Facile construction of boranil complexes with aggregation-induced emission characteristics and their specific lipid droplet imaging applications

Article abstract of DOI:10.1039/c9cc04041b

A rational strategy was reported to construct boranil complexes (DPFB derivatives) with unique aggregation-induced emission effects by installing phenyl rings in the anil ligand as the intramolecular rotors. In view of the good biocompatibility and suitab

Full text of DOI:10.1039/c9cc04041b

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DOI: 10.1039/C9CC04041B
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Facile construction of boranil complexes with aggregation-
induced emission characteristics and their specific lipid droplets
imaging applications
Received 00th January 20xx,
Accepted 00th January 20xx
Na Zhao,*a Chengcheng Ma,a† Weiyao Yang,a† Wei Yin,a Jiahui Weia and Nan Li*a
DOI: 10.1039/x0xx00000x
A rational strategy was reported to construct boranil complexes readily synthesis.11 During the past years, great efforts have
(DPFB derivatives) with unique aggregation-induced emission been put to increase the emission efficiency and extend the
effect by installing phenyl rings in anil ligand as the intramolecular emission wavelength of boranil in solution state.12 However,
rotors. In view of the good biocompatibility and suitable due to the lack of rational molecular design and modification,
lipophilicity, DPFB derivatives can serve as the excellent the strategy to construct the AIE-active boranil is still rare.13
fluorescent probes for specific imaging of lipid droplets in living
cells and yolk lipids in zebrafish.
Lipid droplets (LDs), the dynamic organelles for storage of
neutral lipids, play crucial roles in many biological processes
including lipid metabolism, membrane transport, protein
degradation and signal transduction.14,15 Abnormal lipid
storage in LDs was reported to be associated with metabolic
diseases such as fatty liver and cardiovascular diseases.16
Therefore, development of high-performance fluorescent
probes for imaging of LDs is of great importance for both
biomedical research and early diagnosis of related diseases.
To address these issues, herein, we report a facile strategy
to fabricate AIE-active boranil by installing phenyl ring as the
intramolecular rotor in anil ligand (Scheme 1). DEFB is a well-
known boranil complex and shows ACQ feature. Once two
ethyl groups were replaced by phenyl rings, a new boranil
complex (DPFB) was obtained, which displayed typical AIE
effect. The solid-state emission of AIE-active boranil complexes
were turned from 550 to 610 nm by introducing suitable
substituents. Combining the theoretical calculation with single
crystal analysis, distorted configuration and effective RIR in the
condensed state should be responsible for the AIE
characteristics of DPFB derivatives. Taking advantages of their
good biocompatibility and suitable lipophilicity, DPFB
derivatives could be successfully applied for imaging of LDs in
living cells with fast and wash-free manners as well as staining
of yolk lipids in zebrafish.
Boron complexes based fluorescent materials have received
considerable attentions due to their potential applications in
the field of photoelectric devices, information storage
elements, fluorescent sensors and probes.1-2 However, some
conventional boron complexes, such as difluoroboron
dipyrromethene (BODIPY), suffer from the aggregation-caused
quenching (ACQ) effect because of its coplanar conformation
and intrinsic intermolecular π−π stacking.3 The ACQ
phenomenon limited the practical applications of boron
complexes in solid devices or biological environment.
Fortunately, an opposite phenomenon termed aggregation-
induced emission (AIE) was discovered by Tang’s group in
2001.4 The AIE luminogens (AIEgens) exhibit faint emission in
molecular dissolved state, but give enhanced emission in the
aggregated state with the mechanism of restriction of
intramolecular rotation (RIR).5 It is noted that some novel AIE-
active boron complexes bearing various ligands including
ketoiminate,6 diiminate,7 pyridyl-enamido,8 benzothiazole-
enamide9 and other heteroatom contained ligands10 have
been successfully developed.
Boranil, a kind of boron complex employing anil (also called
salicylaldimine) as ligand, emerged as promising fluorescent
materials in the field of bio-labelling and electroluminescent
devices owing to their excellent optical properties as well as
a.Key Laboratory of Macromolecular Science of Shaanxi Province, Key Laboratory
of Applied Surface and Colloid Chemistry of Ministry of Education, Key Laboratory
of the Ministry of Education for Medicinal Resources and Natural Pharmaceutical
Chemistry, and School of Chemistry & Chemical Engineering, Shaanxi Normal
University, Xi’an 710119, China. nzhao@snnu.edu.cn, nli@snnu.edu.cn.
†C.M. and W.Y. contributed equally to this work.
Scheme 1 Structures of DEFB and DPFB derivatives.
Electronic Supplementary Information (ESI) available: Synthesis and
characterization, single crystal data, the absorption and emission spectra. See
DOI: 10.1039/x0xx00000x
The synthetic routes of all boranil complexes were shown in
Scheme S1. Anil ligands were easily prepared through the
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reaction of 4-(diethylamino)- or 4-(diphenylamino)-2- CN and DPFB-NMe2) were obtained. All three complexes
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hydroxybenzaldehyde with corresponding aniline. Targeted exhibited weak emission in solution, butDgOaIv:e10i.n10te39n/sCe9CeCm0i4s0si4o1nB
complexes were obtained by treating anil with BF3·Et2O at in the aggregated state, indicative of their typical AIE effect
basic condition. All complexes were fully characterized by NMR (Fig. S3−S5). The solid-state emission of these DPFB derivatives
and high resolution mass spectrometry. In DMSO solution, was tuned from 495 to 601 nm (Fig. S1B). Meanwhile, DPFB
both DEFB and DPFB exhibited strong π-π* transition at derivatives exhibited high emission efficiency (Φf > 12.7%)
around 275 nm (Fig. S1A).11 Meanwhile, remarkable after formed nano-aggregates in PBS solution (Fig. S1C and S6).
Table 1 The optical properties of DEFB and DPFB derivatives.
absorption peaks at 400 nm were also observed, which could
be attributed to the intramolecular charge transfer (ICT)
Solutiona
λabs λem
(nm) (nm) [%]
Aggregation
λem
(nm) [%]
transition. Upon photoexcition, DEFB emitted bright blue light
(460 nm) with the quantum yield (Φf) of 4.0%. However, very
faint emission centred at 550 nm (Φf = 0.7%) was obtained for
DPFB. It is noteworthy that the Stokes shift of DPFB (150 nm)
was significantly larger than DEFB (60 nm). Subsequently, the
emission properties of DEFB and DPFB in the aggregated state
were investigated. By adding the poor solvent of water into
DMSO solution, the emission of DEFB was reduced gradually
while the emission intensity at 460 nm dropped to 25% at
water fraction (fw) of 99% with Φf of 2.3% (Fig. 1A and 1B),
suggesting its ACQ nature. In sharp contrast, with increasing of
fw (> 50%), the emission of DPFB was obviously enhanced (Fig.
1C and 1D). Approximately 300-fold enhancement at 567 nm
was obtained when fw reached 99% (Φf = 41.6%), indicative of
its specific AIE effect. The molecular aggregation at fw of 99%
was confirmed by using dynamic light scattering measurement
and the particle sizes were 469.5 and 119.3 nm for DEFB and
DPFB, respectively (Fig. S2). In addition, the solid-state
emission of DEFB and DPFB was detected at 495 nm (Φf = 2.1%)
and 564 nm (Φf = 61.1%), respectively, which was consistent
with their emission behaviour in aqueous solution (Fig. S1B).
c
d
Φfb
Φfb
λem
(nm) [%]
Φfb
Complex
DEFB
401
403
408
419
460
550
535
530
578
4.0
0.7
1.0
0.5
2.1
460
567
550
573
580
2.3
461
1.9
DPFB
DPFB-OMe
DPFB-CN
41.6 559
18.2 556
16.5 574
12.2 576
46.4
20.1
16.0
12.7
DPFB-NMe2 435
aIn DMSO solution. bAbsolute quantum yield determined by using a calibrated
integrating sphere. cIn DMSO/water (1/99) mixtures. dIn DMSO/PBS (1/99)
mixtures.
In order to investigate the different emission properties of
DEFB and DPFB in solution state, optimized structures of two
complexes in their ground (S0) and excited (S1) state were
estimated using DFT and TD-DFT method, respectively. As
shown in Fig. 2, the subtle difference for the structures of
DEFB at S0 and S1 states was observed. Conversely, the
molecular configuration of DPFB exhibited discrepancy from S0
to S1 state. This structure difference in DPFB implied the large
structural relaxation in its S1 state, which probably increased
the ratio of non-radiative decay and resulted in the weak
emission of DPFB in solution state.17 For DEFB, non-radiative
decay was effectively suppressed in the excited state due to its
relative rigid structure. Thus, the bright emission observed for
DEFB in solution state. Meanwhile, the large Stokes shift of
DPFB could be ascribed to its large structural relaxation in the
excited state.
Fig. 2 Optimized structures of DEFB and DPFB in the (A) S0 and (B) S1 state.
The distribution and energy level of frontier orbits for DPFB
derivatives were also calculated (Fig. S7). The highest occupied
molecular orbital (HOMO) mainly located on the N,N-diphenyl
groups and central part (phenyl ring and O-B-N chelate moiety),
whereas the lowest unoccupied molecular orbital (LUMO)
delocalized over the central part as well as aromatic group
linked to the N atom of imine, which suggested the existence
of ICT process in DPFB derivatives. The energy levels of HOMO
were calculated to be -0.77 to -0.65 eV, and the energy levels
Fig. 1 Emission spectra of (A) DEFB and (C) DPFB in DMSO/water mixtures with varied
fw. Plot of I/I0 of (B) DEFB and (D) DPFB versus fw (I0 represents the emission intensity in
DMSO). Inset: photograph of (B) DPFB and (D) DEFB in DMSO/water mixtures with 0
and 99% fw under 365 nm UV irradiation. λex: 401 nm for DEFB and 403 nm for DPFB.
When different substituents including methoxyl, cyano and
N,N-dimethyl groups were introduced into the para-position of
aniline part, a series of boranil complexes (DPFB-OMe, DPFB-
of LUMO varied from -6.59 to -6.30 eV in the order from DPFB
,
2 | J. Name., 2012, 00, 1-3
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DPFB-OMe DFPB-CN to DFPB-NMe2
COMMUNICATION
,
.
The corresponding fluorescent signals from DPFB or DPFB-NMe2 channel were
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DOI: 10.1039/C9CC04041B
energy gaps between HOMO and LUMO were 5.84, 5.82, 5.67 observed, which exactly overlapped with the signals derived
and 5.65 eV, which leaded to their red-shifted absorption from HCS LipidTOX™ Deep Red Neutral Lipid Stain. The overlap
behaviour (from 403 to 435 nm) in solution state.
ratios were calculated to be 97 and 95% for DPFB and DPFB-
Single crystals of DEFB DPFB-CN and DPFB-NMe2 were NMe2, implying that DPFB and DPFB-NMe2 can selectively
,
obtained by slowly evaporating dichloromethane/n-hexane stain the LDs in living cells. The suitable lipophilicities of DPFB
mixtures and characterized using single crystal X-ray diffraction derivatives with high ClogP value (6.24 for DPFB and 6.41 for
(Table S1). As shown in Fig. 3, the aromatic ring linked to the DPFB-NMe2) should be responsible for this specific LDs
imine was not coplanar with the core unit for all crystals and targeting.18
their torsion angles were measured as 42.02, 22.02 and 44.64o
for DEFB, DPFB-CN and DPFB-NMe2, respectively. The
additional torsion angles between two phenyl rings of N,N-
diphenyl group and the central part were 73.45 (79.55) and
48.97 (55.07o) for DPFB-CN and DPFB-NMe2, respectively.
Clearly, the replacement of ethyl group by phenyl ring in DPFB
induced higher twisted configuration. Moreover, the dimer
structures were formed in crystal of DEFB with a π-π stacking
interaction (4.336 Å), which is considered as the primary cause
for ACQ effect. However, the π-π stacking was avoided in
crystal of DPFB-CN and DPFB-NMe2 due to their more
distorted conformation. Meanwhile, weak interactions such as
C−H···N (2.638, 2.617 Å) and C−H···F (2.453, 2.409 Å) were
found in crystal of DPFB-CN and DPFB-NMe2, which helped to
rigidify their configuration (Fig. S8). In addition, the
intramolecular rotation of DPFB derivatives was restricted
once the molecules aggregated, which yielded the high
emission efficiency.
Fig. 4 Co-localization images of HeLa cells stained with DPFB derivatives (5 µM) and
HCS LipidTOX™ Deep Red Neutral Lipid Stain (1:1000 dilution). Scale bar: 10 µm.
Owing to the unique AIE nature, we assessed the imaging
capability of DPFB derivatives without washing step. After
incubated cells with DPFB or DPFB-NMe2 for 30 min, the
images were collected directly (Fig. 5A). It is interesting that
the strong fluorescent signals from LDs with negligible
background were observed, which is nearly no difference
compared to the images after washing. However, the distinct
background from cytoplasm was observed when the cells were
treated with commercial probes BODIPY493/503 or Nile Red.
This high signal-to-noise imaging with wash-free manner for
DPFB derivatives could be attributed to the remarkable
emission increase when molecules aggregated in LDs. This
wash-free imaging mode will not only simplify the protocol for
cell imaging, but also offer a convenient way to track the
morphology of LDs in situ.
Fig. 3 Drawing of the crystal structure, side view and crystal packing of (A) DEPB, (B)
DPFB-CN and (C) DPFB-NMe2.
The excellent optical properties of DPFB derivatives inspired
us to explore their bio-imaging applications. DPFB and DPFB-
NMe2 were chosen for imaging experiments because of their
distinct emission spectra. The biocompatibility was assessed
using
4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium
bromide (MTT) assay (Fig. S9). When the concentration of
DPFB derivatives reached up to 50 μM, cell viabilities were still
more than 90%, demonstrated they possess low cytotoxicity.
Then, the HeLa cells were incubated with DPFB derivatives and
the pre-experiment results suggested that two complexes
penetrated the cell membrane and accumulated in the LDs (Fig.
S10). In order to further prove the intracellular localization of
DPFB derivatives, the co-localization experiments were
performed using commercial LDs marker (HCS LipidTOX™ Deep
Red Neutral Lipid Stain). As illustrated in Fig. 4, the bright
Fig.
5 (A) CLSM images of HeLa cells stained with DPFB derivatives (5 µM),
BODIPY493/503 (500 nM) and Nile red (500 nM) without washing. (B) Time-dependent
images of DPFB (5 µM). Scale bar: 10 µm.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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The time-dependent staining of LDs using DPFB derivatives
was studied. After added the DPFB derivatives into the cell
culture medium, the images were collected with elapse of time.
As described in Fig. 5B, yellow fluorescent signals started to
appear when the incubated time was 1 min. After incubated
cells to 3 min, the fluorescent signals became brighter and
morphology of LDs was observed clearly. Similar short staining
time (< 5 min) was obtained for DPFB-NMe2 (Fig. S11),
indicated that DPFB derivatives can serve as ideal candidates
for fast LDs staining.
In view of the selective LDs imaging of DPFB derivatives,
their capability for imaging of living zebrafish was investigated
because the zebrafish is an ideal model for studying the lipids
related diseases.19 Taking DPFB-NMe2 as example (Fig. 6),
intense orange fluorescent signals originated from the yolk sac
in zebrafish were observed after incubated with DPFB-NMe2
for 30 min. Similarly, yellow fluorescent signals in yolk sac
were also obtained for DPFB (Fig. S12). Lots of neutral lipids
and polar phospholipids exist in yolk, which provide energy for
zebrafish at the initial stage of larval development. Therefore,
above imaging results demonstrated that DPFB derivatives can
stain the yolk lipids in zebrafish and has great potential for
monitoring of lipids transport and metabolism processes.
Conflicts of interest
There are no conflicts of interest to declare.
View Article Online
DOI: 10.1039/C9CC04041B
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2
Fig. 6 CLSM images of zebrafish stained with DPFB-NMe2 (5 µM). Scale bar: 100 µm.
In summary, we reported a facile strategy to construct AIE-
active boranil (DPFB derivatives) by installing phenyl ring as
the intramolecular rotor within anil ligands. Based on the
theoretical calculation and crystal analysis, the AIE nature of
DPFB complexes was attributed to the distorted configuration
as well as effective RIR in the aggregated state. In view of the
good biocompatibility and suitable lipophilicity, DPFB and
DPFB-NMe2 can selectively stain the LDs in living cells with fast
and wash-free manners. Additionally, in vivo staining of yolk
lipids in zebrafish was also successfully obtained. This work not
only provides a convenient method to construct AIE-active
boranil complex, but also extends their bio-imaging
applications.
This work was supported by the National Natural Science
Foundation of China (21672135, 51403122 and 21402115), the
Fundamental Research Funds for the Central Universities
(GK201902006 and GK201702002), Natural Science
Foundation of Shaanxi Province (2018JM5086) and Funded
Projects for the Academic Leaders and Academic Backbones,
Shaanxi Normal University (18QNGG007).
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TOC: A series of boranil complexes with aggregation-induced emission effect were facilVeiew Article Online
DOI: 10.1039/C9CC04041B
constructed, which can be utilized to image lipid droplets in living cells and yolk lipids in
zebrafish.