Fluorescent flavonoids for endoplasmic reticulum cell imaging

Article abstract of DOI:10.1039/C6TB02456D

Visualization of subcellular organelles in vivo is critical for basic biomedical research and clinical applications. Two new flavonoids with an amide substituent were synthesized and characterized. The flavonoids were nearly non-fluorescent in aqueous env

Full text of DOI:10.1039/C6TB02456D

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Fluorescent flavonoids for endoplasmic reticulum
cell imaging†
Cite this:DOI: 10.1039/c6tb02456d
a
a
a
a
Lucas McDonald, Bin Liu,‡ Alexandra Taraboletti, Kyle Whiddon,
ab
a
b
a
Leah P. Shriver, Michael Konopka, Qin Liu and Yi Pang*
Visualization of subcellular organelles in vivo is critical for basic biomedical research and clinical applications.
Two new flavonoids with an amide substituent were synthesized and characterized. The flavonoids were
nearly non-fluorescent in aqueous environment, but exhibited two emission peaks (one lem at 495–536 nm
and the other at 570–587 nm) in organic solvents, which were assigned to the excited normal (N*) and
tautomer (T*) emission. When the dyes were examined on oligodendrocyte cells, they were found to
selectively accumulate in the endoplasmic reticulum (ER), a eukaryotic organelle involved in lipid and
protein synthesis, giving fluorescence turn-on. The ER-selective flavonoids could be a valuable tool due
to its low molecular mass (o500), large Stokes’ shift, low toxicity, and biocompatibility.
Received 20th September 2016,
Accepted 16th November 2016
DOI: 10.1039/c6tb02456d
www.rsc.org/MaterialsB
nervous system (CNS) and promotes rapid conduction of nerve
impulses. Myelin is a poorly hydrated structure containing
Introduction
9
Optical visualization of subcellular organelles and biochemical 40% water, and the dry mass of myelin consists of B70% lipids
processes in vivo is critical for basic biomedical research and and 30% proteins. During the active phase of myelination (the
1–3
the development of novel clinical diagnostics. The endoplasmic process of forming myelin sheath), each oligodendrocyte in the
2
reticulum (ER) plays a central role in the biosynthesis of lipids and CNS must produce as much as B5000 mm of myelin surface area
5
proteins. In the lumen of the endoplasmic reticulum, proteins per day, and B10 myelin-associated protein molecules per
10
fold and mature before being delivered to other compartments of minute. ER stress can disturb lipid and protein synthesis and
cells or released extracellularly. An accumulation of unfolded or this has been linked to oligodendrocyte perikaryon degenera-
11
misfolded proteins causes disturbances in the normal functions tion and myelin sheath disintegration. In addition, ER stress
of the ER, which induces a cellular stress response (or ER stress) in oligodendrocytes has been implicated in dysmyelinating
1
,2
12
that activates the unfolded protein response (UPR).
UPR diseases.
activation increases ER abundance to match needs by mediating
expansion of the ER membrane. Since ER stress is involved in cent probes that can target specific biological tissues/molecules
Considerable interests exist in searching for novel fluores-
3
1
3
the feedback loops that establish and maintain homostasis, it plays to meet the need for advanced bioimaging. Among the known
4
5
a significant role in various diseases such as autoimmunity
probes, few are capable of targeting ER, partly due to the
6
,7
and metabolic disease. Because of the importance of the ER in challenge in controlling ER selectivity. A recent ER-probe uses
1
4
disease, there is significant interest in developing new probes to a nitro-furan group to target ER-associated proteins. The
8
track ER dynamics. An effective ER imaging probe has potential commercial ER-Tracker Green and Red contains a ‘‘gliberclamide’’
to identify perturbations in ER function that are associated with segment (Scheme 1), which binds to the sulphonylurea receptors
+
15,16
17
early disease pathology.
of ATP-sensitive K channels
that are prominent on ER. In
Oligodendrocytes are the myelinating glial cells of the the commercial ER probes, the necessary ‘‘gliberclamide’’ segment
central nervous system. The myelin sheath produced by these for ER selectivity has the mass of 494 Da, whose relative large
cells is a lipid-rich structure that insulates axons in the central moiety could cause significant perturbations to normal cell activity
upon introduction. In order to avoid this potential side effect, an
a
ideal ER probe should have the lowest possible molecular weight
(e.g. Mw below 500 Da). In addition, the probe should be non-toxic
for cells.
Department of Chemistry, University of Akron, Akron, Ohio 44325, USA.
E-mail: yp5@uakron.edu
b
Department of Biology, University of Akron, Akron, Ohio 44325, USA
Electronic supplementary information (ESI) available. See DOI: 10.1039/
†
As a broad class of natural substances normally occurring in
diet, flavonoids exhibit a variety of beneficial health effects,
c6tb02456d
Current address: Shenzhen Key Laboratory of Special Functional Materials,
College of Materials Science and Engineering, Shenzhen University, Shenzhen
18060, China.
‡
1
8,19
including antioxidant properties and anticancer activities.
5
With simple/minor structural modification, the flavonoids can
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product was purified by recrystallization from hexane/ethanol
mixture in good yield.
Optical properties
Both flavonoid compounds showed a similar absorption, revealing
lmax at 406 nm and 411 nm for 2a and 2b, respectively (Fig. 1). The
emission of 2a revealed two peaks with lem at 495 and 570 nm,
which could be attributed to the excited normal form (N*) and the
tautomeric form (T*), respectively. The tautomer emission (from
T*) was associated with the excited state intramolecular proton
25
transfer (ESIPT) (see Scheme 3). In contrast, the emission lem
from the N* and T* of 2b was at 536 and 587 nm, with about equal
intensity. The observed large difference in the ratio of N- and
T-emission indicated that the amino substituent in the
electron-donor segment had a profound impact on the emissive
property of flavonoids. By using rhodamine 6G in ethanol
Scheme 1 Structures of commercial ER-Tracker, where the fluorophore
is shown in blue color.
26
(QY = 95%) as a reference, the fluorescence quantum yield (QY)
of 2a and 2b were determined to be 26% and 47%, respectively.
Both 2a and 2b exhibited large Stokes’ shifts (B170 nm), which
is highly desirable for fluorescence imaging. The observed large
Stokes’ shift could be attributed to a combination of the intra-
molecular charge transfer (ICT) and ESIPT processes occurring
in the flavonoids.
Compound 2 appeared to be non-fluorescent in aqueous
solution, possibly due to excessive hydrogen bonding to chromen-
4-one unit (see 1 in Scheme 2). In a non-aqueous solution, however,
the flavonoid gave a large fluorescence turn-on (Fig. 2). In a less
polar solvent such as THF and toluene, the flavonoid gave a
Scheme 2 Chemical structures of flavonoids 1–2 and synthesis of 2.
also exhibit ‘‘excited-state intramolecular photon-transfer (ESIPT),’’
20
which provides intense fluorescence with large Stokes Shifts. The
combination of the unique optical properties and biocompatibility
makes flavonoid compounds a valuable template for sensor
design. Our recent studies show that the flavonoid 1 (Scheme 2)
2
with a suitable amino substituent (R N–) is an environmentally
sensitive dye, which can be tailored to target various biomolecular
21,22
structures.
Thus, via attaching a functional group at the C-3
position of ‘‘chromen-4-one’’ segment, flavonoid 1a can label
21
mitochondria. Flavonoid 1b shows a large fluorescence turn-on
22
upon selective binding to albumin proteins. The observed protein
selectivity also raises the possibility for other protein binding-
Fig. 1 UV-vis (broken line) and fluorescence spectra (solid line) of 2a and 2b in
CH Cl (10 mM) at room temperature. The fluorescence excitation at 410 nm.
2 2
induced fluorescence turn-on. Herein we disclose that the flavonoid The N* and T* indicate the emission from normal or tautomeric form.
with a small amide group on the C-6 position shows excellent
2
selectivity for ER localization in mammalian cells.
Results and discussion
Synthesis of flavonoids
The new flavonoids 2a–2b include a donor–acceptor (D–A) inter-
action, which retains the desirable fluorescent solvatochromic
property. Flavonoids 2 were synthesized by reaction of N-(3-acetyl-
4-hydroxyphenyl)butyramide 3 with 4-(dialkylamino)benzaldehyde
4
in two steps (Scheme 2), i.e. by Claisen–Schmidt condensation
Scheme 3 The ESIPT process of a representative flavonoid, showing the
The crude structural change from N-tautomer to T-tautomer.
2
3,24
followed by Algar–Flynn–Oyamada reaction.
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Fig. 2 Fluorescence of 2b in different solvents at room temperature.
predominant emission from the excited tautomer (T*). It was
also interesting to note that the T* emission wavelength was
basically not influenced by the solvent polarity, although the
tautomer had a more polar structure. This was in sharp contrast to Fig. 3 Confocal imaging of oligodendrocyte cells incubated with 2b (10 mM)
the N* emission, whose emission wavelength was notably shifted to at o1, 15, 30, and 60 minutes after probe addition. Magnification is 100Â.
a longer wavelength as solvent polarity increased (e.g. from 487 nm
in toluene to 540 nm in DMSO, see Fig. 2). One possible reason
was that the solvent reorganization was not significant during confocal imaging (Fig. 4B) revealed that flavonoid 2 stained the
the short lifetime of the excited tautomer T*.
cell components surrounding the nuclei. The sharp circles in
The environmental response of 2 was further examined on fluorescence image of 2 (Fig. 3) were outer contour of nuclei,
proteins. The weak fluorescence of 2b in aqueous solution was since they are not stained and non-fluorescent.
increased moderately upon addition of 1 equiv. of bovine
The non-uniform stain of 2 on the cells suggests that the
serum albumin (BSA) or human serum albumin (HSA), giving dyes might be selectively binding to intracellular organelles. In
only one emission peak at lem E 532 nm (ESI,† Fig. S2). On the order to confirm the intracellular location and distribution of 2,
basis of the emission wavelength (532 nm), the observed we examined the colocalization of our dye with the commercial
emission from the albumin binding was likely from the N* ER probe, ER-Trackert Red. Incubation of MO3.13 cells with
form (Fig. 2), although it was not clear why the emission from ER–Trackert Red showed robust fluorescence in structures
the T* form was inhibited upon binding proteins.
surrounding the nucleus, a pattern consistent with ER staining
Cytotoxicity of flavonoids was examined by using an MTT (Fig. 4A and D). Staining with both compounds 2a and 2b
assay, showing that the half maximal inhibitory concentration showed similar localization (Fig. 4E and B) and colocalization
(IC50) for 2a–2b was 25 mM. Low cytotoxicity of 2 encouraged us was observed when cells were incubated with both the com-
to further examine the staining of the flavonoids in MO3.13 mercial ER dye and our probes (Fig. 4C and F). The stained cells
cells. This human cell line has the properties of myelinating were scanned repeated at 5 minute interval for 3 hours (ESI,†
oligodendrocytes, and has abundant myelin basic proteins Fig. S12), which revealed good stability in comparison with
2
7
(MBP) which are almost exclusively found in oligodendrocyte.
commercial ER Tracker (ESI,† Fig. S13).
When the cells were incubated with 2 and imaged by confocal
It is well known that ER forms an interconnected network of
microscropy, the dye was found to quickly penetrate into cells, membrane-enclosed sacs which are embedded with transmem-
giving strong fluorescence (Fig. 3). Weak fluorescence could be brane proteins that contain hydrophobic structural motifs. It is
observed immediately after adding dyes into cells and mounting attractive to speculate that the flavonoid dyes were binding to
the sample onto microscope (approximately in less than 1 minute). these types of hydrophobic structures. Such ER binding-induced
Since the dye was relatively non-fluorescent in an aqueous environ- fluorescence turn-on would be valuable for dynamic tracking,
ment, the observed fluorescence was assumed to arise from its since 2 was nearly non-fluorescent in the aqueous environment.
interaction with cellular lipids or hydrophobic pockets of proteins. The assumption was confirmed by observation of clear cell con-
tours without post-staining washing (Fig. 3), as the dyes outside
Colocalization of 2 and ER-Tracker in oligodendrocytes
the cells did not give observable fluorescence. Direct comparison
To the MO3.13 cell media were added ER-Trackert Red (concen- also revealed that flavonoid 2b gave stronger green fluorescence
tration 1 mM) along with flavonoid 2 to be tested (concentration than 2a under the same experimental conditions (Fig. 4B and E),
500 nM). Fluorescent probe DRAQ5 (10 mM) was also added to the illustrating that the ER binding-induced fluorescence turn on was
tissue culture media for staining cell nuclei. The fluorescence also sensitive to substituents on the flavonoid.
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Fig. 4 Confocal imaging of MO3.13 cells incubated with ER-Tracker (1 mM) (panels A and D), flavonoid compounds 2b (panel B) or 2a (panel E) (500 nM,
for each dye respectively). Panels (C) and (F) are the merged images of ER Tracker Red and flavonoid probes. The nuclei of cells were stained with DRAQ5
(
shown in purple).
In order to verify the ER selectivity, flavonoid 2b was further Colocalization of 2b with the commercial ER-Trackert Red
examined by staining in GL261 cells (astrocyte derived glio- revealed the identical patterns (Fig. 5A and B), showing that
blastoma), which is a mouse glioblastoma multiforme (GBM) the flavonoid was indeed selective to ER. The ER selectivity was
2
8
29
cell line and its growth is known to respond to ER stress.
also examined on SIM-A9 cells (semi adherent microglial cells).
Fig. 5 Confocal imaging of GL261 cells (panels A–C) and SIM A9 cells (panels D–F), which were incubated with ER-Tracker (1 mM, panels A and D),
flavonoid 2b (500 nM, panels B and E) for 30 minutes. Panels (C) and (F) are the merged images of ER Tracker Red and flavonoid probes. The nuclei of
cells were stained with DRAQ5 (shown in purple).
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washed with cold ethanol. The product was purified by recrys-
1
tallization from ethanol three times. Yield = 17%. H NMR
(
d6-DMSO, 500 MHz): d = 10.15 (s, 1H, –OH), 9.35 (s, 1H, –NH),
.43 (d, 1H, J = 3.0 Hz), 8.09 (d, 2H, J = 9.0 Hz), 7. 90 (dd, 1H,
J = 9.0 Hz, J = 2.5 Hz), 7.66 (d, 1H, J = 9.0 Hz), 7.38 (t, 4H), 7.16
8
1
2
(
(
1
1
1
m, 6H), 7.04 (d, 2H, J = 9.0 Hz), 2.37 (t, 2H), 1.65 (m, 2H), 0.95
1
3
t, 3H). C NMR (d
6
-DMSO, 125 MHz): d = 172.76, 171.80,
50.88, 149.06, 146.85, 145.92, 138.44, 136.32, 130.23, 129.39,
25.80, 124.65, 124.50, 121.90, 121.19, 119.13, 113.23, 38.77,
+
26 2 4
8.96, 14.07. MS, calcd for [C31H N O + Na] m/z: 513.2;
found: 513.2.
Synthesis of 2a
Compound 2a was prepared from N-(3-acetyl-4-hydroxyphenyl)-
butyramide (50 mmol) and 4-(dimethylamino)benzaldehyde
(50 mmol) in ethanol (100 mL) under the same conditions
Fig. 6 (A) Confocal image of zebrafish (48 hpf) at 10Â magnification, after used for 2b. The product was purified by recrystallization from
1
staining with flavonoid dye 2b. The images were taken from the head and hexane/ethanol (v/v = 1/1). Yield = 21%. H NMR (d -DMSO,
6
tail sections. (B) and (C) Fluorescence confocal images of zebrafish from
5
00 MHz): d = 10.13 (s, 1H, –OH), 9.07 (s, 1H, –NH), 8.40 (d, 1H,
J = 2.5 Hz), 8.11 (d, 2H, J = 9.0 Hz), 7.89 (dd, 1H, J = 9.0 Hz,
= 2.5 Hz), 7.67 (d, 1H, J = 9.0 Hz), 6.85 (d, 2H, J = 9.0 Hz), 3.00
the tail section (approximate position is shown by a red square), at 20Â and
00Â magnification, respectively.
1
1
J
2
1
3
(
s, 6H), 2.34 (t, 2H), 1.66 (m, 2H), 0.95 (t, 3H). C NMR
-DMSO, 125 MHz): d = 157.43, 157.02, 139.20, 138.55,
135.40, 126.91, 125.79, 119.78, 116.29, 113.27, 110.68, 110.24,
and ER stress plays a significant role in the microglial cell 105.65, 104.43, 41.42, 40.27, 22.88, 18.58. ESI-MASS m/z calcd
As a type of glial cell located throughout the brain, microglial (d
cells account for 10–15% of all cells found within the brain,
6
3
0
3
1
+
death. Although the ER distribution in GL261 cells is quite for [C21
different from that in SIM-A9 cells (Fig. 5A and D), colocalization
H N O + H] : 367.2; found: 367.2.
22 2 4
Fluorescence quantum yield
of 2b with the commercial ER-Trackert Red revealed the same
patterns.
The fluorescence quantum yields (QY) were measured using
2
6
rhodamine 6G (sigma) as the standard (F = 0.95, ethanol).
fl
Zebrafish staining
The fluorescence quantum yields were calculated by using the
following equation:
The ability of 2b to penetrate cells (Fig. 3), in addition to its low
toxicity, encouraged us to further examine its potential in vivo
applications. When zebrafish of 48 hours post fertilization (hpf)
was exposed to 2b, bright green fluorescence could be observed
from the entire zebrafish (Fig. 6), indicating that flavonoid dye
could easily penetrate into the fish for in vivo applications.
Interestingly, the detailed cell structure could be clearly visible
from the stained zebrafish (Fig. 6C), as the penetrated flavonoid
dye did not exhibit fluorescence in aqueous solution.
2
2
F
s
= F
r
 (A
r
 n
s
 F
s
)/(A
s
 n
r
r
 F )
where the subscripts s and r refer to the sample and the
standard (rhodamine 6G), respectively. F is the quantum yield,
F is the integrated emission intensity, A is the absorbance, and
n is the refractive index of the solution.
Live imaging
Imaging was performed on a Nikon A1 confocal system with a
1
00Â Plan Apo l, NA = 1.45 oil objective with both GaAsP
dectectors and high sensitivity low noise PMTs for detection.
The excitation used for our dyes was 405 nm with standard
DAPI, FITC, and Texas Red filters. The ER-Trackert was excited
Experiment section
Synthesis of 2b
N-(3-Acetyl-4-hydroxyphenyl)butyramide (10 mmol) was added using 560 nm and with a 600/50 nm bandpass filter used for
to a solution of 4-(diphenylamino)benzaldehyde (10 mmol) in emission. The nuclear dye, DRAQ5, was excited at 620 nm and
ethanol (50 mL), then 50 mL of aqueous NaOH (4 g, 100 mmol) 680/75 nm bandpass filter was used for emission. All imaging
solution was added slowly to the solution at 0 1C over 1 hour was done in an Okolab Bold Cage Incubator at 37 1C, and
period. The mixture was stirred at reflux (B80 1C) for 6 hours, images were processed using NIS Elements or ImageJ Pro
and then cooled to room temperature for another 48 hours. imaging software.
H O solution (10 mL of 30%) was slowly added into the
reaction solution over 1 hour while cooled in an ice-water bath.
2
2
Cell culturing and staining
After stirring at room temperature for 96 hours, the mixture was MO3.13 cells (oligodendrocytes), GL261 cells (astrocyte derived
poured into ice water and then placed into the refrigerator glioblastoma) or SIM A9 cells (semi adherent microglial) were
5
overnight. The precipitate was collected via filtration, and plated on microscopy dishes at a density of 5 Â 10 cells per mL
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in DMEM media supplemented with 10% FBS and 1% penicillin/ References
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1
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2
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along with a final concentration at 500 nM of the flavonoid dye
to be tested. Cells were left to incubate for 2 hours, and then
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imaging.
4
5
Zebrafish breeding and staining
Zebrafish were maintained as described in the Zebrafish Book
by the University of Oregon. They were kept in 28.5 1C water for
mating, male and female zebrafish were maintained in one
tank on a 12 h light/12 h dark cycle. Spawning was triggered by
light stimulation in the morning and eggs were immediately
fertilized. All embryos were maintained in E3 medium (15 mM
6
7
8
9
NaCl, 0.5 mM KCl, 1 mM MgSO
4
, 1 mM CaCl
2
, 0.15 mM
À5
KH PO , 0.05 mM Na HPO , 0.7 mM NaHCO
2
4
2
4
3
, and 10
%
methylene blue at 7.5 pH). All animal related procedures were
approved by the Care and Use of Animals in Research Committee
at The University of Akron.
A stock solution of the dye was prepared in DMSO at 1 mM.
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the fish were washed 3 times with E3 medium to limit potential
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with 0.5 mL of MS-2,2,2 20–30 minutes prior to imaging to limit
movement. Once imaging was completed all the fish were
euthanized using excess MS-2,2,2 before disposal.
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Conclusion
In summary, we report the first example of flavonoid-based
probes for ER sensing in living eukaryotic cells. The D–A type
flavonoid 2 exhibits many attractive features, which include
fluorescence turn-on, protein interaction, low toxicity, good
biocompatibility, efficient cellular uptake, and ER-targeting.
The peculiar ER-targeting has been independently observed
in all the cell lines examined. The low molecular mass of 2
1
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(o500 Dalton), in addition to its low toxicity, is anticipated to
cause less perturbation to cellular function during imaging. It
appears that the amide group plays an important role in the
observed ER selectivity, as the control compounds without the
amide side chain do not exhibit good selectivity to ER (ESI,†
Fig. S3). The observed ER selectivity from 2 could be attributed
to flavonoid’s binding to specific proteins. Further study will be
carried out to fine tune the ER selectivity.
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This work was supported by NIH (Grant No. 1R15EB014546-01A1). 17 A. J. Smith, T. K. Taneja, J. Mankouri and A. Sivaprasadarao,
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