Piezochromism, acidochromism, solvent-induced emission changes and cell imaging of D-π-A 1,4-dihydropyridine derivatives with aggregation-induced emission properties

Article abstract of DOI:10.1016/j.dyepig.2016.06.008

Three D-π-A 1,4-dihydropyridine derivatives with aggregation-induced emission characteristics were synthesized. The molecules comprise of a 4-(dimethylamino)styryl group as the electron donor group and different end groups, dicyanomethylene, vinylcyanoacetate, and 2-methylene-1H-indene-1,3(2H)-dione, as the electron acceptor, respectively. These target compounds display different stimulus-responsive fluorescent properties in the solid state. The original samples with the dicyanomethylene and vinylcyanoacetate groups do not show fluorescence color changes in response to external force stimuli, whereas the compound containing the 2-methylene-1H-indene-1,3(2H)-dione unit exhibits reversible piezochromism and solvent-induced emission changes due to the transformation between the crystalline and amorphous states, which can be ascribed to their different molecular stacking mode in the solid state. Furthermore, these compounds show different acid/base-induced solid-state fluorescence switching properties due to the different sites of the protonation. Additionally, all of the derivatives can be fabricated into biocompatible fluorescent nanoparticles and used for MCF-7 cell imaging.

Full text of DOI:10.1016/j.dyepig.2016.06.008

Dyes and Pigments 133 (2016) 261e272
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Piezochromism, acidochromism, solvent-induced emission changes
and cell imaging of D-p-A 1,4-dihydropyridine derivatives with
aggregation-induced emission properties
Yunxiang Lei a, Dongliang Yang b, Huaijie Hua c, Chunhui Dai d, Lianhui Wang b,
,
, **
Miaochang Liu a, Xiaobo Huang a *, Yang Guo d, Yixiang Cheng d, Huayue Wu a
a College of Chemistry and Materials Engineering, Wenzhou University, Wenzhou, 325035, PR China
b Key Laboratory for Organic Electronics and Information Displays and Institute of Advanced Materials, Nanjing University of Posts and
Telecommunications, Nanjing, 210023, PR China
c School of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, PR China
d School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three D-p-A 1,4-dihydropyridine derivatives with aggregation-induced emission characteristics were
Received 24 March 2016
Received in revised form
3 June 2016
Accepted 4 June 2016
Available online 6 June 2016
synthesized. The molecules comprise of a 4-(dimethylamino)styryl group as the electron donor group
and different end groups, dicyanomethylene, vinylcyanoacetate, and 2-methylene-1H-indene-1,3(2H)-
dione, as the electron acceptor, respectively. These target compounds display different stimulus-
responsive fluorescent properties in the solid state. The original samples with the dicyanomethylene
and vinylcyanoacetate groups do not show fluorescence color changes in response to external force
stimuli, whereas the compound containing the 2-methylene-1H-indene-1,3(2H)-dione unit exhibits
reversible piezochromism and solvent-induced emission changes due to the transformation between the
crystalline and amorphous states, which can be ascribed to their different molecular stacking mode in
the solid state. Furthermore, these compounds show different acid/base-induced solid-state fluorescence
switching properties due to the different sites of the protonation. Additionally, all of the derivatives can
be fabricated into biocompatible fluorescent nanoparticles and used for MCF-7 cell imaging.
© 2016 Elsevier Ltd. All rights reserved.
Keywords:
1,4-Dihydropyridine derivatives
Aggregation-induced emission
Piezochromism
Acidochromism
Solvent-induced emission changes
Cell imaging
1. Introduction
also be developed as stimuli-responsive fluorescent solid-state
materials for pH determination or solvent sensing by the intro-
Since aggregation-induced emission (AIE) [1,2] phenomenon
were discovered, many organic molecules with these properties
have been developed for the application of fluorescent sensors, cell
imaging, and organic light-emitting diodes [3e8]. In general, AIE-
active molecules are characterized by the strongly twisted confor-
mations, which not only enable them to emit more efficiently in the
aggregated state by hindering intermolecular close stacking and
intense pep interaction, but also result in some of them exhibiting
piezochromic property by the change of molecular packing mode in
response to external force [9,10]. Additionally, these molecules can
duction of appropriate functional units [11e14]. Thus, AIE com-
pounds have opened up a new path for the development of novel
multifunctional stimuli-responsive fluorescent materials. However,
there are only limited reports on multi-stimuli responsive fluo-
rescent materials because of the lack of clear guidelines on the
structureeproperty relations of the molecules [11e16].
Dicyanomethylene-4H-pyran (DCM) derivatives have been used
as excellent fluorophores in red fluorescence materials because of
their long emission wavelengths and high fluorescent yields arising
from ultra-fast intramolecular charge transfer (ICT) [17]. Recently,
some DCM derivatives have also been found to exhibit obvious AIE
activity because of a twisted conformation that results in the re-
striction of intramolecular rotation (RIR) in the aggregated state
[18e22]. However, DCM-1 (Scheme 1), which contains 4-(dime-
thylamino)styryl group, exhibits aggregation-caused quenching
because the strong pushepull interactions associated with the
* Corresponding author.
** Corresponding author.
E-mail addresses: xiaobhuang@wzu.edu.cn (X. Huang), huayuewu@wzu.edu.cn
(H. Wu).
http://dx.doi.org/10.1016/j.dyepig.2016.06.008
0143-7208/© 2016 Elsevier Ltd. All rights reserved.

262
Y. Lei et al. / Dyes and Pigments 133 (2016) 261e272
Scheme 1. Chemical structures of DCM-1, DCMP-1, and synthetic route to DHP derivatives.
aminoecyano pairs lead to its fluorescence emission quenching
owing to nonradiative relaxations of its ICT states [19]. Very
recently, we reported a dicyanomethylene-1,4-dihydropyridine
derivative DCMP-1 with remarkable AIE property resulting from
the replacement of the oxygen atom of the DCM skeleton with an
N-ethyl group, which could effectively promote the distortion of
the molecular structure and avoid the fluorescence quenching in
the aggregation state [23]. Furthermore, a series of indene-1,3-
been further fabricated into biocompatible fluorescent nano-
particles and used for MCF-7 cell (Human breast adenocarcinoma
cell line) imaging.
2. Experimental part
2.1. Measurements and materials
dionemethylene-1,4-dihydropyridine
derivatives
with
Melting points were recorded on a Buchi Melting Point B-540
(uncorrected). The 1H and 13C NMR spectra were recorded in so-
lution of CDCl3 on a Bruker DRX 500 NMR spectrometer with tet-
ramethylsilane (TMS) as the internal standard. Electrospray
ionization-mass spectrometry (ESI-MS) spectra were recorded on
a Finnigan DECAX-30000 LCQ Deca mass spectrometer. C, H and N
of elemental analyses were performed on an Elementar Vario MI-
CRO analyzer. Fourier transform infrared (FT-IR) spectra were ob-
tained on a Nexus 870 FT-IR spectrometer. UVevis absorption
spectra were recorded on a Perkin-Elmer Lambda 25 spectrometer.
Fluorescence spectra were recorded on a HITACHI F-7000 fluo-
triphenylamine/bis(diphenyl-amino)triphenylamine end groups
were found to exhibit AIE characteristics and multifunctional
stimuli-responsive fluorescent behavior in the solid state [24].
In this work, we report our recent progress in the fabrication
and application of stimuli-responsive fluorescent materials based
on 1,4-dihydropyridine (DHP) unit. A series of D-p-A DHP de-
rivatives, namely DHP-1, DHP-2, and DHP-3, were designed and
synthesized using a 4-(dimethylamino)styryl group as the electron
donor and different end groupsddicyanomethylene, vinyl-
cyanoacetate, and 2-methylene-1H-indene-1,3(2H)-dionedas the
electron acceptor, respectively (Scheme 1). The introduction of
different electron-withdrawing end groups was used to investigate
the effect of the substituents on the solid-state optical properties.
With regard to the design of the D-p-A structures, on the one hand,
we wanted to obtain the emitters with long wavelengths for the
application in cell imaging; on the other hand, some studies have
rometer. Fluorescence quantum yields (ФF) were determined by a
FluoroMax-4 (Horiba Jobin Yvon) fluorometer equipped with an
integrated sphere. Confocal laser confocal scanning microscope
images were taken on an Olympus Fluo-view 1000. X-ray diffrac-
tion (XRD) measurements were performed by using a Bruker X-ray
diffractometer (D8 Advance). The average particle size and the
polydispersity index (PDI) of the aggregates were measured using a
Zetasizer Nano ZS laser particle size analyzer. Crystal X-ray
diffraction intensity data were collected at 100 K on a Bruker-
Nonius Smart Apex CCD diffractometer with graphite-
suggested that D-p-A structure molecules are always advantageous
for the realization of fluorescence change under external stimuli
[25e30]. The introduction of a long octyl side chain onto the DHP
ring not only improves the dissolution properties of these com-
pounds, but also might have a significant effect on their aggregation
behaviors in the solid state by changing the molecular backbone
conformation and the molecular packing mode, leading to AIE or
piezochromism [31e38]. Additionally, when a dimethyl amino
group, 1H-indene-1,3(2H)-dione unit and DHP ring were intro-
duced into the molecular structures [24], the acidochromic prop-
erties of these compounds was expected through the basic
nitrogen/oxygen atoms as the binding sites for the protons. As a
result, these target compounds exhibited obvious AIE characteris-
tics, and interestingly, the electron-withdrawing end groups
endowed them with different stimuli-responsive fluorescent be-
haviors in the solid state. Additionally, all of the compounds have
monochromated MoKa radiation. The intensity data was pro-
cessed by using the SAINT and SADABS routines, and the structure
solution and refinement were carried out by the SHELXTL suite of
X-ray programs (version 6.10). 2,6-Dimethyl-4-pyrone (1), malo-
nonitrile (2), ethyl 2-cyanoacetate (3), and 2H-indene-1,3-dione (4)
were purchased from Sigma-Aldrich. 1,2-Distearoyl-sn-glycero-3-
phosphoethanolamine-N-[Methoxy(polyethylene-glycol)-2000]
(DSPE-PEG2000) was obtained from Avanti Polar Lipids, Inc. 3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
was purchased from Sigma-Aldrich and used without further pu-
rification. Milli-Q water was supplied by Milli-Q Plus System
(Millipore Corporation, Breford, USA).

Y. Lei et al. / Dyes and Pigments 133 (2016) 261e272
263
2.2. Synthesis of compounds 6aꢀc
67.7, 59.0, 49.4, 40.1, 31.7, 29.6, 29.1, 29.0, 26.5, 22.5, 14.8, 14.0. FT-IR
(KBr, cmꢀ1): 2914, 2852, 2174, 1657, 1586, 1525, 1471, 1363, 1329,
1279, 1187, 1168, 1057, 966, 808, 740. MS (ESI, m/z): 593.30 (MþþH).
Anal. Calcd for C38H48N4O2: C, 76.99; H, 8.16; N, 9.45. Found: C,
76.90; H, 8.22; N, 9.35.
2-(2,6-Bis((E)-4-(dimethylamino)styryl)-1-octylpyridin-4(1H)-
ylidene)-1H-indene-1,3(2H)- dione (DHP-3). Orange solids
(450 mg), 72% yield; m. p. 237e238 ꢁC. 1H NMR (CDCl3, 500 MHz):
A mixture of compound 5a/5b/5c (5 mmol) and 1-octylamine
(10 mL) in acetonitrile (15 mL) was heated under argon at 80 ꢁC
for 24 h. The reaction mixture was allowed to reach room tem-
perature and then poured into ice methanol, a large amount of
solids separated out in the mixture. After filtration, the crude
product was washed with methanol three times, and then dried to
afford pure compounds 6a¡c.
d
9.00 (s, 2H), 7.60 (dd, 3J ¼ 4.5 Hz, 4J ¼ 3.0 Hz, 2H), 7.46 (dd,
2-(2,6-Dimethyl-1-octylpyridin-4(1H)-ylidene)malononitrile
(6a). White solids (1.06 g), 75% yield; m. p. 154e155 ꢁC. 1H NMR
3J ¼ 4.5 Hz, 4J ¼ 3.0 Hz, 2H), 7.39 (d, J ¼ 8.5 Hz, 4H), 7.31 (d,
J ¼ 15.5 Hz, 2H), 6.72 (d, J ¼ 15.5 Hz, 2H), 6.66 (d, J ¼ 8.5 Hz, 4H),
4.08 (t, J ¼ 8.0 Hz, 2H), 3.00 (s, 12H), 1.83e1.78 (m, 2H), 1.38e1.24
(CDCl3, 500 MHz):
d
6.58 (s, 2H), 3.90 (t, J ¼ 8.5 Hz, 2H), 2.46 (s, 6H),
1.71e1.65 (m, 2H), 1.39e1.26 (m, 10H), 0.87 (t, J ¼ 7.0 Hz, 3H). 13
C
(m, 10H), 0.86 (t, J ¼ 7.0 Hz, 3H). 13C NMR (CDCl3, 125 MHz):
d 192.7,
NMR (CDCl3, 125 MHz):
d
155.7, 147.5, 147.4, 118.7, 113.8, 49.1, 44.7,
151.3, 149.4,149.2, 140.5,139.6,131.4, 128.9,123.5,119.6,113.9,113.7,
112.0, 102.7, 49.8, 40.1, 31.7, 29.4, 29.1, 29.0, 26.5, 22.6, 14.1. MS (ESI,
m/z): 626.30 (MþþH). FT-IR (KBr, cmꢀ1): 2923, 1710, 1625, 1600,
1580, 1524, 1489, 1393, 1329, 1185, 1092, 980, 948. Anal. Calcd for
44.6, 31.6, 29.6, 29.0, 28.9, 26.6, 22.5, 20.5, 13.9. MS (ESI, m/z):
284.10 (MþþH). FT-IR (KBr, cmꢀ1): 2926, 2192, 2171, 1625, 1550,
1506, 1483, 1352, 1317, 1185, 1062, 874, 725.
Ethyl 2-cyano-2-(2,6-dimethyl-1-octylpyridin-4(1H)-ylidene)-
C42H47N3O2: C, 80.60; H, 7.57; N, 6.71. Found: C, 80.73; H, 7.48; N,
6.59.
acetate (6b). White solids (1.29 g), 78% yield; m. p. 105e106 ꢁC. 1H
NMR (CDCl3, 500 MHz):
d
8.14 (d, J ¼ 2.5 Hz, 1H), 6.75 (d, J ¼ 2.0 Hz,
1H), 4.17 (q, J ¼ 7.0 Hz, 2H), 3.86 (t, J ¼ 7.0 Hz, 2H), 2.43 (s, 3H), 2.41
(s, 3H), 1.67e1.61 (m, 2H), 1.38e1.24 (m, 13H), 0.87 (t, J ¼ 7.0 Hz,
2.4. Synthesis of the AIE nanoparticles based on DHP-1ꢀDHP-3
3H). 13C NMR (CDCl3, 125 MHz):
d 167.5, 154.4, 146.6, 146.4, 122.1,
A
THF solution (6 mL) containing DHP-1/DHP-2/DHP-3
(0.06 mg) and DSPE-PEG2000 (0.12 mg) was stirred at room tem-
perature for 30 min. Then the solution was purified with a 0.22
116.0, 114.9, 67.8, 59.1, 48.9, 31.6, 29.8, 29.0, 28.9, 26.6, 22.5, 20.7,
20.3, 14.7, 14.0. MS (ESI, m/z): 331.15 (MþþH). FT-IR (KBr, cmꢀ1):
2927, 2180, 1666, 1627, 1501, 1354, 1260, 1120, 1062, 857, 764.
2-(2,6-Dimethyl-1-octylpyridin-4(1H)-ylidene)-1H-indene-
1,3(2H)-dione (6c). Yellow solids (1.49 g), 82% yield; m. p.
mm
syringe filter. A 2 mL quantity of the above solution was injected
rapidly into 8 mL Milli-Q Water under sonication. The dilute AIE
nanoparticle suspension was obtained after THF evaporation under
reduced pressure. The AIE nanoparticle suspension was further
concentrated to 2 mL and filtered with a syringe filter (0.22
obtain the AIE nanoparticles based on DHP-1ꢀDHP-3.
219e220 ꢁC. 1H NMR (CDCl3, 500 MHz):
d 8.50 (s, 2H), 7.59e7.58 (m,
2H), 7.46e7.45 (m, 2H), 3.72 (t, J ¼ 8.5 Hz, 2H), 2.46 (s, 6H),
1.59e1.53 (m, 2H), 1.25e1.20 (m, 10H), 0.87 (t, J ¼ 7.0 Hz, 3H). 13
C
mm) to
NMR (CDCl3, 125 MHz):
d 192.7, 149.5, 148.3, 140.1, 131.7, 119.8,
116.9, 101.9, 49.3, 31.6, 29.3, 29.0, 28.7, 26.5, 22.5, 20.6,14.0. MS (ESI,
m/z): 364.15 (MþþH). FT-IR (KBr, cmꢀ1): 2925, 2369, 1713, 1616,
1578, 1507, 1359, 1341, 1188, 1096, 952, 885, 738.
2.5. Cell culture
MCF-7 cells were cultured in Dulbecco’s modified Eagle’s me-
dium (DMEM) containing 10% fetal bovine serum (FBS) and anti-
2.3. Synthesis of DHP derivatives
biotics (100 units/mL penicillin and 100
mg/mL streptomycin),
A solution of compound 6a/6b/6c (1.0 mmol), 4-(dimethyla-
mino)benzaldehyde (1.49 g, 10.0 mmol), piperidine (1 mL) and
DMSO (7 mL) was refluxed under argon for 48 h. After cooling to
room temperature, the reaction mixture was poured into methanol
(50 mL) and suction filtered to give the crude product. The crude
product was washed with methanol three times, respectively, and
then dried to give the pure DHP derivatives.
maintained at 37 ꢁC under a humidified atmosphere of 95% air and
5% CO2. The cells were pre-cultured prior to experiments until 90%
confluence was reached.
2.6. Cytotoxicity evaluation
2-(2,6-Bis((E)-4-(dimethylamino)styryl)-1-octylpyridin-4(1H)-
ylidene)-malononitrile (DHP-1). Orange solids (371 mg), 68% yield;
The cytotoxicity of the AIE nanoparticles based on DHP-1ꢀDHP-
3 against MCF-7 cells was estimated using MTT assays, respectively.
MCF-7 cells were seeded in 96-well plates at a density of
4 ꢂ 104 cells mLꢀ1. After 24 h incubation, the medium was replaced
by the AIE nanoparticles at concentrations of 0.08, 0.12, 0.24, 0.3,
0.4, 0.6, and 1.2 ppm and cultured at 37 ꢁC for additional 48 h. The
sample wells were washed twice with 1 ꢂ PBS (Phosphate buffer
m. p. 231e232 ꢁC. 1H NMR (CDCl3, 500 MHz):
d
7.38 (d, J ¼ 8.5 Hz,
4H), 7.12 (d, J ¼ 15.5 Hz, 2H), 6.91 (s, 2H), 6.70e6.65 (m, 6H), 4.02 (t,
J ¼ 8.0 Hz, 2H), 3.03 (s, 12H), 1.84e1.78 (m, 2H), 1.40e1.27 (m, 10H),
0.87 (t, J ¼ 7.0 Hz, 3H). 13C NMR (CDCl3, 125 MHz):
d 155.3, 151.5,
148.7, 139.7, 129.0, 122.9, 119.6, 113.2, 112.0, 110.4, 49.6, 44.9, 40.1,
31.7, 29.5, 29.1, 29.0, 26.4, 22.5, 14.0. MS (ESI, m/z): 546.30 (MþþH).
FT-IR (KBr, cmꢀ1): 2931, 2360, 2187, 1588, 1532, 1507, 1491, 1473,
1329, 1178, 963, 818, 753. Anal. Calcd for C36H43N5: C, 79.23; H,
7.94; N, 12.83. Found: C, 79.34; H, 7.85; N, 12.65.
saline) and 100 mL of freshly prepared MTT (0.5 mg/mL) solution in
culture medium was added into each well. The MTT medium so-
lution was removed after 3 h incubation. Freshly prepared DMSO
(150 mL) was then added into each well and the plate was gently
Ethyl 2-(2,6-bis((E)-4-(dimethylamino)styryl)-1-octylpyridin-
4(1H)- ylidene)-2-cyanoacetate (DHP-2). Orange solids (414 mg),
shaken for 10 min at room temperature to dissolve all the pre-
cipitates formed. The absorbance of individual wells at 570 nm was
then monitored by the microplate Reader (BioTek, PowerWave XS2,
Vermont, USA). The absorbance of MTT in the sample well was
determined by the differentiation between the absorbance of the
sample well and that of the corresponding control well. Cell
viability was expressed by the ratio of the absorbance of MTT in the
sample wells to that of the cells incubated with culture medium
only.
70% yield; m. p. 181e182 ꢁC. 1H NMR (CDCl3, 500 MHz):
d 8.56 (d,
J ¼ 2.5 Hz, 1H), 7.37 (d, J ¼ 7.5 Hz, 4H), 7.22 (d, J ¼ 15.5 Hz, 1H), 7.14
(d, J ¼ 2.5 Hz, 1H), 7.12 (d, J ¼ 15.5 Hz, 1H), 6.70e6.66 (m, 6H), 4.22
(q, J ¼ 7.0 Hz, 2H), 4.01 (t, J ¼ 8.0 Hz, 2H), 3.014 (s, 6H), 3.011 (s, 6H),
1.82e1.76 (m, 2H), 1.36e1.25 (m, 13H), 0.87 (t, J ¼ 7.0 Hz, 3H). 13
C
NMR (CDCl3,125 MHz): d 167.9,154.0,151.3,151.2,148.2,148.1,139.1,
139.0, 128.8, 123.4, 123.2, 122.8, 114.2, 113.9, 112.8, 112.1, 112.0, 111.8,

264
Y. Lei et al. / Dyes and Pigments 133 (2016) 261e272
2.7. Cell staining and imaging
3.2. AIE properties of DHP derivatives
MCF-7 cells were seeded into a glass bottom dish with a density
of 1 ꢂ 105 cells per dish, and incubated for 24 h. Then the solution
was removed and the adherent cells were washed with PBS buffer
(2 mL ꢂ 3). After co-incubated with the AIE nanoparticles for 2 h,
the culture medium was removed and the treated cells were
washed three times with PBS (2 mL ꢂ 3) before observation.
Fluorescence imaging was performed with confocal laser scanning
microscopy, and the images were captured using a photomultiplier.
The AIE properties of the DHP derivatives were examined. The
compounds were dissolved in aqueous THF solutions with different
fractions of water and their absorption and fluorescence spectra
were recorded. Taking DHP-1 as an example, it was found that its
absorption spectra exhibited no significant change until the water
volume fraction (fw) was increased to 70% where the entire ab-
sorption spectra showed dramatic decrease (Fig. 2a), meanwhile,
there was an obvious leveled-off tail in the visible region due to
light-scattering effect because of the formation of nanoscopic ag-
gregates [40]. The pure THF solution of DHP-1 emitted weak green
light, which was ascribed to the free rotation of the phenyl ring and
DHP ring around the CeC bond facilitating a non-radiative relaxa-
tion process [1,23]. As water was gradually added to the THF so-
lution, the dissolved molecules began to aggregate with a RIR
process resulting in the enhancement of the fluorescence (Fig. 2b).
When fw was up to 70e90%, the fluorescence intensity of the so-
lution abruptly increased, and reached the maximum intensity at
90% water fraction, showing an approximately 3.6-fold enhance-
ment compared with that in pure THF, which was related with the
formation of a large number of aggregates. At the same time, the
aggregation of the fluorescent molecules forced them to adopt
more planar conformation and the color of the solution changed
from green to yellow under 365-nm UV illumination. Furthermore,
when fw ¼ 99%, the fluorescence intensity exhibited an obvious
decrease and the emission peak red-shifted to 612 nm. Similar AIE
phenomena could be observed in DHP-2 and DHP-3 (Figs. S1 and
S2, see Supporting Information). It should be pointed that the
emission intensities of these DHP derivatives at high water frac-
tions showed complex changes which were often observed in other
reports of AIE compounds [32,41,42], but the reasons remained
unclear. A possible reason for this phenomenon was explained as
follows: at a high water fraction, the solute molecules might
aggregate into more emissive, bluer crystal particles and less
emissive, redder amorphous particles. Because the formation of
nanoparticles was hard to control at high water content, the
measured fluorescence intensity and emission peak often exhibited
a complex change. The average particle size and the polydispersity
index (PDI) of the aggregates of three compounds in the THF/water
mixtures were measured using a laser particle size analyzer
(Figs. S3eS5 and Table S1, see Supporting Information). It was
found that the average diameters of DHP-1 in the THF/water mix-
tures (fw ¼ 70%, 80% and 90%) were ~968 nm, ~399 nm, and
~241 nm, and the corresponding PDI was 0.325, 0.04, and 0.057,
respectively. The average diameters of the aggregates of DHP-2 and
3. Results and discussion
3.1. Synthesis and features of DHP derivatives
Scheme 1 illustrates the synthetic route to DHP derivatives. 2-
(2,6-Dimethyl-4H-pyran-4- ylidene)malononitrile (5a) [23], ethyl
2-cyano-2-(2,6-dimethyl-4H-pyran-4-ylidene)acetate (5b) [39],
and
2-(2,6-dimethyl-4H-pyran-4-ylidene)-2H-indene-1,3-dione
(5c) [24] were synthesized by the reaction of 2,6-dimethyl-4-
pyrone (1) with malononitrile (2), ethyl 2-cyanoacetate (3), and
2H-indene-1,3-dione (4) in acetic anhydride according to the pre-
vious literature, respectively. The intermediates 6a¡c were syn-
thesized by the reaction of compounds 5a¡c with n-octylamine in
CH3CN at 80 ꢁC. Then the DHP derivatives were obtained from the
Knoevenagel condensation between compounds 6a¡c and 4-
(dimethylamino)benzaldehyde in 68e72% yields. The chemical
structures of DHP-1¡DHP-3 were characterized by 1H and 13C
NMR, mass spectrometry, and elemental analysis. These com-
pounds are soluble in common organic solvents such as CHCl3,
CH2Cl2, and THF, but insoluble in methanol and water. Fig. 1 shows
the UVevis absorption and emission spectra of these compounds in
THF solution. As shown in Fig. 1a, DHP-1¡DHP-3 show one distinct
absorption band in the range of 350e500 nm and their maximum
absorption wavelengths (lmabasx) exist at 404, 413, and 443 nm,
respectively, which can be regarded as the absorption of the con-
jugated structure arisen from the ICT band between electron-rich
donor and electron-deficient acceptor. DHP-1¡DHP-3 exhibit
green or yellow-green fluorescence situated at 531, 532, and
540 nm, respectively. The results indicate that DHP-3 containing
max
max
IDM unit shows longer
compared with DHP-1 and DHP-2. In addition, the fluorescence
quantum yields (
l
and emission wavelengths (
l
)
abs
em
Ф
F) of DHP-1¡DHP-3 are determined to be 0.31%,
0.29%, and 0.39% by a FluoroMax-4 fluorometer in the THF solution,
respectively.
Fig. 1. UVevis absorption (a) and fluorescence spectra (b) of the DHP derivatives in THF solution.

Y. Lei et al. / Dyes and Pigments 133 (2016) 261e272
265
Fig. 2. (a) UVevis spectra of DHP-1 (10
mM) in THF/water mixtures with different fw. (b) Fluorescence spectra of DHP-1 (10
mM) in THF/water mixtures with different fw. Insets:
Visible fluorescence of DHP-1 (10 mM) in 0%, 90% and 99% water under a 365-nm UV lamp. (c) The effect of fw on the maximum emission intensity of DHP-1 (10 mM) in THF/water
mixtures.
DHP-3 in the THF/water mixtures at high water fractions were also
in the range of 329 nme948 nm. The results indicate the aggregates
of the DHP derivatives were AIE nanoparticles.
sample showed a 38 nm red shift to 636 nm compared with that of
the original sample (598 nm) (Fig. 5 and Table 1), indicating that
DHP-3 exhibited distinct piezochromic property. After the ground
sample was annealed at 100 ꢁC for 30 min or fumed with ethyl
acetate (EA) vapour, the fluorescence color was converted to that of
the emissive original sample, demonstrating good reversibility of
the fluorescence color.
Herein, the RIR mechanism of the AIE could be confirmed by
solution thickening and cooling experiments. It was found that the
emission spectra of the pure THF and methanol/glycerol mixtures
of DHP-1 both showed a notable enhancement with decreasing
solution temperatures from 20 ꢁC to ꢀ78 ꢁC (Fig. S6, see Supporting
Information) and increasing amounts of glycerol (Fig. S7, see
Supporting Information), respectively. These results indicated that
the cooling and thickening processes could effectively hamper the
intramolecular rotations and then enhance the emission of DHP-1,
suggesting that the AIE property of DHP-1 should be caused by the
RIR process [43].
When the original sample of DHP-3 was dissolved in chloroform
and then evaporated to dryness using vacuum rotary evaporation, a
max
strong red-emitting solid was obtained (
l
¼ 626 nm), which was
em
similar to the sample showing piezochromic property and could be
ascribed to solvent-induced emission change caused by a simple
dissolutionedesolvation process (DDP) [24]. By the same method,
max
red emissive solids were achieved using CH2Cl2 (
l
¼ 619 nm) as
em
a solvent, but THF had little influence on the color and the lmemax of
max
the DDP-THF sample only showed 4-nm red shift (
l
¼ 602 nm).
em
3.3. Piezochromism and solvent-induced emission changes
The results indicated that different aggregates of DHP-3 were ob-
tained by the DDP in different solvent systems. The solid samples
obtained by the DDP using chloroform as a solvent could also be
nearly converted to the original sample by fuming (EA vapour) or
annealing. The results indicate that DHP-3 in the solid state has
reversible color change features and can be used as a promising
candidate for temperature, vapour, or pressure sensing as well as
optical recording materials. However, DHP-1 and DHP-2 did not
show similar solvent-induced emission changes.
The stimuli-responsive fluorescent behavior of DHP derivatives
in response to external pressure was investigated. When the orig-
inal solid samples of DHP-1 and DHP-2 with orange fluorescence
were ground with a pestle in a mortar, it was found that fluores-
max
cence color changes were not obvious (Fig. 3). The
l
of their
em
original samples were 609 nm and 600 nm, respectively, and those
of the corresponding ground samples only showed 8-nm red shift
to 617 nm and 608 nm (Table 1 and Fig. S8, see Supporting
Information), respectively. In sharp contrast, the orange emitting
XRD measurements for the DHP solid samples in various situ-
ations were performed to obtain an insight into the origin of the
piezochromic effect and the solvent-induced emission change
solid in DHP-3 could be converted into a red emissive solid when
max
the original sample was ground (Fig. 4). The
l
of the ground
em

266
Y. Lei et al. / Dyes and Pigments 133 (2016) 261e272
Fig. 3. Fluorescence images of the solid samples of DHP-1 and DHP-2 taken under a 365-nm UV lamp: (a) original sample; (b) ground sample; (c) DDP sample: the sample obtained
using chloroform as a solvent.
functional theory (DFT) calculations (Fig. S10, see Supporting
Information) [45]. The twisted conformations of these com-
pounds effectively weakened the fluorescence quenching caused
Table 1
Peak emission wavelengths and fluorescence quantum yields of DHP derivatives in
the solid state.
by the formation of pep stacking interactions in the concentrated
Sample
Type
lem (nm)
ФF
solution and solid state, and thus boosted the emission. Addition-
ally, it was found that the molecules of DHP-2 adopted a stacking
mode with J-type aggregation and there were practically no over-
DHP-1
Original
Ground
DDP-chloroform
Original
Ground
DDP-chloroform
Original
Ground
DDP-chloroform
DDP-CH2Cl2
DDP-THF
Annealed
Fumed
609
617
617
600
608
605
598
636
626
619
602
602
598
4.3%
e
e
DHP-2
DHP-3
5.2%
e
lapping between different ring planes, but
pep stacking existed
between the ester group (COOEt) and one of the phenyl rings with a
distance of 3.31 Å between the structures (Fig. 7). The molecules of
DHP-2 with a solvent molecule CHCl3 were further restricted by
multiple CeH%/N and CeH/O hydrogen bonds and the distances
of these H$$$N and H$$$O bonds were in the range of 2.23 Å to
2.45 Å (Table S4, see Supporting Information). The tight intermo-
lecular packing of DHP-2 might account for its poor piezochrom-
ism. Although we could not obtain the single crystal data for DHP-3,
based on the results of XRD measurements we could conclude that
its crystalline state should adopt a loose molecular packing mode,
which caused the crystal to become more easily destroyed under
external stimuli, such as pressure and solvent, and then increased
molecular conjugation and promoted the obvious red shifts of the
fluorescence spectra of DHP-3 compared with that of DHP-1 and
DHP-2.
e
3.2%
1.6%
2.5%
e
e
3.0%
2.9%
caused by the DDP. In the case of DHP-3, the diffraction curves of
the original sample revealed many sharp and intense reflections,
indicative of a well-ordered microcrystalline structure (Fig. 6). After
grinding or a DDP using chloroform or CH2Cl2 as a solvent, the
diffraction peaks of the treated samples disappeared or weakened
considerably, indicating a decrease in crystalline order and the
transformation from the crystalline state to the amorphous state,
which caused the large red shift in the fluorescence spectra. The
remaining crystalline features in the DDP samples could probably
be attributed to the incomplete phase transition. Meanwhile, the
treated samples showed a longer emission wavelength and a lower
3.4. Acidochromism of DHP derivatives
Another interesting phenomenon was that the pronounced
acidochromism of the DHP derivatives in the solid state. After the
original sample of DHP-1 was exposed to trifluoroacetic acid (TFA)
vapour for 5 min, the fluorescence color changed from orange to
green under a 365-nm UV lamp (Fig. 8). And the maximum emis-
sion showed 91-nm blueshift from 608 nm to 517 nm (Fig. S11, see
Supporting Information). The emission of the acid-fumed sample
could be nearly switched back to the original form by exposing it to
triethylamine (TEA) vapour for about 6 min or just standing at room
temperature for about 60 min because of the volatilization of TFA.
Therefore, the two-channel emissions of DHP-1 in the solid state
could be easily switched by treatment with TFA/TEA. Similar to that
of DHP-1, the fluorescence color of DHP-2 also exhibited a change
from orange (600 nm) to green (510 nm) when the original sample
was exposed to TFA vapour, and the acid-exposed solid reverted to
its original state upon TEA-exposure (Fig. 9 and Fig. S11, see
Supporting Information). However, the difference was that the re-
covery of the emission of DHP-2 acid-fumed sample could not be
realized by standing for 60 min or longer. For DHP-3, the fluores-
cence of the original sample was completely quenched by TFA
ФF than those in the original state (Table 1). Similar XRD signals to
those in the original samples appeared after fuming and annealing,
which resulted in a blueshift in the fluorescence spectra and the
recovery of fluorescence. Conversely, diffraction patterns of DHP-1
and DHP-2 changed little from their original forms after grinding or
a DDP (Fig. S9, see Supporting Information).
To better understand the relationship between the optical
properties and the molecular conformation and packing mode, we
obtained single crystals of DHP-2 from CHCl3/MeOH solution which
were suitable for X-ray structural analysis (Fig. 7 and Table S2, see
Supporting Information) [44]. The dihedral angles between the
central DHP ring and the substituted phenyl rings C13eC18 and
C23eC28 were 30.4ꢁ and 31.3ꢁ, respectively. The torsion angles
between the two ethylene units and the DHP ring were 34.8ꢁ and
23.9ꢁ, respectively (Table S3, see Supporting Information). More-
over, the N-octyl group had a torsion angle of 84.0ꢁ from the DHP
ring. The results indicated that DHP-2 had a twisted conformation,
which was consistent with the results obtained from density

Y. Lei et al. / Dyes and Pigments 133 (2016) 261e272
267
Fig. 4. Fluorescence images of DHP-3 solid samples taken under a 365-nm UV lamp: (a) original sample; (b) ground sample; (c) annealed sample; (d) fumed sample; (e), (f) and (g)
DDP samples: the sample obtained using chloroform, CH2Cl2 and THF as a solvent, respectively.
Fig. 5. Fluorescence changes of DHP-3 solid samples under various conditions.
Fig. 6. XRD curves of DHP-3 solid samples under different conditions.
vapour, but then restored by exposure to a base (Fig. 10). Addi-
tionally, when the DHP-3 acid-fumed sample was left standing for
60 min, a red emitting solid was obtained, and then changed to
orange emitting solid after exposure to TEA vapour. The results
indicated the solid-state emissions of the DHP derivatives could be
reversibly tuned by exposure to acidic and basic vapour. In addition,
it was found that TFA vapour could blue-shift the absorption
spectra of the original samples of DHP-1, DHP-2, and DHP-3, and
then TEA vapour could recover the absorption spectra of acid-
fumed samples to a certain extent (Fig. S12, see Supporting
Information).
To verify the cause of the color transformation of the DHP de-
rivatives in response to TFA, the changes of the 1H NMR spectra of
DHP-1 and DHP-3 before and after the addition of TFA were
investigated. For DHP-1, it was found that the addition of TFA
caused the signals of H4 and H5 in the phenyl ring, H2 and H3 in the

268
Y. Lei et al. / Dyes and Pigments 133 (2016) 261e272
Fig. 7. (a) Single-crystal structure of DHP-2 with a solvent molecule CHCl3. Hydrogen atoms are omitted for clarity. (b) The molecular packing showing various hydrogen bonds in
the single-crystal structure of DHP-2 (green dashed lines). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)
Fig. 8. Fluorescence images of DHP-1 solid samples taken under a 365-nm UV lamp: (a) original sample; (b) fumed sample by TFA vapour; (c) exposed to TEA vapour for about
3 min; (d) exposed to TEA vapour for more 3 min; (e) obtained from the protonated sample by standing 60 min at room temperature.
Fig. 9. Fluorescence images of DHP-2 solid samples taken under a 365-nm UV lamp: (a) original sample; (b) fumed sample by TFA vapour; (c) exposed to TEA vapour for about
3 min; (d) exposed to TEA vapour for more 3 min; (e) and (f) fumed sample stood 60 min at room temperature and then was soaked with TEA.

Y. Lei et al. / Dyes and Pigments 133 (2016) 261e272
269
Fig. 10. Fluorescence images of DHP-3 solid samples taken under a 365-nm UV lamp: (a) original sample; (b) fumed sample by TFA vapour; (c) exposed to TEA vapour for about
3 min; (d) exposed to TEA vapour for more 3 min; (e) and (f) fumed sample stood 60 min at room temperature and then was soaked with TEA.
ethylene unit, and the hydrogen atoms of the NeCH3 group to
display a low-field shift (Fig. S13, see Supporting Information),
which was mainly attributed to the hydrogen ions bound to the
nitrogen atom of the NeCH3 group. Compared with the nitrogen
atom in the DHP ring, it was found that the NeCH3 group exhibited
stronger ability bound to proton based on the calculation results of
the proton affinities of nitrogen atoms in the DHP-1 and DHP-2
(Table 2). The protonation of the NeCH3 group led to the formation
of a stronger acceptor and weakening of the ICT effect in the
structure, resulting in the blueshift of the emission in the solid state.
With regard to DHP-3, considering that the signals of H1 in the DHP
ring and H6 in the phenyl ring displayed obvious high-field shifts
(Fig. S14, see Supporting Information) and high proton affinities of
the oxygen atom of carbonyl group (Table 2), it was concluded that
there was also the protonation of the carbonyl groups in the
structure in addition to the protonation of the NeCH3 group.
The DHP-derivative-loaded AIE nanoparticles were synthesized
through a matrix-encapsulation method using DSPE-PEG2000 as the
encapsulation matrix [46]. The DHP-1/DHP-2/DHP-3@DSPE-
PEG2000 nanoparticles were formed spontaneously upon diluting
the mixture of the corresponding DHP derivative and DSPE-PEG2000
from THF into water under sonication, respectively. After incuba-
tion with the AIE suspensions (~0.08 ppm) at 37 ꢁC for 2 h, the MCF-
7 cells were stained. The confocal images were taken upon exci-
tation at 405 nm with a 458 nm long pass barrier filter (Fig. 11). It
was clearly found that the AIE nanoparticles based on DHP-1, DHP-
2, and DHP-3 were taken up by MCF-7 cells and the fluorescent dots
accumulated in the edge of cytoplasm. Moreover, the cell viability
after incubation with the nanoparticles at different concentrations
was estimated (Fig. 12). It was found that the cell viabilities
remained above 90% upon incubation with the nanoparticles based
on DHP-2 or DHP-3 for 48 h at a concentration of 0.3 ppm, indi-
cating the low cytotoxicity. For those based on DHP-1, the cyto-
toxicity was very low at 0.08 ppm, but began to increase gradually
with the further increase of the concentration. These results
demonstrate that the nanoparticles based on the DHP derivatives
are effective fluorescent imaging reagents for live cell imaging with
high biocompatibility at low concentrations.
3.5. Cell imaging application of DHP derivatives
To demonstrate the potential application of the DHP derivatives
in bioimaging, these compounds were tested as fluorescent re-
agents for fabrication of AIE nanoparticles for MCF-7 cell imaging.
Table 2
Proton affinitiesa of nitrogen atom and oxygen atom in the DHP derivatives in gas phase.
þHþ b (Kcal/mol)
þHþ b (Kcal/mol)
þHþ b (Kcal/mol)
þHþ b (Kcal/mol)
O
Compound
N
N
N
DHP-1
DHP-2
DHP-3
213.636
217.845
219.497
218.886
221.686
222.370
e
e
221.163
e
e
249.238
a
The optimized configurations and Gibbs free energies were obtained from the calculations at B3LYP/6-31G** and B3LYP/6-311 þ G** level, respectively.
Proton affinities: N and O in different colors are points of protonation.
b

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Y. Lei et al. / Dyes and Pigments 133 (2016) 261e272
Fig. 11. Confocal laser scanning microscopy images of MCF-7 cells stained with the nanoparticles based on DHP-1 (a), DHP-2 (c), and DHP-3 (e). Bright-field images for the
nanoparticles based on DHP-1 (b), DHP-2 (d), and DHP-3 (f). The fluorescence images were taken upon excitation at 405 nm with a 458 nm long pass barrier filter.
4. Conclusion
Three D-
2 containing vinylcyanoacetate, DHP-3 based on 1H-indene-
1,3(2H)-dione unit exhibited reversible piezochromism and
solvent-induced emission changes due to the transformation be-
tween the crystalline and amorphous states, which should be
ascribed to their different molecular stacking mode in the solid
state, which should be attributed to their different molecular
stacking mode in the solid state. Although all of the DHP derivatives
showed reversible acidochromism, the colors of the emission of
DHP-1 and DHP-2 were changed from orange to green when their
original samples were exposed to TFA vapour, whereas, the fluo-
rescence of DHP-3 solids was directly quenched. For DHP-3, the
difference could be attributed to the fact that the protonation of the
p
-A DHP derivatives with different electron-
withdrawing end groups were designed and synthesized. The
target compounds showed twisted conformations because of the
introduction of a long octyl side chain onto the DHP ring, as
revealed by crystallographic data and theoretical calculations. The
new molecules exhibited AIE characteristics caused by the RIR
process. It was noted that they displayed different stimuli-
responsive fluorescent properties in the solid state because of
different electron-withdrawing terminal substituent. Being
different from DHP-1 containing dicyanomethylene unit and DHP-

Y. Lei et al. / Dyes and Pigments 133 (2016) 261e272
271
Fig. 12. Metabolic viability of MCF-7 cells after incubation with the nanoparticles based on DHP-1 (a), DHP-2 (b), and DHP-3 (c) at different concentrations for 48 h, respectively.
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carbonyl groups in addition to the protonation of the NeCH3 group
occurred. Additionally, we found all of these DHP derivatives could
be developed as safe fluorescent probes for live cell imaging at low
concentrations. The current study will help researchers to develop
more AIE-active fluorescent materials by the fine tuning of mo-
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Acknowledgements
This research work was supported by National Natural Science
Foundation of China (No. 21272176, 21572165, 81273409), Zhejiang
Provincial Natural Science Foundation (No. LY16B040005), and
Graduate Innovation Foundation of Wenzhou University (No.
3162014040).
Appendix A. Supplementary data
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phase transition with remarkable fluorescence variation. Chem Mater
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2016.06.008.
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