Far-Red and Near-IR AIE-Active Fluorescent Organic Nanoprobes with Enhanced Tumor-Targeting Efficacy: Shape-Specific Effects

Article abstract of DOI:10.1002/anie.201501478

The rational design of high-performance fluorescent materials for cancer targeting in vivo is still challenging. A unique molecular design strategy is presented that involves tailoring aggregation-induced emission (AIE)-active organic molecules to realize preferable far-red and NIR fluorescence, well-controlled morphology (from rod-like to spherical), and also tumor-targeted bioimaging. The shape-tailored organic quinoline-malononitrile (QM) nanoprobes are biocompatible and highly desirable for cell-tracking applications. Impressively, the spherical shape of QM-5 nanoaggregates exhibits excellent tumor-targeted bioimaging performance after intravenously injection into mice, but not the rod-like aggregates of QM-2.

Full text of DOI:10.1002/anie.201501478

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201501478
German Edition: DOI: 10.1002/ange.201501478
Aggregation-Induced Emission
Far-Red and Near-IR AIE-Active Fluorescent Organic Nanoprobes
with Enhanced Tumor-Targeting Efficacy: Shape-Specific Effects**
Andong Shao, Yongshu Xie, Shaojia Zhu, Zhiqian Guo,* Shiqin Zhu, Jin Guo, Ping Shi,
Tony D. James, He Tian, and Wei-Hong Zhu*
Abstract: The rational design of high-performance fluorescent
materials for cancer targeting in vivo is still challenging. A
unique molecular design strategy is presented that involves
tailoring aggregation-induced emission (AIE)-active organic
molecules to realize preferable far-red and NIR fluorescence,
well-controlled morphology (from rod-like to spherical), and
also tumor-targeted bioimaging. The shape-tailored organic
quinoline–malononitrile (QM) nanoprobes are biocompatible
and highly desirable for cell-tracking applications. Impres-
sively, the spherical shape of QM-5 nanoaggregates exhibits
excellent tumor-targeted bioimaging performance after intra-
venously injection into mice, but not the rod-like aggregates of
QM-2.
role of particle shape, size, and surface chemistry (such as
polymers, liposomes, dendrimers, immunoconjugates, carbon
nanotubes, porphysomes, and inorganic particles),[6] methods
to rationally tailor small molecules to afford organic nano-
structures with the desired morphology, and therefore per-
forming ideal function in diagnosis or therapy in vivo is less
understood.
Several impressive photoelectronic materials with well-
defined morphologies can be obtained using tailor-made
small molecules,[7] but these functional self-assembled mate-
rials remain unexplored for biomedical application, which is
mainly due to several limitations: 1) inherent fluorescence
quenching, which is common for most organic fluorophores
during their aggregation in aqueous media; 2) lacking high-
performance near-infrared (NIR) emission; and 3) the
unclear relationship between tailored morphologies and
targeting efficacy for in vivo diagnostics. Fortunately, since
the concept of aggregation-induced emission (AIE) was
originally reported by Tang et al.,[8a] AIE-active molecules
exhibit highly bright fluorescence when aggregated, and weak
fluorescence when separated in solution,[8] making them ideal
for biosensing and imaging in vivo. However, mapping the
nature of AIE-active organic molecules to finely control the
morphologies and sizes of organic aggregated nanostructures
is uncharted territory, especially the influence of substituents
on the shape, and therefore the excellent optical properties
for bioimaging in vivo.
As is well-known, far-red and NIR emission could
minimize photo-damage to living cells, enable deep tissue
penetration, and circumvent the spectral overlap with bio-
substrate autofluorescence.[9] While great efforts have been
made towards the development of high-performance AIE-
active systems for long-term non-invasive bioimaging in
vivo,[8b] the majority of AIE luminogens has emission wave-
lengths below 650 nm. Particularly, it is not clear whether AIE
nanoaggregates with specific morphologies are suitable for
targeted imaging in vivo. Herein we set out to construct
a tailor-made far-red and NIR AIE-active system (Figure 1A)
employing the quinoline–malononitrile (QM) as AIE build-
ing block,[8f] wherein the morphology of organic nanostruc-
tures could be controlled by changing the electron donor
groups and thiophene p-bridge. Different shapes of these
AIE-active QM derivatives with red to NIR emission were
carefully evaluated under an aggregated microenvironment,
thus taking insight into the effect of specific shape on both
real-time cell tracing and tumor-targeted imaging in vivo.
We performed a series of experiments to examine the
photoluminescence properties of QM derivatives. As
expected, all QM compounds exhibit red to NIR AIE-active
T
he development of bioimaging probes that can differentiate
tumors from normal tissues are highly desirable for cancer
diagnosis and therapy in vivo.[1,2] Fluorescent materials that
provide dynamic and quantitative information of imaging
biomolecules have become indispensable for biological
analysis and clinical diagnosis.[3,4] In particular, organic nano-
materials with excellent synthetic flexibility for chemical
modification are advantageous for real-time cell visualiza-
tions, diagnosis, and treatment of diseases in vivo.[5] While
nanomaterial bioimaging displays a critical interdependent
[*] A. Shao,[+] Prof. Dr. Y. Xie,[+] S. Zhu, Dr. Z. Guo, S. Zhu,
Prof. Dr. P. Shi, Prof. Dr. H. Tian, Prof. Dr. W. Zhu
Key Laboratory for Advanced Materials and Institute of Fine
Chemicals, Shanghai Key Laboratory of Functional Materials
Chemistry, State Key Laboratory of Bioreactor Engineering, Collab-
orative Innovation Center for Coal Based Energy (i-CCE)
East China University of Science & Technology
Shanghai 200237 (China)
E-mail: guozq@ecust.edu.cn
whzhu@ecust.edu.cn
Dr. J. Guo
PerkinElmer Instruments (Shanghai) Co.
Zhangheng Road 1670 Shanghai 201203 (China)
Prof. Dr. T. D. James
Department of Chemistry, University of Bath, Bath BA2 7AY (UK)
[+] These authors contributed equally to this work.
[**] This work was supported by NSFC for Creative Research Groups
(21421004) and Distinguished Young Scholars (21325625),
National 973 Program (2013CB733700), NSFC/China, Oriental
Scholarship, Shanghai Pujiang Program (13PJD010), Fok Ying Tong
Education Foundation (142014), Fundamental Research Funds for
the Central Universities (222201313010), and Catalysis and Sensing
for our Environment (CASE) network. AIE=aggregation-induced
emission.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201501478.
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Figure 1. QM derivatives and AIE properties: A) Molecular structures and the single crystal configurations of QM-1, QM-2, and QM-3.
Photoluminescence spectra and plot of the relative PL intensity of QM derivatives (10À5 m): B) QM-1, C) QM-2, D) QM-4, and E) QM-5 in THF/
H2O mixtures with different volume fractions of water (fw); lex =480 nm. Inset: Fluorescent photoimages in pure THF solvent and THF/H2O
solution of B) QM-1 (fw =90%), C) QM-2 (fw =70%), D) QM-4 (fw =70%) and E) QM-5 (fw =90%) under 365 nm illumination. F) Normalized
fluorescent spectra of QM-1 (fw =90%), QM-2 (fw =70%), QM-3 (fw =90%), QM-4 (fw =70%), QM-5 (fw =90%), and QM-6 (fw =90%) in THF/
H2O solution. G) Normalized fluorescent spectra of QM derivatives in the solid state.
characteristics along with an increasing volume fraction of
water in tetrahydrofuran/water (THF/H2O) mixtures
(Figure 1; Supporting Information, Figure S1 and Table S1).
Moreover, their fluorescent properties are dependent upon
the aggregated microenvironment. Specifically for QM-1,
a strong fluorescence was not observed until the volume
fraction of water (fw) in THF/H2O solutions up to 80%
(Figure 1B). Its fluorescence quantum yield (FF) exhibited
AIE light-up enhancement by 178-fold in the mixed fw = 90%
THF/H2O solution compared with that in pure THF solution.
When strong donor groups are introduced into the
thiophene moiety elongating the p-conjugated systems of
the QM molecules, their emission spectra are extended to
deep red, and even to the NIR region. As shown in Figure 1C,
with a gradual addition of water into THF, QM-2 molecules
containing the triphenylamine donor clustered into nano-
aggregates and the emission was dramatically enhanced with
an fw increase, showing an obvious AIE effect. Successively,
the emission spectra showed a little decrease when fw > 80%,
which might be attributed that QM-2 molecules aggregate
and precipitate quickly at higher water fraction, leading to
amorphous agglomerate formation with lower fluorescence
intensity.[10] Moreover, the fluorescence quantum yield (FF)
of QM-2 was increased sharply by about 183-fold from pure
THF solution to the mixed THF/H2O (fw = 70%) solution.
Furthermore, when a stronger alkoxytriphenylamine donor
moiety was introduced into QM-3, the emission peak located
at 672 nm in pure THF was observed in its molecularly
dissolved state. When water was mixed with THF (fw > 50%),
its emission spectra was bathochromically shifted to 705 nm
with intensified AIE-active emission.
into QM-2 to give QM-4, in which the steric hindrance of
EDOT can change the initial D-p-A structure into more twist
structure. In fact, the structural and conformational differ-
ences of QM derivatives are responsible for the different
aggregated microenvironment, thus resulting in different
AIE-active spectral features. As demonstrated by the X-ray
diffraction (XRD) patterns (Supporting Information, Fig-
ure S2), QM-4 aggregates are mainly in amorphous state,
which might affect its fluorescent quantum yield.[10] Indeed,
QM-4 exhibited 17-fold enhancement in FF value from fw = 0
to 70% in a mixed THF/H2O solution, and concomitantly the
emission peak was red-shifted from 642 to 669 nm (Fig-
ure 1D).
Following this line of thought, a combinational molecular
strategy was employed in the design of QM-5, wherein the
alkoxy-substituted triphenylamine moiety as a stronger donor
would extend the NIR emission spectra, and EDOT-substi-
tuted thiophene would disrupt the initial D-p-A structure for
tailoring the aggregate formation. Similar to QM-4, with an
increase in water fraction, the emission spectra of QM-5
exhibited a large red shift from 668 nm in pure THF solution
to 721 nm in fw = 90% of the mixed THF/H2O solution
(Figure 1E). To further verify our molecular design strategy,
QM-6 was designed and synthesized, which also endowed
similar AIE-active fluorescence properties. As a consequence,
we are able to extend the long wavelength AIE-active
luminescence of QM derivatives from 612 to 721 nm in
THF/H2O solution (Figure 1F), and even from 651 to 738 nm
in their solid state (Figure 1G).
To explore the influence of substituents on the nano-
structures, we employed a solution evaporation approach to
fabricate QM aggregates. The progressive transformation of
morphology for QM aggregates were successfully observed,
To further take insight into the substituent influence,
a 3,4-ethylene-dioxythiophene (EDOT) unit was introduced
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Figure 2. Multiple morphologies of micro/nanoaggregates fabricated from QM derivatives (10À5 m). TEM, SEM, and confocal laser scanning
microscope (CLSM) images recorded for the micro/nanoaggregates of QM-1 (A–C), QM-2 (D–F), QM-3 (G–I), QM-4 (J–L), QM-5 (M–O), and
QM-6 (P–R), prepared by adding different content of water into pure THF solution to afford the mixed solution of THF/H2O then still standing for
1 h.
characterized with transmission electron microscopy (TEM),
scanning electron microscopy (SEM), and confocal laser
scanning microscopy (CLSM). As shown in Figure 2A–I, the
aggregates of QM-1, QM-2, and QM-3 were well-defined
microrods with different sizes and diameters. For example,
uniform 1D microrods of QM-2 were observed by SEM with
the size of about 10 mm in length and 0.5–1 mm in diameter
(Figure 2E). The TEM and CLSM images in Figure 2D and
2F further verified rod-like microstructures in the aggregates
of QM-2. Similar morphological characteristics could also be
observed in QM-1 and QM-3 aggregates, demonstrating
strong AIE-active fluorescence and easily linear self-aggre-
gation when thiophene was utilized as a p-conjugated bridge
in the QM derivatives. Fortunately, single crystals of QM-1,
QM-2, and QM-3 were obtained by the slow evaporation
approach (Supporting Information, Table S2 and Figure S3).
All QM-1, QM-2, and QM-3 display twisted conformations in
their crystal structures with large torsional angles of 85.4–
87.08 between the N-ethyl and the quinoline units (Fig-
ure 1A). Furthermore, moderate interplanar angles of 32.2–
35.38 are observed between the ethylene and quinoline units,
and no obvious p–p stacking interactions can be found in the
crystals, which may be ascribed to the twisted molecular
structures. As a result, their aggregation states display
enhanced emission.
from SEM images (90 Æ 10 nm). Under the same fabricating
conditions, the TEM images of QM-5 had the smallest
diameters and smoothest spherical morphologies with respect
to QM-4 and QM-6 (Supporting Information, Figure S4).
Based on the rational molecular design, the flexibility and
electron-rich properties of the epoxy ethyl groups in QM
chemical structures play important roles in the morphological
formation during the aggregation process when EDOT is
introduced as the p-conjugated bridge. It is expected that the
incorporation of both alkoxytriphenylamine group and
EDOT unit in QM derivatives is a preferable design strategy
to generate AIE-active spherical nanostructures, resulting in
a distinct change in morphology from QM-1 to QM-6
(Figure 2).
Considering nanoprobes for bioimaging in vivo, it is
necessary to assess the external influence on aggregation and
photostability of AIE materials. As shown in the Supporting
Information, Figures S5,S6, once QM derivatives have
formed into aggregation state under the optimized fw, external
factors have little effect on the morphology of QM aggregates.
The photostability of AIE-active QM materials and commer-
cial ICG dye (approved by FDA for NIR clinical imaging
agents) was also evaluated by time-course fluorescence
measurement. After exposure to high density light, the half-
life time of QM derivatives was about 20-fold longer than
ICG dye, demonstrating that QM derivatives are more
photostable materials.
However, for QM-4, QM-5, and QM-6 (Figure 2J–R),
instead of the microrod structures, the resulting morphology
was predominated with spherical shaped nanoparticles in
diameter of about 80–200 nm. This might be ascribed to the
attachment of an epoxyethyl group in the EDOT p-bridge,
leading to a totally different intermolecular interaction in the
aggregation state. Moreover, the average diameters of QM-5
aggregates measured by laser light scattering (LLS) were
about 85(Æ 10) nm, which is exactly consistent with the data
Among all of the QM derivatives, QM-2 and QM-5 are
typical representatives of rod-like and spherical shapes,
respectively. Thus, we chose these two compounds to further
explore their potential shape effects on in vitro and in vivo
applications in living system. QM-2 and QM-5 exhibited low
toxicity against both cancer cells and normal cells, which is
highly preferable for cell imaging or tracking applications
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(Supporting Information, Figures S7–S10). Flow cytometric
studies were conducted to evaluate the kinetics of the cell
uptake process of QM-2 and QM-5 in the HeLa cells.
Although the relative uptake ratio of both compounds after
48 h incubation was close to 100% (ꢀ 99.5%), the initial
uptake rate of QM-5 was much faster compared to that of
QM-2. Additionally, the CLSM images presented a direct
observation of the spherical shape of QM-5 in cells.
could predominately be detected in the tumor at 30 min,
rather than other organs of the mouse, and retained in tumor
tissue even at 24 h after injection. The long retention of the
QM-5 nanoaggreates in tumors makes this system very
promising for tumor labeling and chemotherapy. However,
even at 24 h after the injection, the QM-2 aggregates were still
present in the whole mouse body biodistribution without
producing tumor-targeted fluorescence signals in vivo. A
similar phenomenon was also observed for another rod-like
nanostructure QM-3 (Supporting Information, Figure S11),
further confirming the non-specific in vivo imaging biodistri-
bution of organic rod-like QM assemblies.
The ex vivo fluorescence images of the internal organs of
mice sacrificed at 24 h post-injection in Figure 3D also
indicated that QM-5 aggregates accumulated in the tumor
and liver tissue, whereas the fluorescence of QM-2 aggregates
in tumors was much weaker than other organs such as liver or
lung (Figure 3C). Furthermore, the 3D fluorescence imaging
of tumor-bearing mice in Figure 3E at 24 h post-injection of
QM-5 (0.15 mgkgÀ1) further confirmed the NIR fluorescence
accumulation in tumors. Accordingly, the semi-quantitative
analysis data of the average fluorescence intensity distribu-
tion in organs (Figure 3F) also demonstrated that the
spherical shape of QM-5 aggregates exhibited much higher
tumor-targeting ability than the rod-like aggregates of QM-2.
Undoubtedly, in contrast with the fast degradation in
aqueous media and quick clearance from the body of small
molecular imaging agent ICG dye[11] (control; Supporting
Information, Figure S12), the shape-specificity of QM aggre-
gates contributes a direct benefit for long-term retention and
bioimaging in vivo. Actually, from the TEM images of cells
and tissues, we clearly observed that QM aggregates almost
maintained their initial aggre-
The high-brightness emission for AIE-active organic
nanomaterials could be used as efficient long-term cell
tracers. For instance, QM-2, QM-5, and commercial ICG
dye as control showed bright fluorescence after incubation
with HeLa cells for 24 h despite the different retention in the
cytoplasm of HeLa cells (with the potential as long-term cell
tracers). For QM-2 and QM-5, even after four passages of
incubation with living cells, the fluorescence of QM aggre-
gates staining in HeLa cells was still emissive. In contrast,
there was almost no fluorescence signal using ICG at two
passages of incubation. Therefore, the formation of highly
biocompatible and photostable AIE-active QM aggregates
with different shape and size is beneficial to retain fluores-
cence in the cells, which is desirable for long-term cell tracing.
The promising long-term cell tracing results of AIE-active
QM derivatives inspired us to further explore their feasibility
as NIR bioprobes in vivo. Upon intravenous injection of QM-
2 at a dose of 0.15 mgkgÀ1, we immediately monitored the
fluorescence distribution in mice at different periods of time.
As shown in Figure 3A, the fluorescence was clearly observed
in mice at 30 min, which is indicative of the rapid distribution
of QM-2 aggregates via the blood circulation. Surprisingly,
upon the paralleled intravenous injection of QM-5
(0.15 mgkgÀ1, Figure 3B), the distinct NIR fluorescence
gated morphologies in situ
(Supporting Information,Fig-
ure S13). While the fluores-
cence of rod-like aggregates
by QM-2 exhibited the whole
body biodistribution in mice,
the spherical QM-5 aggre-
gates enhanced tumor-target-
ing capacity, which could be
ascribed to the “passive”
tumor-targeting by enhanced
permeability and retention
(EPR) effect.[12] Apparently,
here the particle geometry
plays an important role in
the tumor-targeted bioimag-
ing in vivo. Another potential
possibility for this disparity
could be the difference in
shape and shape-related fac-
tors such as curvature and
Figure 3. In vivo non-invasive imaging of tumor-bearing mice after intravenous injection of A) QM-2
(0.15 mgkgÀ1), B) QM-5 (0.15 mgkgÀ1) at different periods of time (0.5, 1.5, 3 and 24 h), and ex vivo
fluorescence images of the internal organs of mice sacrificed at 24 h post-injection with C) QM-2 and
D) QM-5. E) The 3D fluorescence imaging of tumor-bearing mice after intravenous injection of QM-5
(0.15 mgkgÀ1) for 24 h. F) Average fluorescence intensity distribution for tumor and internal organs from
mice sacrificed at 24 h post-injection with QM-2 and QM-5 (n=3).
aspect ratio, which affect
cell-particle interactions, par-
ticle transport characteristics
and margination dynam-
ics.[6k,12] Therefore, based on
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c) W. M. Xuan, C. Q. Sheng, Y. T. Cao, W. H. He, W. Wang,
the TEM images of HeLa cells and tissues, the shape
differences of QM aggregates formed by tailoring AIE-
active organic molecules are of great value for tumor-targeted
bioimaging in vivo.
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In summary, far-red and NIR AIE-active fluorescent
organic QM nanoprobes have been rationally designed. We
specifically focused on the modulation of long emitting
wavelength and the aggregated morphologies via essentially
tailoring p-bridge and donor unit in molecular structures. QM
derivatives from rod-like to spherical morphology were well
confirmed by SEM, TEM and CLSM images. In vitro
experiments have verified that these tailor-made long wave-
length AIE-active organic QM nanomaterials are biocom-
patible and retained in the cytoplasm of living cells. The most
striking feature of NIR spherical QM-5 nanoaggregates is
their excellent tumor-targeting performance in mice. Con-
versely, the same is not true for the rod-like aggregates of
QM-2, which do not display any tumor targeting properties.
To the best of our knowledge, this is the first report of shape-
specific tumor targeting using bare NIR AIE-active nanop-
robes. Our strategy generates high-performance long wave-
length AIE-active organic nanomaterials with ideal biological
geometries for tumor-targeted bioimaging in vivo, providing
a promising platform for in situ and in vivo tumor imaging
agents.
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Keywords: aggregation-induced emission · fluorescent probes ·
morphology effects · near infrared · tumor targeting
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Received: February 14, 2015
Revised: March 29, 2015
Published online: && &&, &&&&
6
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 7
These are not the final page numbers!

Angewandte
Chemie
Communications
Aggregation-Induced Emission
A. Shao, Y. Xie, S. Zhu, Z. Guo,* S. Zhu,
J. Guo, P. Shi, T. D. James, H. Tian,
W. Zhu*
&&&& — &&&&
Far-Red and Near-IR AIE-Active
Fluorescent Organic Nanoprobes with
Enhanced Tumor-Targeting Efficacy:
Shape-Specific Effects
Tailoring long-wavelength aggregation-
induced emission-active molecules
affords organic nanoaggregates with
desired morphology, from rod-like to
spherical. The latter are preferable for
enhanced tumor-targeted bioimaging in
vivo.
Angew. Chem. Int. Ed. 2015, 54, 1 – 7
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7
These are not the final page numbers!