A highly selective AIE fluorogen for lipid droplet imaging in live cells and green algae

Article abstract of DOI:10.1039/c3tb21675f

Lipid droplets (LDs) are subcellular organelles for energy storage and lipid metabolism regulation. Here we report an aggregation-induced emission-active fluorogen, TPE-AmAl, for specific LD imaging. TPE-AmAl is cell-permeable: upon entering the live cells, the dye molecules can selectively accumulate in the LDs and turn on the fluorescence. TPE-AmAl possesses twisted intramolecular charge transfer properties as well: the emission colour in the hydrophobic LDs is blue-shifted by >100 nm than that in aqueous buffers. Compared with the commercial lipid droplet dye, TPE-AmAl demonstrates the advantages of low background, short staining time, high selectivity, excellent biocompatibility, and good photostability. The utilization of TPE-AmAl for LD staining in green algae is also demonstrated, indicating their potential application in the high-throughput screening of high-value microalgae as a preferential biofuel source. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c3tb21675f

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Materials Chemistry B
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A highly selective AIE fluorogen for lipid droplet
imaging in live cells and green algae†
Cite this: J. Mater. Chem. B, 2014, 2,
2013
Erjing Wang,‡ab Engui Zhao,‡ab Yuning Hong,ab Jacky W. Y. Lamab
abc
*
and Ben Zhong Tang
Lipid droplets (LDs) are subcellular organelles for energy storage and lipid metabolism regulation. Here we
report an aggregation-induced emission-active fluorogen, TPE-AmAl, for specific LD imaging. TPE-AmAl is
cell-permeable: upon entering the live cells, the dye molecules can selectively accumulate in the LDs and
turn on the fluorescence. TPE-AmAl possesses twisted intramolecular charge transfer properties as well:
the emission colour in the hydrophobic LDs is blue-shifted by >100 nm than that in aqueous buffers.
Compared with the commercial lipid droplet dye, TPE-AmAl demonstrates the advantages of low
background, short staining time, high selectivity, excellent biocompatibility, and good photostability. The
utilization of TPE-AmAl for LD staining in green algae is also demonstrated, indicating their potential
application in the high-throughput screening of high-value microalgae as a preferential biofuel source.
Received 27th November 2013
Accepted 27th January 2014
DOI: 10.1039/c3tb21675f
www.rsc.org/MaterialsB
affect the normal level of LDs. For example, LDs appearing in
peripheral blood leukocytes are indicative of lipid storage
myopathy.14 The increase of LDs in myobers is frequently
observed during the treatment of inammatory myopathy,15
while the decrease of LD volume in adipocytes is indicative of a
successful weight loss to some extent.16
Introduction
Lipid droplets (LDs) are the main reservoir for neutral lipid
storage and extensively exist in eukaryotic and prokaryotic
cells.1,2 The rst discovery of LDs can be traced back to as early
as the 17th century3 and for a long period of time since then, LDs
had been simply regarded as a place for energy storage and
deemed as an inert cell inclusion.4 However, the emergence of a
series of LD proteins implies that the functions of LDs are more
than lipid storage.5,6 They are dynamic organelles involved in
regulating lipid metabolism and storage, membrane transfer,
protein degradation, and signal transduction.7–11 Failure of the
regulation may lead to metabolic diseases such as fatty liver
disease, atherosclerosis, type II diabetes, and hyperlipidemia.12
A recent study has revealed another function of LDs as a key
organelle for the production and assembly of infectious hepa-
titis C virus, a causative agent of chronic liver diseases.13 The
abnormality of LDs in cells is a critical biomarker for the related
diseases. Moreover, the treatment of certain diseases may also
Monitoring and localizing the accumulation and abnormal
level of LDs is thus of particular importance for biomedical
research and early diagnosis of the related diseases. In the
human body, the liver is the main organ that controls the
dynamic balance of lipid metabolism.17 To assess the LD level
and diagnose hyperliposis-related liver diseases, computerized
tomography scanning and B-type ultrasound examination are
oen used. However, these methods are relatively insensitive
and only above 25 wt% of the fat content in liver can be detected
with reasonable accuracy, which could be diagnosed as the late-
stage of severe liver diseases.18 Diagnosis of early-stage liver
diseases including mild fatty liver still relies on transhepatic
biopsy to achieve high sensitivity and accuracy.19 Hence, direct
detection of LDs and lipid accumulation in liver tissue still play
an important role. In addition to the biomedical signicance,
the LD is a kind of alternative biofuel for sustainable energy
research.20 Algae are among the best potential sources of biofuel
with high efficiency. Therefore, methods for screening algae
with high contents of LDs are in high demand.21
aHKUST-Shenzhen Research Institute, No. 9 Yuexing 1st RD, South Area, Hi-tech Park,
Nanshan, Shenzhen, 518057, China. E-mail: tangbenz@ust.hk; Fax: +86-852-2358
1594; Tel: +86-852-2358 7375
bDepartment of Chemistry, Division of Biomedical Engineering, Division of Life Science,
State Key laboratory of Molecular Neuroscience, Institute of Molecular Functional
Materials and Institute for Advanced Study, The Hong Kong University of Science &
Technology, Clear Water Bay, Kowloon, Hong Kong, China
cGuangdong Innovative Research Team, SCUT-HKUST Joint Research Laboratory,
State Key Laboratory of Luminescent Materials and Device, South China University
of Technology, Guangzhou, 510640, China
Fluorescence techniques are powerful tools for studying LDs
in living cells and tissues owing to their advantages such as high
sensitivity, visibility and tunability in the design of bio-probes.22
Nile red23 and BODIPY dyes24 are widely used for LD imaging.
Nile red is a cell-permeable dye that stains most intracellular
structures.25 It emits yellow uorescence in the nonpolar LDs
and red emission in the polar cytosol and other organelles, thus
† Electronic supplementary information (ESI) available: Experimental section,
UV-vis and PL emission spectra, and bright eld and uorescent images of
living cells. See DOI: 10.1039/c3tb21675f
‡ These authors contributed equally.
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enabling the discrimination of LDs from others. However, it coupling reaction between 4,40-bis(dimethylamino) benzophe-
suffers from low sensitivity because of the high background none (1) and 4-bromobenzophenone (2) generated TPE-AmBr,
noise and signal overlap between the two channels. BODIPY lithiation and formylation of which gave the designed product
dyes, on the other hand, usually have small a Stokes shi.26 TPE-AmAl. The nal product was fully characterized by mass
Other stains, such as Oil Red O stain xed cells only.27 Besides and NMR spectroscopies, which conrmed the acquisition of
these drawbacks, traditional uorophores also encounter the the compounds. TPE-AmAl is completely soluble in common
concentration-caused quenching effect.28 Accumulation of the organic solvents such as THF, CHCl3, CH2Cl2, DMSO and DMF,
dye molecules in certain organelles will increase the local dye but poorly soluble in water.
concentration and result in the self-quenching problem.
We then investigated the photophysical properties of TPE-
To overcome the concentration-quenching problem, a group AmAl. From the UV-vis spectrum, the absorption maximum of
of uorogens possessing the aggregation-induced emission TPE-AmAl is at ꢀ410 nm in THF solution, which is about 50 nm
(AIE) characteristic are developed.29 An AIE luminogen is almost red-shied as compared to that of its precursor TPE-AmBr
non-emissive in the solution state but emits strongly in the (Fig. S1†). With the absorption maximum in the visible-light
aggregate or solid state. Restriction of intramolecular motions region, TPE-AmAl could be used in many biological applications.
has been suggested as the main cause for the AIE phenom- This red-shi could be ascribed to the presence of the donor–
enon.30 Our research group has developed a library of AIE acceptor structures: the dimethylamino group is a strong
molecules31 and extensively explored their applications in electron-donating group while the formyl group is an electron-
organic light-emitting diodes, chemo-sensing, bio-probing, accepting group. The electron push–pull effect narrows the
etc.32 For bio-imaging, the AIE luminogens exhibit excellent energy gap for electronic transition, corresponding to the bath-
photostability.33 Hydrophobic AIE luminogens can form ochromic shi in the absorption spectrum. Increasing the solvent
nanoaggregates in an aqueous environment. Such nano- polarity causes a slight red-shi of the absorption maximum
aggregates are more resistant to photo-bleaching than a single (from ꢀ400 to 415 nm) but a large red-shi of the emission
molecule: even when the molecules on the surface are maximum (from ꢀ470 to 610 nm), which is a typical phenom-
damaged, they could protect the inner molecules against enon of twisted intramolecular charge transfer (TICT) (Fig. S2†).
further photo-bleaching and photo-oxidation. Owing to the
TPE-AmAl is faintly uorescent in THF and becomes highly
superior photostability and biocompatibility, the AIE dyes have emissive in THF–H2O mixtures with the water fraction larger
been successfully used for specic mitochondria imaging,33a than 80%, showing a characteristic AIE phenomenon (Fig. 1).
full-range intracellular pH mapping,34 and long-term cell Interestingly, with the increase of water fraction from 0 to 70 vol
tracking,35 suggesting them as promising candidates for the %, the emission maximum is gradually red-shied (from 525 m
new generation of uorescent dyes for cell research.
to 590 nm) (Fig. 1B). As the polarity of water is much higher than
From the structural point of view, LD dyes such as Nile red that of THF, increasing the water content leads to a higher
and Seoul-Fluor-based dyes contain both donor (D) and polarity of the solvent system and thus red-shis the emission
acceptor (A) moieties and experience solvatochroism.23a,36 due to the TICT attribute. Further increase of the water fraction
Inspired by the structures of these molecules, in this work, we (>70 vol%), however, results in the blue-shi of the emission
design and synthesize a tetraphenylethene (TPE) derivative peak, due to the increasing hydrophobicity of the local envi-
(TPE-AmAl) substituted with alkylamino (D) and carbonyl (A) ronment aer the formation of nanoaggregates. The solid-state
groups. TPE is an archetypical AIE-active luminogen which quantum yield of TPE-AmAl is 22% measured with the integral-
emits in the blue region. With the D–A moieties, TPE-AmAl sphere technique, which is appreciable for a dye emitting in the
emits orange colour in its aggregate state. Systematic studies on orange-to-red region.
the staining of HeLa cells, liver LO2 cells, and green algae are
performed, which demonstrate the high selectivity, photo-
stability and biocompatibility of TPE-AmAl for LD imaging.
Results and discussion
Synthesis and optical properties
TPE-AmAl was synthesized according to the synthetic route
shown in Scheme 1 in moderate yield. Briey, McMurry cross-
Fig. 1 (A) Emission spectra of TPE-AmAl in THF–water mixtures with
different water fractions (fw). Concentration: 10 mM; excitation wave-
length: 410 nm. (B) Plot of the relative PL intensity (I/I0) and the
emission peak wavelength of TPE-AmAl versus the water fraction in
THF–H2O mixtures. I0 ¼ emission intensity in pure THF solution. Inset:
photographs of TPE-AmAl (10 mM) in THF–water mixtures with 0 and
Scheme 1 Synthetic route to TPE-AmAl.
2014 | J. Mater. Chem. B, 2014, 2, 2013–2019
95 vol% of water taken under UV illumination.
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accommodating the hydrophobic tails of the lipids. Therefore,
the polarity inside the LDs is much lower. Because of the TICT
characteristics, the emission of TPE-AmAl would be at the
shorter wavelength region in the LDs environment with low
polarity.38 To verify the hypothesis, the emission spectra of TPE-
AmAl in dichloromethane–n-hexane mixtures with different
polarity were recorded. With an increase of n-hexane fraction,
the emission peak gradually blue-shied from 560 to 500 nm
(Fig. S5†), showing the polarity-dependent emission of
TPE-AmAl.
Oleic acid is known to stimulate cells to produce LDs.39 Upon
treatment with oleic acid for 6 h, the amount of LDs is signi-
cantly increased (Fig. 3). The increase of the oleic acid
concentration leads to the emergence of more greenish-blue
spots in the intracellular region, indicating that more LDs are
produced in the cytoplasm. Meanwhile, in the untreated cells,
the LDs are located closely to the plasma membrane, while the
oleic acid treated cells, the LDs, are trafficked more closely to
the nucleus. TPE-AmAl can thus be used to monitor the health
of the cells.
Photostability, which is a key parameter for evaluating a
uorescent imaging agent, was examined on TPE-AmAl upon
staining the oleic acid-treated HeLa cells. Continuous scanning
using a confocal microscope (Zeiss laser scanning confocal
microscope LSM7 DUO) was adopted for quantifying the pho-
tostability of TPE-AmAl. A dish of oleic acid-treated HeLa cells
was incubated with 10 mM TPE-AmAl for 15 min. The initial
intensity, which refers to the rst scan of HeLa cells stained
with TPE-AmAl, was normalized and the remaining uores-
cence signals were calculated as a percentage of the initial
intensity. As shown in Fig. 4 and S6,† TPE-AmAl selectively
enters the LDs in HeLa cells. Even aer 55 scans with a total
irradiation time span of ꢀ10 min, the signal loss of TPE-AmAl in
the cells was less than 20%. No signicant difference was
Live cell imaging
Before exploring the application for live cell imaging, we
examined the biocompatibility of TPE-AmAl. The cytotoxicity of
the dye was evaluated using 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyltetrazolium bromide (MTT) assay (Fig. S3†). The
results show that no detectable inhibitory effect is observed on
the growth of both cervical cancer HeLa cells and liver LO2 cells
when up to 100 mM TPE-AmAl is added to the culture media.
The excellent biocompatibility of TPE-AmAl motivated us to
further investigate their application in cell imaging.
TPE-AmAl was then systematically assessed for its capability
to localize in LDs by uorescence microscopy. HeLa cells were
incubated with 10 mM TPE-AmAl for 15 min and the excess dye
was washed away with phosphate buffered saline. As shown in
Fig. 2A–C, TPE-AmAl enters the live HeLa cells spontaneously
and lights up the spherical LDs specically within a short time
(10–15 min is sufficient). The spherical objects in cytosol with
distinctly bright uorescence could be easily identied as LDs.
TPE-AmAl is a lipophilic dye which is prone to accumulate in
the hydrophobic LDs. Thanks to the AIE property, increasing
the concentration of TPE-AmAl in the staining process can
enhance the emission intensity in the LDs monotonically
without sacricing the specicity (Fig. S4†).34,37
Parallel experiments were performed with Nile red as the
stain. Compared with TPE-AmAl which stains the LDs only, Nile
red stains not only the LDs (yellow spots) but also other intra-
cellular structures like mitochondria (red emission) (Fig. 2E).
The poor specicity makes it difficult to use Nile red as a stain to
differentiate LDs from other organelles, not to mention to
observe dynamic motions of LDs.
On the other hand, although TPE-AmAl emits an orange
color when dispersed in aqueous media, it exhibits a greenish-
blue emission in the LD regions (Fig. 1B, inset vs. Fig. 2B). This
is understandable as the emission of TPE-AmAl is sensitive to
the polarity of the environment. The LD is surrounded by a
monolayer of phospholipids with the inner part
Fig. 2 Images of HeLa cells after incubation with (A–C) TPE-AmAl (10
mM) for 15 min and (D–F) Nile red (100 ng mLꢁ1, 0.314 mM) for 5 min
under (A and D) bright field and (B and E) photoexcitation by fluores- Fig. 3 Fluorescent images of HeLa cells stained with TPE-AmAl
cence microscopy. (C and F) Merged images of the corresponding (10 mM) for 15 min after incubation in the presence of (A) 0, (B) 12.5, (C)
bright-field and fluorescence images. Excitation wavelength: 330– 25 and (D) 50 mM oleic acid for 6 h. Excitation wavelength 330–
385 nm for TPE-AmAl and 460–490 nm for Nile red.
385 nm.
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Fig. 4 Signal loss (%) of fluorescence intensity of TPE-AmAl in oleic
acid-treated HeLa cells with the scanning time. Inset: fluorescent
images of oleic acid-treated HeLa cells stained with TPE-AmAl (10 mM)
with increasing numbers of scans (1–55 scans; the number of scans
were shown in the upper left corner). Staining time: 15 min; excitation
wavelength: 405 nm; emission filter: 449–520 nm; irradiation time:
11.22 s per scan; scale bar: 20 mm.
Fig. 5 Fluorescent images of liver LO2 cells stained with TPE-AmAl (10
mM) after incubation in the presence of (A) 0, (B) 12.5, (C) 25 and (D) 50
mM oleic acid for 6 h. Staining time: 15 min; excitation wavelength:
330–385 nm.
Nile red can also image the increase amount of LDs when
treated with oleic acid, the background noise from other
membrane-bound organelles makes it difficult to be used for LD
observed between the rst and 50th scans (Fig. 4, inset). The
photostability with Nile red (0.314 mM) was also investigated
(Fig. S7†). The signal of emission intensity of Nile red in cells quantication (Fig. S12†).
decreases about 20% in 2 min irradiation and remains constant
thereaer. It is noteworthy that a 0.7% laser power was used for
scanning of Nile red experiments while a laser power of 2% was
Green alga imaging
Algae biofuel research is ascending in the recent decade
used for TPE-AmAl. In general, the photostability of TPE-AmAl
is better than that of Nile red under the same imaging
conditions.
because of their potential to be an environment-friendly alter-
native to fossil fuels. The content of lipids is an important
parameter to assess the value of the algae. The high selectivity
and light up property of TPE-AmAl inspired us to utilize it for
the imaging of LDs in algae.
Green algae Nannochloropsis sp. was chosen as model algae.
To facilitate the diffusion of TPE-AmAl into the algae, different
fractions of DMSO containing the same concentration of TPE-
AmAl were used in culture media. Higher contents of DMSO
result in shorter diffusion times of TPE-AmAl to enter the algal
body. When green algae were incubated with 2.5 mM TPE-AmAl
at 40 ꢂC for 10 min in the presence of 20% DMSO, a clear image
with the lipid content identied by the greenish blue emission
of the dye can be acquired (Fig. 6). Note that the red emission
colour originates from the chloroplast, which can be differen-
tiated from the LDs under a uorescence microscope. The
The excellent photostability of TPE-AmAl can be attributed to
the formation of nanoaggregates and the inert atmosphere
inside the hydrophobic LDs. In the culturing medium (MEM)
containing 0.1 vol% DMSO – the same condition for HeLa cell
imaging – lipophilic TPE-AmAl forms nanoparticles with an
average size of ꢀ178 nm, as revealed by particle size analysis
(Fig. S8†). The particles can enter the cells and accumulate in
the nearly neutral LDs; the hydrophobic and inert environment
of LDs could protect the substance against further damage by
the external stimuli.
Liver cells are rich in LDs because of the unique function of
the liver in lipid metabolism. Aer treatment of liver LO2 cells
with oleic acid, all the cells in a wide view range gave a bluish
green emission (Fig. S9†). Similar to the results obtained on
HeLa cells, TPE-AmAl could also selectively target the LDs in
liver LO2 cells, giving a bluish green emission (Fig. 5A).
Compared with HeLa cells, liver LO2 cells produce much more
LDs even without the treatment of oleic acid (Fig. S10†).
Exposure of the LO2 cells to increased concentrations of
oleic acid can trigger the cells to generate more LDs (Fig. 5).
TPE-AmAl can be used to visualize the increase of the LDs,
implying its potential application for in vitro transhepatic
biopsy to assist the diagnosis of early-stage liver diseases. To
further verify if all the LDs are stained by TPE-AmAl, Nile red
was used for co-staining experiments. As shown in Fig. S11,† all
the spherical spots stained by Nile red can be visualized by TPE-
Fig. 6 Fluorescent images of green algae stained with TPE-AmAl
(2.5 mM) in the presence of (A) 10 vol% and (B) 20 vol% DMSO at 40 ꢂC
for 10 min. Excitation wavelength: 330–385 nm. The blue emission is
AmAl, indicative of the specicity of TPE-AmAl to LDs. Although from the LDs; the red emission is from the chloroplast.
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diameter of green emission region reects the size of LDs, while Particle size analysis was determined at room temperature
the overall brightness of the image could be correlated with the using a ZetaPlus Potential Analyzer (Brookhaven Instruments
total lipid content in the LD body. These results demonstrate Corporation, USA).
the feasibility of using TPE-AmAl for high-throughput screening
of algae species with high bio-fuel contents.
Synthesis
Preparation of TPE-AmBr.40 To an oven-dried two-necked
ask 4,40-bis(dimethylamino)benzophenone (1) (3 g, 11.2
mmol), 4-bromobenzophenone (2) (3.78 g, 14.5 mmol) and zinc
dust (4.18 g, 64 mmol) were added. The mixture was degassed
while purging nitrogen at least three times, and then freshly
distilled fresh THF (80 mL) was injected into the ask. The
reaction system was cooled to ꢁ78 ꢂC in a dry ice-acetone bath
for at least 15 min. TiCl4 (6.11 g, 32.3 mmol) was added drop-
wise, aer which the reaction was continued for about 8 h under
reuxing. The excess zinc was ltered aer cooling to room
temperature. Aqueous potassium carbonate was added, the
mixture was extracted with dichloromethane, and the ltrate
was dried with anhydrous magnesium sulfate. Aer removing
the solvent with a rotary evaporator, and the residue was puri-
ed with a silica gel chromatographic column using petroleum
ether/ethyl acetate (5 : 1) as the eluent to afford a yellow solid as
the product 2.33 g. Yield 42%. HR-MS (MALDI-TOF): calcd for
Conclusion
In summary, an AIE-active TPE derivative, TPE-AmAl, is
synthesized and utilized as a uorescent agent for LD imaging
in live HeLa cells, liver LO2 cells and green algae. With the
advantages of high selectivity, excellent biocompatibility, high
photostability, and simplicity, TPE-AmAl represents an excel-
lent probe for selective imaging and tracking of LDs. The reason
for the high affinity to LDs is proposed to be the hydrophobic
interaction between TPE-AmAl and LDs. Once entering LDs, the
intramolecular motion of TPE-AmAl will be restricted and the
emission from TPE-AmAl will be turned on; because of the low
polarity inside LDs, TPE-AmAl emits a greenish blue colour
distinct from the extracellular orange emission. Collectively,
TPE-AmAl exhibits a much lower background noise and higher
sensitivity when compared with commercial LD dyes. TPE-AmAl
could nd a wide range of applications such as in auxiliary
diagnosis of early-stage fatty liver disease, monitoring of
macrophage foam cells, screening of high-throughput algae for
biofuels, searching for oil-producing bacteria, and other
aspects.
C
30H29BrN2 496.1514, found: 496.1523. 1H-NMR (400 MHz,
CDCl3) d 7.23–7.17 (m, 2H), 7.14–7.05 (m, 3H), 7.05–6.99
(m, 2H), 6.94–6.91 (m, 1H), 6.91–6.83 (m, 5H), 6.51–6.40
(m, 4H), 2.90 (d, J ¼ 11.0 Hz, 12H). 13C-NMR (100 MHz, CDCl3) d
148.14, 148.08, 144.01, 143.54, 141.16, 134.63, 132.45, 131.88,
131.85, 131.74, 131.71, 131.11, 131.01, 130.73, 129.90, 126.90,
126.75, 124.92, 118.50, 110.73, 110.62, 110.53, 110.46, 39.56.
Preparation of TPE-AmAl.41 TPE-AmBr (1.5 g, 0.3 mmol) was
added to a 100 mL two-necked ask with nitrogen purging. Dry
THF (40 mL) was injected into the reactor and the mixture was
Materials and methods
General
Tetrahydrofuran (THF) was distilled from a sodium–benzo-
phenone system in a nitrogen atmosphere under normal pres-
sure immediately prior to use. DMF was puried using an ion-
exchanged resin purication system. Other solvents were
directly used without further purication. The reagents, 4,40-
ꢂ
cooled to ꢁ78 C in a dry ice-acetone bath for at least 15 min.
Then 1.8 mL n-BuLi (2 M in cyclohexane) was slowly added
dropwise and the temperature was kept at ꢁ78 ꢂC for 2 h. Dried
DMF (2 mL) was added and the mixture was slowly recovered to
room temperature and continued stirring for 2 h. Aer using
saturated ammonium chloride to quench the reaction, the
mixture was extracted with dichloromethane and dried with
magnesium sulfate. The organic solvent was removed through
rotary evaporation under reduced pressure. The residue was
puried on a silica column using petroleum ether and
dichloromethane for gradient eluting to afford an orange yellow
solid product 0.85 g. Yield 63%. HR-MS (MALDI-TOF) calcd for
bis(dimethylamino)benzophenone,
4-bromobenzophenone,
zinc, titanium(IV) chloride, n-butyl lithium (2.0 M in cyclo-
hexane), oleic acid, Nile red, and ammonium chloride were
purchased from commercial companies and directly used
without purication. For the biological study, minimum
essential medium (MEM), fetal bovine serum (FBS), and RPM
1640 medium were purchased from Invitrogen. The green algae
in culture medium was a present from Prof. Ka Ming Ng's group
in HKUST.
C
31H30N2O 446.2358, found 446.2350. 1H-NMR (400 MHz,
CDCl3) d 9.88 (s, 1H), 7.60 (d, J ¼ 6.8 Hz, 2H), 7.18 (d, J ¼ 7.6 Hz,
2H), 7.13–7.08 (m, 3H), 7.04–7.02 (m, 2H), 6.91–6.87 (m, 4H),
6.44 (d, J ¼ 8.8 Hz, 4H), 2.90 (s, 12H). 13C-NMR (100 MHz,
CDCl3) d 192.27, 152.81, 149.48, 149.33, 144.80, 144.04, 135.68,
133.65, 132.96, 132.86, 132.72, 132.37, 131.82, 131.67, 131.60,
129.36, 128.08, 126.16, 111.75, 111.40, 40.50.
Instrumentation
1H and 13C-NMR spectra were recorded on a Bruker ARX 400
spectrometer using CDCl3 as the solvent and tetramethyl silane
(TMS) as the internal standard. High-resolution mass spectra
(HRMS) were recorded on a GCT premier CAB048 mass spec-
trometer operated in a MALDI-TOF mode. UV-vis absorption
spectra were recorded on a Milton Roy Spectronic 3000 Array
Cell viability evaluated by MTT assay
spectrometer. Photoluminescence spectra were recorded on a Cells were seeded in 96-well plates at a density of 5000 cells per
Perkin-Elmer LS 55 spectrouorometer. The FF,f value of solid well. Aer overnight culturing, medium in each wells were
powder was determined using an integrating sphere method. replaced by fresh medium containing different concentrations
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of TPE-AmAl. Aer 24 hours of treatment, into each well, 10 mL Research Grants Council of Hong Kong (604711, 604913,
MTT solution (5 mg mLꢁ1 in phosphate buffer solution) was HKUST2/CRF/10 and N_HKUST620/11), and the University
added. Aer 4 hours of incubation at 37 ꢂC, 100 mL SDS–HCl Grants Committee of Hong Kong (AoE/P-03/08). B.Z.T. thanks
solution (10% SDS and 0.01 M HCl) was added to each well. the Guangdong Innovative Research Team Program for support
ꢂ
Aer incubation at 37 C for 6 hours, the absorbance of each (201101C0105067115).
well at 595 nm was recorded using a plate reader (Perkin-Elmer
Victor3™). Each of the experiments was performed at least
5 times as a parallel test.
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