhibited that curcumin and the Dyes 1-3 possessed significant increases
of quantum yield after mixing with the Aβ plaques. Curcumin showed a 15.7-fold
increase in quantum yield and a Stokes shift (98 nm) in fluorescence upon binding to
11

ACCEPTED MANUSCRIPT
Aβ plaques, but the maximum emission wavelength was observed only at 521 nm.
Among the compounds, Dye 2 with methoxy and methyl substituents showed the
most notable fluorescence properties (λem, 635 nm, 136 nm of Stokes shift and
1
9.5-fold quantum yield increase), which indicated that it had the desired optical
properties of a useful fluorescence probe for Aβ plaques. However, Dyes 1 and 3
displayed relatively small increase in fluorescence intensities and quantum yields
upon mixing with Aβ plaques. Compared with the low fluorescence quantum yield (<
0
.2%) of the free compound in DMF, the curcumin-based NIR probes exhibited
significant fluorescence increase upon binding to Aβ1-40 plaques (Supporting
Information). The fluorescence enhancement might be attributed to the restricted
rotation of the photo-excited dyes upon binding to Aβ plaques, resulting in a large
reduction in the fluorescence decay [32]. The results suggested that these dyes would
be “turned on” upon interacting with a host.
On the other hand, comparison of the fluorescence spectra of NIR probes upon
binding to Aβ plaques provided a clue for understanding this interesting
structure–property relationship. The compound with small fluorescence increases
(Dye 1 and 3) shared a common feature: there is electron-withdrawing groups in the
molecular structure. In particular, electron withdrawing ability of the nitryl group in
Dye 3 was stronger than that of methoxy group in Dye 1.
In order to quantitatively evaluate the binding affinity of Dyes 1-3 with Aβ plaques,
an in vitro saturation-binding assay was conducted by the fluorescence titrations and
the K constants were measured by using saturation assays as reported previously [33].
d
As shown in Fig. 1b, the nonlinear curve fitting indicated that these Dyes possessed
stronger binding affinities toward the Aβ plaques, and the fluorescence enhancement
upon binding to Aβ plaques is in the order of FDye 2 (K ,1.36 µM) > FDye 1 (K ,2.68
d
d
µM) > FDye 3 (K ,3.43 µM). Interestingly, Dyes 2 bound to Aβ plaques more favorably
d
than Thioflavin-S, a widely used agent for detecting protein and Aβ aggregation (Kd,
1
.90 µM) [34].
1
2

ACCEPTED MANUSCRIPT
−
5
Fig. 1. (a) Absorption and fluorescence emission spectra of Dyes 1-3 (1.0 × 10
mol/L). (b) Saturation binding curves of Aβ1-40 plaques (50 µM) to the concentration
of each dye (0-100 µM) (c) Fluorescence intensity changes of Dyes 1-3 during
irradiation time. (d) Cytotoxicity of obtained compounds against MCF-7 cell line (24
h). (e) Cytotoxicity of obtained compounds against MCF-7 cell line (48 h) at
different concentrations from the MTT assay.
3
.5. Cytotoxicity and stability studies
In order to evaluate the potential application of Dyes 1-3, the cytotoxicity of these
dyes towards human breast cancer (MCF-7) cells was evaluated using the MTT assay.
Different concentrations in the range of 0-100 µM of Dyes 1-3 were given to cells
upon exposure for 24 h and for 48 h. The results were shown in Fig. 1c. No obvious
cell viability decrease was observed and the cell viability(≤ 40 µM) was still greater
than 85% when the concentration of the compound reached up to 80 µM, which is 4
times of the concentration used for cell imaging in our living cell imaging. The low
cytotoxicity of the obtained target compound indicates its suitability for biological
imaging applications.
We investigated too the in vitro stability of Dyes 1-3 in mouse plasma by
1
3

ACCEPTED MANUSCRIPT
monitoring their fluorescence intensity for 3 h (Fig. 1d). Dyes 1-3 existed as a hardly
degradation form, suggesting these compounds were relatively stable in mouse
plasma. On the whole, Dyes 2 showed promising potential application for the imaging
of Aβ plaques.
3
.6. Fluorescent staining of AD brain tissue sections
In order to assess bioimaging, Dye 2 was selected to monitor intracellular Aβ
plaques. Post-mortem human frontal cortex tissue from a subject diagnosed with
clinical Alzheimer's disease were sliced into 5 µm by a microtomeand, and then
stained with Dye 2 (20 µM). With confocal microscopy observation, the intracellular
expression of Aβ plaques was confirmed by yellow green fluorescence stained with
Dye 2 (Fig. 2b). It is spacing needed that the same circular vacuole-like subcellular
structures were also observed when stained with the silver reagent (white arrows in
Fig. 2a). In contrast, the adjacent section was uniformly stained in the negative control
(normal group) (Fig. 2c). The results of the colocalization in the in vitro fluorescence
images suggested that Dye 2 specifically detected Aβ plaques in live cells.
Fig. 2. Fluorescent staining of adjacent brain tissue sections of AD patients. a) silver
staining, b) Dye 2 staining, c) the negative control. The white arrows indicate Aβ
plaques. The scale bars are 50 μm.
3
.7. Curcumin binding in control and AD brain tissue
It was observed that curcumin binding on human brain tissue presented no
background staining and auto-fluorescent signals in neurons. In brain tissue of EOAD,
LOAD and AD cases with CAA (CAA type 1), the compounds strongly bound to
amyloid plaques and CAA (Fig. 1). Curcumin staining revealed different types of
1
4

ACCEPTED MANUSCRIPT
plaques. We observed intense staining of primitive/compact plaques and moderate
staining of the cores in cored plaques. In AD cases, neurofibrillary tangles (NFTs)
showed a weak detection by the Dyes. Adjacent sections were assessed by
immunohistochemistry for the presence of Aβ and pTau in a corresponding area,
which confirmed the presence of these pathological structures. The binding of the
dyes to these pathological structures were investigated by co-stainings for dyes and
Aβ (IC-16) or pTau (AT8). Primitive/compact plaques showed almost a complete
overlap of dyes signal and immunodetection by Aβ (Fig. 2). Classic cored plaques
presented co-labeling of the core, while the corona of these plaques were
immuno-labelled by anti-Aβ and not by dyes. No binding of the dyes were observed
in diffuse Aβ deposits. CAA affected blood vessels were stained most intensively by
dyes and showed a complete overlap with the detection of CAA by Aβ. While
compact plaques showed moderate neuritic changes, as presented by anti-pTau
immuno-labeling, no co-labelling of anti-pTau and dyes were present in these plaques
(Fig. 3). Neuritic cored plaques showed a strong immunodetection of pTau around the
core. The core of neuritic plaques showed a strong binding of the dyes while
co-labelling with anti-pTau were absent. A strong binding of dyes were observed in
CAA, however, no co-labelling with anti-pTau, observed around CAA affected
capillaries, was detected. Co-labeling with antipTau confirmed that curcumin weakly
detects NFTs, they showed slightly less apparent staining of fibrillar,
primitive/compact plaques and NFTs compared to Thioflavin-S (Fig. 2).
3
.8. In vivo imaging
To investigate whether Dye 2 had the capacity to detect Aβ plaques in the mouse
brain, APP/PSI mice, which most studied transgenic AD mouse model [35,36], were
used as a NIR imaging of Dye 2. Previous reports shown that the APP/PSI mouse
carried double humanized APP/PSI genes and develops significant amyloid plaques
and displays memory deficits around 10 - 12 months of age [24,37]. In this studied,
we use 17- month of age mouse model for in vivo NIR imaging. As shown in Fig. 3,
one can be seen that the Aβ plaques site was recognizable within 1 h after injection of
the compound Dye 2. Interestingly, as time progresses, the probe increasingly
accumulated in the Aβ plaques and the fluorescence reached the highest intensity at
1
5

ACCEPTED MANUSCRIPT
about 2 h after injection. However, the fluorescence in the Aβ plaques position began
to wear off, and then the fluorescence almost completely disappeared from the Aβ
plaques at about 4 h after injection. The results suggest that the compound is able to
detect the Aβ plaques in AD.
Fig. 3. Representative images of APP/PSI mice and control littermates at different
time points before and after intravenously injection with Dye 2
4
. Conclusions
In summary, three curcumin-based NIR dyes 1-3 for Aβ plaques were synthesized
which exhibited obvious fluorescence enhancement after binding to Aβ plaques.
Interestingly, Dye 2 with methoxy and methyl substituents shows the intense
fluorescence, low cytotoxicity and high quantum yield, good stability in mouse serum,
as well as fluorescent imaging of Aβ plaques reveal its potentially suitable for use in
the early diagnoses of Aβ plaques.
Acknowledgements
This research was financially supported by the National Natural Science
Foundation of China (No. 21071001), The Natural Science Foundation of Anhui
Province (No. 1808085MB47), Department of Education Committee of Anhui
Province and the Center for Anhui Province Engineering of Modern Chinese
traditional medicine (No. KJ2017A291), and the Opening Foundation of Anhui
Province Key Laboratory of Environment-friendly Polymer Materials (No. 2018001)..
References
[
1] J. Hardy, D.J. Selkoe, The amyloid hypothesis of Alzheimer's disease: progress
and problems on the road to therapeutics. Science 2002, 297: 353–356.
1
6

ACCEPTED MANUSCRIPT
[
2] J. Praet, C. Bigot, J. Orije, M. Naeyaert, D. Shah, Magnetization transfer contrast
imaging detects early white matter changes in the APP/PS1 amyloidosis mouse model.
NeuroImage: Clinical 2016, 12: 85–92.
[
3] L. Zhu, K. Ploessl, H.F. Kung, PET/SPECT imaging agents for neurodegenerative
diseases. Chem. Soc. Rev. 2014, 43: 6683-6691.
4] Y. Cheng, M. Ono, H. Kimura, M. Ueda, H. Saji, Technetium-99m labeled pyridyl
[
benzofuran derivatives as single photon emission computed tomography imaging
probes for beta-amyloid plaques in Alzheimer's brains. J. Med. Chem. 2012, 55:
2
279-2286.
[
5] S. Li, H. He, W. Cui, B. Gu, J. Li, Z. Qi., Detection of Aβ plaques by a novel
specific MRI probe precursor CR-BSA-(Gd-DTPA)n in APP/PS1 transgenic mice.
Adv. Integr. Anat. Evol. Biol. 2010, 293: 2136-2143.
[
-
-
[
6] Di Wu, Yuanzhi Shen, Jianhua Chen, Guotao Liu, Haiyan Chen, Jun Yin, Naphth
alimide-modified near-infrared cyanine dye with a large stokes shift and its applica
tion in bioimaging, Chin. Chem. Lett. 2017, 28, 1935-1942.
7] Zhiqiang Xu, Jianhua Chen, Liu-Li Hu, Ying Tan, Sheng-Hua Liu, Jun Yin,
Recent advances in formaldehyde-responsiive fluorescent probes, Chin. Chem. Lett.
017, 28, 1935-1942.
8] Yanzhu Liu, Liucheng Mao, Saijiao Yang, Meiying Liu, Hongye Huang,
2
[
Yuanqing Wen, Fengjie Deng, Yongxiu Li, Xiaoyong Zhang, Yen Wei, Synthesis and
biological imaging of fluorescent polymeric nanoparticles with AIE feature via the
combination of RAFT polymeric nanoparticles polymerization modification, Dyes
and Pigments, 2018, 158, 79-87.
[
9] Ruming Jiang, Meiying Liu, Hongye Huang, Long Huang, Qiang Huang,
Yuanqing, Wen, Qianyong Cao, Jianwen Tian, Xiaoyong Zhang, Yen Wei,
Microwave-assisted multicomponent tandem polymerization for rapid preparation of
biodegradable fluorescent organic nanoparticles with aggregation-induced emission
feature and their biological imaging applications Dyes and Pigments, 2018, 149,
5
81-587,
[
10] Hongye Huang, Meiying Liu, Qing Wan, Ruming Jiang, Dazhuang Xu, Qiang
1
7

ACCEPTED MANUSCRIPT
Huang, Yuanqing Wen, Fengjie Deng, Xiaoyong Zhang, Yen Wei, Facile fabrication
of luminescent hyaluronic acid with aggregation-induced emission through formation
of dynamic bonds and their theranostic applications, Materials Science and
Engineering: C, 2018, 91, 201-207
[
11] Ruming Jiang, Han Liu, Meiying Liu, Jianwen Tian, Qiang Huang, Hongye
Huang, Yuanqing Wen, Qianyong Gao, Xiaoyong Zhang, Yen Wei, A facile one-pot
Mannich reaction for the construction of fluorescent polymeric nanoparticles with
aggregation-induced emission feature and their biological imaging,
Science and Engineering: C, 2017, 81, 416-421.
Materials
[
12] Xiaoyong Zhang, Shiqi Wang, Liangxin Xu, Lin Feng, Yan Ji, Lei Tao, Shuxi Li
and Yen Wei, Biocompatible polydopamine fluorescent organic nanoparticles: facile
preparation and cell imaging, Nanoscale, 2012, 4 (18), 5581-5584.
[
13] G.Y. Xu, D. Wei, J.F. Wang, B. Jiang, M.H. Wang, X. Xue, S.S. Zhou, B.X. Wu,
M.H.Jiang, Crystal structure, optical properties and biological imaging of
twocurcumin derivatives. Dyes Pigments, 2014, 101: 312–317.
[
14] U. Subudhi, K. Das, B. Paital, S. Bhanja, G.B.N. Chainy, Supplementation of
curcumin and vitamin E enhances oxidative stress, but restores hepatic
histoarchitecture in hypothyroid rats. Life Sciences 2009, 84: 372-379.
[
15] Zhiqiang Xu, Xiaoting Huang, Xie Han, Di Wu, Bibo Zhang, Ying Tan, Meijiao
Gao, Shenghua Liu, Jun Yin, Juyoung Yoon, A visible and Near-infrared,
Dual-Channel Fluorescence on probe for Selectively Tracking Mitochondrial GI
Utathione, Chem 2018, 7, 1609-1628
[
16] S.S. Zhou, X. Xue, J.F. Wang, B. Jiang, D. Wei, Y. Jia, Synthesis, optical
properties and biological imaging of the rare earth complexes with curcumin and
pyridine. J. Mater. Chem. 2012, 22: 22774–22780.
[
17] M. Garcia-Alloza, L.A. Borrelli, A. Rozkalne, B.T. Hyman and B.J. Bacskai,
Curcumin labels amyloid pathology in vivo, disrupts existing plaques, and partially
restores distorted neuritis in an Alzheimer mouse model. J. Neurochem. 2007, 102:
1
095-1101.
[
18] F. Yang, G.P. Lim, A.N. Begum, O.J. Ubeda, M.R. Simmons, S.S. Ambegaokar,
1
8

ACCEPTED MANUSCRIPT
P.P. Chen, R. Kayed, C.G. Glabe, S.A. Frautschy and G.M. Cole, Curcumin inhibits
formation of amyloid beta oligomers and fibrils, binds plaque, and reduces amyloid in
vivo. J. Biol. Chem. 2005, 280: 5892-5898.
[
19] K.S. Park, Y. Seo, M.K. Kim, K. Kim, Y.K. Kim, H. Choob and Y. Chong, A
curcumin-based molecular probe for near-infrared fluorescence imaging of tau fibrils
in Alzheimer’s disease. Org. Biomol. Chem. 2015, 13: 11194–11199.
[
20] M. Albota, D. Beljonne, L. Br´edas, J.E.J. Ehrlich, J.Y. Fu, A.A. Heikal, S.E.
Hess, T. Kogej, M.D. Levin, S.R. Marder, D. McCord-Maughon, J.W. Perry, H.
Rcckel, M. Rumi, C. Subramaniam, W.W. Webb, X.L. Wu and C. Xu, Science 1998,
2
81: 1653–1655.
[
21] M. Rumi, J.E. Ehrlich, A.A. Heikal, J.W. Perry, S. Barlow, Z. Hu, D.
McCord-Maughon, T.C. Parker, H. Rcckel, S. Thayumanavan, S.R. Marder, D.
Beljonne and J.L. Brecdas, Structure−Property Relationships for Two-Photon
Absorbing Chromophores: Bis-Donor Diphenylpolyene and Bis(styryl)benzene
Derivatives J. Am. Chem. Soc. 2000, 122: 9500–9510.
[
22] C. Ran, X. Xu, S.B. Raymond, B.J. Ferrara, K. Neal, B.J. Bacskai, Z. Medarova
and A. Moore, Design, synthesis, and testing of difluoroboron-derivatized curcumins
as near-infrared probes for in vivo detection of amyloid-β deposits. J. Am. Chem. Soc.
2
009, 131: 15257-1562.
[
23] X. Zhang, Y. Tian, Z. Li, X. Tian, H. Sun, H. Liu, A. Moore and C. Ran, Design
and synthesis of curcumin analogues for in vivo fluorescence imaging and inhibiting
copperinduced cross-linking of amyloid beta species in Alzheimer’s disease. J. Am.
Chem. Soc. 2013, 135: 16397-16401.
[
24] W.E. Klunk, Y.M. Wang, G.F. Huang, M.L. Debnath, D.P. Holt, C.A. Mathis,
Uncharged thioflavin-T derivatives bind to amyloid-beta protein with high affinity
and readily enter the brain. Life science 2001, 68: 1471-1484.
[
25] D.D. Nan, C.S. Gan, C.W. Wang, J.P. Qiao, X.M. Wang, J.N. Zhou,
-Methoxy-indanone derivatives as potential probes for β-amyloid plaques in
Alzheimer's disease. European Journal of Medicinal Chemistry 2016, 124: 117-128.
26] C.Z. Ran, X.Y. Xu, S.B. Raymond, B. J. Ferrara, K. Neal, B. J.Bacskai, Z.
6
[
1
9

ACCEPTED MANUSCRIPT
Medarova, A. Moore, Design, synthesis, and testing of difluoroboron derivatized
curcumins as near infrared probes for in vivo detection of amyloid-β deposits, J. Am.
Chem. Soc. 2009, 131(42): 15257–15261.
[
27] Xiaoyong Zhang, Ke Wang, Meiying Liu, Xiqi Zhang, Lei Tao, Yiwang Chen
and Yen Wei, Polymeric AIE-based nanoprobes for biomedical applications: recent
advances and perspectives, Nanoscale, 2015, 7 (27), 11486-11508.
[
28] G.Y. Xu, J.F. Wang, T. Liu, M.H. Wang, S.S. Zhou, M.H. Jiang, Synthesis and
crystal structure of a novel copper(II) complex of curcumin-type and its application in
in vitro and in vivo imaging, J. Mater. Chem. B, 2014, 2, 3659 - 3666.
[
29] C.Z. Ran, X.Y. Xu, S.B. Raymond, B. J. Ferrara, K. Neal, B. J.Bacskai, Z.
Medarova, A. Moore, Design, synthesis, and testing of difluoroboron derivatized
curcumins as near infrared probes for in vivo detection of amyloid-β deposits, J. Am.
Chem. Soc. 2009, 131(42): 15257–15261.
[
30] H. Chao, R.H. Li, C.W. Jiang, H. Li, L.N. Ji and X.Y. Li, Mono-, di- and
tetra-nuclear ruthenium(II) complexes containing
,2′-p-phenylenebis(imidazo[4,5-f ]phenanthroline): synthesis, characterization and
third-order non-linear optical properties. J. Chem. Soc., Dalton Trans., 2001,
920-1926.
31] C.Z. Ran, X.Y. Xu, S.B. Raymond, B.J. Ferrara, K.N. Brian, Z. Medarova, and A.
2
1
[
Moore, Design, synthesis, and testing of difluoroboron derivatized curcumins as near
infrared probes for in vivo detection of amyloid-β deposits. J. Am. Chem. Soc. 2009,
1
31(42): 15257–15261.
[
32] H.P. Zhou, D.M. Li, J.Z. Zhang, Y.M. Zhu, J.Y. Wu, Z.J. Hu, Crystal structures,
optical properties and theoretical calculation of novel two-photon polymerization
initiators. Chem. Phys. 2006; 322: 459-470.
[
33] H.J. Tong, K. Loua, W. Wang, Near-infrared fluorescent probes for imaging of
amyloid plaques in Alzheimer's disease. Acta Pharmaceutica Sinica B 2015, 5(1):
5–33.
34] Y.H. Li, D. Xu, S.L. Ho, H.W. Li, R.H. Yang, A theranostic agent for in vivo
near-infrared imaging of β-amyloid species and inhibition of β-amyloid aggregation.
2
[
2
0

ACCEPTED MANUSCRIPT
Biomaterials 2016, 94: 84-92.
35] B. Delalour, M. Guegan, A.Volk, M. Dhenain, Neurobiology of aging, 2006, 27:
35-839.
36] X.L. Zhang, Y.L. Tian, Z. Li, X.Y. Tian, A. Moore, C.Z. Ran, Design and
[
8
[
synthesis of curcumin analogues for in vivo fluorescence imaging and inhibiting
copper-induced cross linking amyloid beta species in Alzheimer's disease. J. Am.
Chem. Soc. 2013, 135 (44): 16397 – 16409.
[
37] Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, Yang F,
Cole G. Correlative memory deficits, A beta elevation, and amyloid plaques in
transgenic mice. Science 1996, 274: 99–102.
2
1

ACCEPTED MANUSCRIPT
Research highlights
*
Three novel compounds curcumin-based was synthesized
*
*
*
These compounds shown obvious fluorescence change after binding to Aβ plaques.
These compounds have Cytotoxicity..
Imaging strained with Dye 2 confirmed that it could be to detect Aβ plaques in AD.