Robust Colloidal Nanoparticles of Pyrrolopyrrole Cyanine J-Aggregates with Bright Near-Infrared Fluorescence in Aqueous Media: From Spectral Tailoring to Bioimaging Applications

Article abstract of DOI:10.1002/chem.201604741

Colloidal nanoparticles (NPs) containing near-infrared-fluorescent J-aggregates (JAGGs) of pyrrolopyrrole cyanines (PPcys) stabilized by amphiphilic block co-polymers were prepared in aqueous medium. JAGG formation can be tuned by means of the chemical structure of PPcys, the concentration of chromophores inside the polymeric NPs, and ultrasonication. The JAGG NPs exhibit a narrow emission band at 773 nm, a fluorescence quantum yield comparable to that of indocyanine green, and significantly enhanced photostability, which is ideal for long-term bioimaging.

Full text of DOI:10.1002/chem.201604741

A Journal of
Accepted Article
Title: Robust Colloidal Nanoparticles of Pyrrolopyrrole Cyanines J-
Aggregates with Bright Near Infrared Fluorescence in Aqueous
Media: From Spectral Tailoring to Bioimaging Application
Authors: Cangjie Yang, Xiaochen Wang, Mingfeng Wang, Keming Xu,
and Chenjie Xu
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201604741
Link to VoR: http://dx.doi.org/10.1002/chem.201604741
Supported by

10.1002/chem.201604741
Chemistry - A European Journal
FULL PAPER
Robust Colloidal Nanoparticles of Pyrrolopyrrole Cyanines J-
Aggregates with Bright Near Infrared Fluorescence in Aqueous
Media: From Spectral Tailoring to Bioimaging Application
Cangjie Yang†, Xiaochen Wang†, Mingfeng Wang*, Keming Xu, Chenjie Xu
Abstract: We report a new class of colloidal nanoparticles called
JAGG containing near-infrared fluorescent J-aggregates of
pyrrolopyrrole cyanines (PPcys) stabilized by amphiphilic block
copolymers in aqueous media. The formation of such JAGG can be
tuned by the chemical structure of PPcys, the concentration of the
chromophores inside the polymeric nanoparticles and ultrasonic
treatment. These JAGG nanoparticles exhibit a narrow emission
band at 773 nm, a fluorescence quantum yield comparable to that of
indocyanine green, and significantly enhanced photostability that is
ideal for long-term bioimaging.
fluorescence, have attracted considerable attention for various
applications.[9] Such
resonant fluorescence band of J-
a
aggregates with quite narrow bandwidth is highly desirable for
simultaneous detection of multiple targets to minimize the
spectral overlapping and interference. However, the bioimaging
application of existing J-aggregates of dyes is limited by
relatively low FL quantum yield, short emission wavelength and
poor solubility in aqueous media. For instance, J-aggregates of
ICG associated by van der Waals forces in water have been
10]
observed.[9c,
But the colloidal stability of these aggregates
without stabilization by surfactants was susceptible to changes
of environmental factors such as concentration, ionic strength,
and temperature. Recently, Zheng and coworkers[11] reported
that porphyrin J-aggregates formed in synthetic liposomes (also
called porphysomes) showed dual photoacoustic and
fluorescent properties for cancer imaging applications. Würthner
and coworkers[12] reported the J-type aggregation of H-bonding
perylene bisimide derivatives with far-red fluorescence and high
quantum yield, close to unity. But such self-assembly took place
in nonpolar solvents which are not suitable for bioimaging.
Introduction
Fluorescence imaging in vivo in the near-infrared (NIR)
window is important to enable deep tissue penetration with high
optical contrast.[1] To date there are only two clinically approved
NIR fluorophores, indocyanine green (ICG) and methylene blue
(MB), both of which are small molecules that can be rapidly
excreted during circulation in physiological systems.[2] In contrast
to small molecular imaging probes, fluorescent nanoparticles
(NPs) with sizes in the range of 10-100 nm usually show
prolonged intracellular retention and enhanced permeation
retention (EPR) effect,[3] making them suitable for long term cell
tracking[4] and tumor targeting.[5] One common strategy to
prepare fluorescent colloidal NPs is to encapsulate the
molecular fluorophores in a polymer matrix. However, the
significant fluorescence (FL) quenching caused by the
intermolecular aggregation of most organic fluorophores within
NPs remains a challenge for bioimaging, with an exception of a
group of special chromophores with strong emission in
[6]
aggregated state
and a series of fluorescent dyes covalently
tethered with polymers.[4b, 7]
Since the first discovery in 1936 by Jelley and Scheibe,[8] J-
aggregates (also called Scheibe aggregates), which exhibit
strongly red-shifted light absorption and a nearly resonant
Scheme 1 Schematic diagram of encapsulation of the PPcy derivatives into H-
type and J-type aggregated nanoparticles (HAGG and JAGG): (a) molecular
structures of PC, PCBF, PCBFB and the routes to formation of JAGG and
HAGG nanoparticles; (b-c) schematic representation of J-aggregate (b) and H-
aggregate (c); (d) chemical structure of the amphiphilic block copolymer PCL-
b-POEGMA.
[¶]
C. Yang,[†] Dr. X. Wang,[†] Prof. Dr. M. Wang, Dr. K. Xu, Prof. Dr. C.
Xu
School of Chemical and Biomedical Engineering
Nanyang Technological University
62 Nanyang Drive, Singapore 637459, Singapore
E-mail: mfwang@ntu.edu.sg (MW)
Dr. C. Xu
Herein, we present colloidal NPs of J-aggregates (denoted as
JAGG) in water with bright NIR fluorescence and narrow
bandwidth for bioimaging applications. The key component of
the JAGG is a brightly fluorescent molecule, PCBF (as shown in
Scheme 1), a pyrrolopyrrole cyanines (PPcy) derivative. PPcys
containing a pyrrolopyrrole unit as the central element of a
cyanine-type chromophore with emission in the NIR region up to
1000 nm and higher quantum yields than those of other organic
NIR dyes were previously reported by Ficsher and coworkers.[13]
NTU-Northwestern Institute for Nanomedicine
Nanyang Technological University
50 Nanyang Avenue, Singapore 639798, Singapore
Dr. X. Wang (Present address)
National Center for Nano Science and Technology
Zhongguancun Beiyitiao, Beijing 100190, China.
These two authors contribute equally.
[†]
Supporting information for this article including detailed synthetic
and preparation procedure and supporting figures is given.
This article is protected by copyright. All rights reserved.

10.1002/chem.201604741
Chemistry - A European Journal
FULL PAPER
To render the hydrophobic dye PCBF dispersible in water, a
occur in the corresponding NPs. These NPs exhibited bright NIR
fluorescence, which is mainly contributed by J-aggregating
components, and robust photostability that is important for
bioimaging application.
synthetic amphiphilic block copolymer PCL-b-POEGMA (Mn
=
31,100; Mw/Mn = 1.37) was employed to encapsulate PCBF into
the hydrophobic core of NPs via a process of nanoprecipitation
(Scheme 1).[7c] Both H-aggregation and J-aggregation of PCBF
Scheme 2 Synthetic route to Compound PC, PCBF and PCBFB.
absorption maximum of monomeric PCBF appears at 700 nm
(absorption coefficient: ε = 1.55×105 cm-1M-1, even higher than
that of porphyrin[14]) while the FL emission spectrum exhibits a
maximum at 715 nm. Thus PCBF in CHCl3 shows a Stokes shift
of 15 nm and a quite high FL quantum yield of 0.69 using ICG in
water (QY~0.01) as a standard. The emission band is relatively
broad with full-width-at-half-maximum (FWHM) value of 6910
cm-1 than that of absorption band (3849 cm-1) (Figure 1a). The
optical properties of PCBF observed here are consistent with
what was reported by Fischer and coworkers.[13]
Results and Discussion
The synthetic route to PCBF is illustrated in Scheme 2, using
a modified method reported by Fischer and coworkers.[13a] 3,6-
Bis(4-octyldodecyloxyphenyl)-2,5-dihydropyrrolo [3.4-c]pyrrole-
1,4-dione (Compound 3, Scheme 2) was prepared by using a
condensation
of
diisopropyl
succinate
4-
octyldodecyloxybenzonitrile in the solution of potassium tert-
butoxide and 2-methyl-2-butanol, where octyldodecyl branch
group can provide DPP with
Subsequently, condensations of C-H acidic methylene groups
from 2-cyanomethylpyridine at the carbonyl group of
a
relatively good solubility.
In order to prepare colloidal NPs of PCBF in water, a mixture
of PCBF and PCL-b-POEGMA codissolved in THF was rapidly
injected into large excess water under ultrasonication, followed
by evaporation of the organic solvent, resulting in colloidally
stable NPs. Our previous work has demonstrated that the size of
nanoparticles prepared by this nanoprecipitation method can be
facilely tuned from 50 to 400 nm by varying the weight ratio of
fluorophore/PCL-b-POEGMA.[7c] In this work, a dispersion of
NPs based on PCBF was obtained at a weight ratio of
PCBF/PCL-b-POEGMA at 3.5 and a concentration of PCBF at
0.4 mM. As shown in Figure 1c, roseberry-like spherical NPs
3
proceeded in toluene with an excess of phosphoryl chloride to
give compound PC, respectively. In order to stiffen the
chromophores to activate NIR emission, trifluoroborane etherate
was added to the dichloromethane solution of PC at the
presence of Huning base to give PCBF with a quantitative yield.
The UV-Vis-NIR absorption and FL emission spectra of PCBF
in CHCl3 (Figure 1a) show all the intrinsic features of PPcy. The
This article is protected by copyright. All rights reserved.

10.1002/chem.201604741
Chemistry - A European Journal
FULL PAPER
with an average diameter of 30.0 ± 2.5 nm were observed in the
transmission electron microscopy (TEM) image of JAGG NPs
(Figure 1c). Beneficial from the higher electron density of PCBF
than the PCL-b-POEGMA matrices, dye aggregation inside the
NPs gave a clear contrast of PCBF aggregates as darker dots in
the grey matrices of the polymeric NPs. The hydrodynamic
diameter of JAGG NPs measured by dynamic light scattering
(DLS) is 47.5 nm (Figure 1d), which is larger than that from TEM
result due to the hydration of POEGMA corona.
In striking contrast to the optical absorption of monomeric
PCBF in CHCl3, the JAGG NPs in water exhibit two new
absorption bands as shown in Figure 1b. The blue-shifted band
versus the monomeric band (M-band) appears as a typical
signal of the formation of H-type and/or dimer aggregation (both
are included as H-band here for simplification).[15] The maximum
of the H-aggregate absorption band (H-band) is centered at 647
nm. As compared to the M-band, another narrow and intense
band in the absorption transition shows a 61 nm red-shift to 768
nm (Figure 1b), which is a spectral signature of ordered J-
aggregates. Furthermore, the FWHM of J-band is narrower
(2235 cm-1) than that of PCBF in CHCl3 (3849 cm-1).This
Figure 1 (a) UV-Vis-NIR absorption and FL emission spectra of PCBF in
chloroform. (b) UV-Vis-NIR absorption and FL emission spectra of PCBF
based JAGG NPs with the concentration of 0.4 mM in aqueous media. (c)
TEM image of JAGG NPs of PCBF with the concentration of 0.4 mM in
aqueous media; (d) DLS result shows the size distribution of JAGG NPs of
PCBF in aqueous media.
spectral-narrowing phenomenon is
a
known property of
coherently coupled J-aggregates. It has been predicted that the
absorption spectral width of the aggregate is narrower than that
of the monomer by a factor N1/2, where N is the number of
monomers in direct communication with each other.[16] The
average J-aggregation number N calculated from the spectral
narrowing of JAGG NPs is 7.4.
The FL emission spectrum of JAGG NPs dispersion shows a
characteristically small Stokes shift of 5 nm, a λmax,em of 773 nm
and a smaller FWHM of 2114 cm-1 than that of PCBF in
chloroform (6910 cm-1) as shown in Figure 1b, which provides
further evidence of J-aggregate formation. Evidently, the
emission of monomeric molecules was not observed in Figure
1b. It can be explained by the intrinsic Föster resonance energy
transfer (FRET) from monomer to J-aggregates in confined
space due to the well overlapping between emission band of
monomer (Figure 1a) and absorption band of J-aggregates
(Figure 1b). The excitation spectrum (Figure S2) of JAGG
dispersion at the emission wavelength of 773 nm shows
emission maxima at the excitation wavelength of 769 nm, with
an intensity approximately 35 times higher than that excited at
650 nm. This result suggests that the J-aggregates are the most
active component that contributes to the fluorescence. While the
calculated the FL quantum yield (0.012) of JAGG NPs excited at
650 nm in water is much lower than that (0.69) of PCBF in
A contol experiment was also conducted to monitor the
formation of PCBF aggregates by varying the volume ratio of
water/THF mixture in the absence of PCL-b-POEGMA. As
shown in Figure S3a, with the gradual increase water fraction (fw,
by volume) from 0 to 40%, there is no significant change in the
absorption spectra. Further increase of fw to 50% leaded to the
appearance of J-band, accompanied with a transition from
transparent solution to suspension of aggregates which
precipated after standing overnight, implying the poor colloidal
stability in the absence of the polymeric surfactant. The DLS
histogram (Figure S3b) of the formed aggregated shows an
average diameter of 10 µm, signficantly larger than the size of
the JAGG NPs stabilized by PCL-b-POEGMA. When fw was
increased to 60%, the J-band became stronger and sharper.
The absence of H-band in the absorption spectra of the neat
PCBF aggregates suggests the preferable formation of J-
aggregation under the present experimental condition. The
presence of the relatively high fraction of good solvent (i.e. THF)
in the THF/water mixture might enable PCBF molecules to self-
assemble to reach the more thermodynamically stable state, i.e.
J-aggregation. In contrast, the aforementioned JAGG NPs were
prepared by a rapid injection of a small amount of PCBF/THF
solution (1 mL) into water (10 mL). The flash precipitation
process may not allow all of the PCBF molecules to form J-
aggregation immediately, resulting in the mixture of H-
aggregates, J-aggregates and some PCBF monomers in JAGG
NPs. In other words, the JAGG NPs described above represent
CHCl3, it is comparable with those of water-soluble PPcy
[17]
derivatives (0.01)[13c] and ICG (0.01)
in water. It should be
noted that the actual FL quantum yield of JAGG is
underestimated here, given the fact that only a small fraction of
light at 650 nm (i.e. the exciation wavelength) is absorbed by the
J-aggregates. Due to the small Stokes shift (5 nm) of the
emission band versus the absorption band of the J-aggregates,
it is difficult to estimate the FL quantum yield of JAGG when the
J-absorption band is excited. Nevertheless, the effect of the
excitation wavelength on the FL intensity of the J-aggregates is
reflected from the FL excitation spectrum shown in Figure S2.
The photostability of JAGG NPs under continuous irradiation
with
a 770-nm laser (power density ~
0.22 W/cm2) was
investigated using ICG in water as the control, where the
absorbance at 770 nm of both JAGG and ICG was kept at 1.4.
With the increase of irradiation time, the maximal absorbance
a
kinetically trapped or instantly frozen state in the
nanoprecipitation process.
This article is protected by copyright. All rights reserved.

10.1002/chem.201604741
Chemistry - A European Journal
FULL PAPER
and FL emission intensity of ICG aqueous solution decreased
quickly, accompanied by the color change of ICG solution from
green to colorless (Figure 2a & 2b). In contrast, only negligible
decrease of absorbance could be observed in JAGG solution
under the same condition of irradiation (Figure 2c). After 55 min
of continuous irradiation, the decrease of the FL intensity at 771
nm of JAGG solution was ~12% (Figure 2d), much lower than
that (97%) at 805 nm of ICG solution, indicating relatively high
photostability of JAGG NPs. The excellent photostability could
be attributed to the intrinsic structural stability of PPcy[13a] and its
of PCBF/PCL-b-POEGMA on the spectral characteristics were
studied. We gradually decreased the feeding amount of PCBF
when the amount of surfactant was kept constant. The
concentration-dependent UV-Vis-NIR spectroscopy revealed
that the J-aggregation in JAGG NPs was enhanced with the
increase of the concentration of PCBF from 0.04 to 0.4 mM,
accompanied by a gradually sharpening J-band and increasing
ratio between J-band and H-band absorbance (Figure 3a & 3c).
This result, as expected, indicates that formation of the J-
aggregate was favoured by increased concentration of PCBF.
With the increase of J-aggregate proportion, a slight 4 nm red-
shift in the absorption from 764 to 768 nm was observed (Figure
3c). A shoulder peak at longer wavelength can be found in the J-
aggregate absorption band of the JAGG dispersions at 0.04,
0.08 and 0.16 mM, respectively. Interestingly, the J-band in the
absorption spectrum of the NPs at 0.24 mM exhibits two
overlapping peaks at 766 and 771 nm, respectively (Figure 3c).
This is a clear indication of the two overlapping bands of J-
aggregation presumably due to two populations of J-aggregates
with different geometrical configurations.[20] Evidently, with the
increase of PCBF concentrations from 0.04 to 0.4 mM, the
intensity of the emission band with λmax,em at 773 nm gradually
increased and the bandwidth turned narrower (Figure 3b), which
is attributed to the increasing proportion of J-aggregation and
configuration corresponding to the longer absorption wavelength.
18]
isolation from unfavorable influences of the environment.[4b,
Hence, these JAGG NPs with such a large size are preferable
for long-term tracking due to the increase of circulation half-life
and excellent photostability compared to water-soluble dyes.[18b,
19]
Figure 2 The evolution of UV-Vis-NIR absorption spectra (a) and FL emission
spectra (b) of ICG solution in water under continuous irradiation by a 770 nm
laser for ~1h at a power density of 0.22 W/cm2; UV-Vis-NIR absorption spectra
(c) and FL emission spectra (d) of PCBF based JAGG dispersion under
continuous irradiation by a 770 nm laser for ~1h at a power density of 0.22
W/cm2; (e) NIR absorbance ratio A/A0 of ICG solution and JAGG dispersion
plotted as a function of irradiation time. A0 is the initial absorption maximum
and A is the absorption maximum of the sample at different time points after
illumination. (f) NIR fluorescence intensity ratio A/A0 of ICG solution and JAGG
dispersion plotted as a function of irradiation time. F0 is the initial fluorescence
maximum and F is the fluorescence maximum of the sample at different time
points after illumination.
Figure 3 Concentration-dependent absorption spectra (a) and FL emission
spectra (b) of PCBF based JAGG NPs upon the encapsulation of PCL-b-
POEGMA. (c) Concentration-dependent normalized absorption spectra and
zoom-in J-band variation of PCBF based JAGG NPs. CPCBF corresponds to
concentration of PCBF.
We envisioned that such unusual optical property of the J-
aggregate of PCBF was encoded in its molecular structure: a
planar conjugated core stiffened by difluoroborane group. To
confirm this presumption, PC, the precursor of PCBF, was
encapsulated by PCL-b-POEGMA to give colloidal dispersion as
The effect of the concentration of PCBF and the weight ratio
This article is protected by copyright. All rights reserved.

10.1002/chem.201604741
Chemistry - A European Journal
FULL PAPER
by changing the environmental temperature.[12, 21] In contrast, as
shown in Figure S10, only a slight change around J-band was
observed in the UV-Vis-NIR absorption spectra of JAGG
dispersion (0.4 mM) with the gradually increased temperature
from 25 to 60 oC.
a control. In contrast to PCBF, the absorption spectra of PC
dispersion (Figure 4c) with 0.4 mM exhibited two peaks at 648
and 705 nm, which could be typically assigned to H-band and M-
band, respectively. The same optical feature was observed at
different concentrations of PC (Figure 4c & S8), suggesting no
formation of J-aggregates. Such dramatically different
aggregation behaviours between PC and PCBF may be
rationalized by the more rigid and planar aromatic core and the
resonance structure of a merocyanine in the latter, which favors
the formation of J-aggregates.
We previously reported that ultrasonication played a role to
induce the colloidal crystallization of an indigo derivative.[22]
Here, the colloidal NPs of PCBF were also treated with
ultrasonication. Upon the ultrasonic treatment for 200 mins, the
absorbance of J-band at 774 nm gradually increased,
accompanied with the decrease of H-band absorbance (Figure
5a). The change can be observed with naked eyes, as the bluish
solution of JAGG NPs turned green upon ultrasonication (Figure
5a). These results clearly indicate the transition from H-
aggregate to J-aggregate.[10] Moreover, the J-band in the
absorption spectra exhibits a red-shift of 6 nm (from 768 to 774
nm) upon ultrasonication for 200 mins while the FL emission
spectrum shows a λmax,em of 774 nm and negligible Stokes shift
(Figure 5b & 5c). The FL quantum yield of JAGG NPs after
ultrasonication for 200 mins was increased to 0.025, consistent
with the increase of J-aggregate population. It is noted that there
was no obvious change of the morphology and the size of JAGG
after the ultrasonication (Figure 5d & Figure S11).
To further understand how the structural details determine the
aggregating state, PCBFB extended with two benzene rings was
synthesized (Scheme 2). In contrast to PCBF, PCBFB showed a
higher λmax.abs of 729 nm and λmax.em of 743 nm due to the
extension of conjugation (Figure 4b). A high FL quantum yield
(0.56) of PCBFB in CHCl3 was also achieved. After
encapsulation with PCL-b-POEGMA in water, the absorption
spectra of PCBFB NPs (0.4 mM) exhibited the typical feature of
H-aggregation (Figure 4d). PCBFB preferentially formed
sandwich-type H-aggregates with significantly quenched
fluorescence (quantum yield of 0.0011). The twist between the
benzene ring and the PPcy core in PCBFB may reduce the
planarity of the aromatic component as compared to PCBF.
These results imply that the planarity and rigidity of the aromatic
cores in PCBF could possibly facilitate the formation of J-
aggregates. Nevertheless, a clear picture how PCBF molecules
stack inside the colloidal nanoparticles remains unclear, and
warrants further experimental study, for example, of the
crystalline structures of PPcy derivatives and some simulation
study in the future.
Figure 5 The evolution of UV-Vis-NIR spectra (a) and FL emission spectra (b)
of PCBF-based JAGG NPs (diluted by eight folds from 0.4 mM stock solution
with water) after ultrasonication over different periods; arrows indicate the
spectroscopic changes with increasing ultrasonication time. (c) UV-Vis-NIR
absorption and FL emission spectra of PCBF based JAGG NPs after
sonication for 200 mins. (d) TEM image of PCBF based JAGG NPs after
sonication for 200 mins.
Figure 4 (a) UV-Vis-NIR absorption and FL emission spectra of PC in
chloroform. (b) UV-Vis-NIR absorption and FL emission spectra of PCBFB in
chloroform. Concentration-dependent absorption spectra of (c) PC and (d)
PCBFB based HAGG NPs upon the encapsulation of PCL-b-POEGMA. CPC
and CPCBFB correspond to concentration of PC and PCBFB in stock solutions.
(All the samples were diluted by eight folds before test)
The optical properties of JAGG NPs described above, meeting
most requirements of a FL imaging probe, encouraged us to
examine their potential for in vivo imaging. Firstly, the
cytotoxicity of JAGG NPs against mouse fibroblast cells (L929)
was studied by PrestoBlue assays. As summarized in Figure 6a,
there was no obvious change of the metabolic viability of L929
cells after incubation with JAGG for 72 h at a series of
concentrations of 0.01, 0.02, 0.05, 0.1, 0.2 mM, respectively,
The optical properties of several kinds of J-aggregates based
on supramolecular assembly have been reported to be tunable
This article is protected by copyright. All rights reserved.

10.1002/chem.201604741
Chemistry - A European Journal
FULL PAPER
indicating the low cytotoxicity of JAGG NPs. In in vivo
experiment, subcutaneous and intramuscular injections of JAGG
dispersions (50 µL aliquots, 1 mg/mL) into the left flank (position
II in Figure 6b) and left leg (position III in Figure 6b) of a nude
mouse, respectively, were administered. The mouse was
imaged in a fluorescence mode (excitation filter, 605-640 nm,
and emission filter, 740-800 nm). The FL signals collected from
both injected regions were clearly observed and the FL intensity
emanating from deeper tissue (ROI (III) = 9.856 × 1010, ROI
stands for regions of interest) is comparable with that of the
near-skin fluorescence (ROI (I) = 1.029 × 109) (Figure S12). As a
control, the HAGG dispersion of PCBFB was subcutaneously
and intramuscularly injected to right flank (position IV) and right
leg (position V), respectively (Figure 6b). Obviously, the weak
fluorescence signals (ROI (IV) = 5.340 × 108; ROI (V) = 5.492 ×
108) observed here were only slightly stronger than that of the
blank (ROI (I) = 1.571 × 108), due to the quite low FL quantum
yield of the PCBFB NPs.
Materials
10×phosphate buffer saline (PBS) buffer with pH = 7.4 (ultrapure
grade) is a commercial product of 1st BASE Singapore. MilliQ
water (18.2 MQ) was used to prepare the buffer solution from
the 10×PBS stock buffer. 1×PBS consists of NaCl (137 mM),
KCl (2.7 mM), Na2HPO4 (10 mM) and KH2PO4 (1.8 mM).
Chloroform-D (99%) was purchased from Cambridge Isotope
Laboratories, Inc. All other chemicals and reagents were
purchased from Aldrich or Merck and used as received unless
other specified. Block copolymers PCL-b-POEGMA was
obtained according to our previous work.[7c]
Characterization
The samples were dissolved with chloroform-d for 1H NMR
and 13C NMR measurements on a Bruker AV300 MHz NMR
spectrometer. Emission spectra of solutions were measured by
fluorescence spectrophotometry on
a
Horiba Fluolog
3
spectrofluorometer at 25 oC. The fluorescence quantuam yield of
these materials were measured using indocyanine green (ICG)
solution in water as the standard (ФF =0.01). UV-Vis-NIR spectra
of the samples were measured on a SHIMADZU UV-2450
spectrophotometer. The photostability of ICG solution and JAGG
dispersion subjected to the continuous irradiation by a 770 nm
laser (power density ~ 0.22 W/cm2) over different periods was
monitored by the UV-Vis spectrometer and fluorometer. TEM
measurements were performed with a TEM Carl Zeiss Libra 120
Plus at an acceleration voltage of 120 kV. A 5 µL droplet of
diluted samples was directly dropped onto a copper grid (300
mesh) coated with a carbon film, followed by drying at room
temperature. The size distribution of resulting nanoparticles
were determined by dynamic light scattering (DLS) using a BI-
200SM (Brookhaven, USA) with angle detection at 90o.
Ultrasonic treatment of JAGG dispersion was performed in an
ultrasonic cleaner (SB-120 DTN, power: 120 W) bath.
Temperature-dependent variation on absorption spectra of
JAGG dispersions was monitored by the UV-Vis spectrometer
from 25 to 60 oC. Each temperature point was kept for 1 h to
treat the sample.
Figure 6 (a) Metabolic viability of L929 cancer cells after incubation with
JAGG NPs at different concentrations. (b) In vivo fluorescence image of JAGG
and HAGG NPs (50 µL of
1 mg/mL) injected subcutaneously and
intramuscularly on each flank of a mouse. Position I: blank control; II: JAGG
NPs subcutaneous; III: JAGG NPs intramuscular; IV: HAGG NPs
subcutaneous; V: HAGG NPs intramuscular.
Conclusions
Synthesis of Compound 1
In summary, we have presented a novel type of colloidal NPs
based on the J-aggregation of PPcy dyes stabilized with a
polymeric surfactant in water. The packing configuration of PPcy
in NPs is determined by factors including the chemical structure,
the concentration of the dye and the ultrasonication treatment.
The outstanding spectroscopic properties of JAGG NPs, such as
large light-absorption coefficient, relatively high FL quantum
yield, as well as narrow emission band, enable them to be
promising candidates for in vivo imaging in the near infrared
range. These JAGG NPs may also be useful for multimodal
imaging (fluorescence and photoacoustic)[11a] and therapeutic
(photothermal therapy) applications.[23]
Triphenylphosphine (26 g, 0.1 mol) was dissolved in
dichloromethane (100 mL). Then, bromine (16 g, 0.1 mol) was
added dropwise at 0 oC. After completion of adding bromine, the
reaction mixture was stirred half an hour at room temperature. 2-
octyl-1-dodecanol (30 g, 0.1 mol) was added slowly. The
reaction mixture was stirred at room temperature overnight.
Then the reaction mixture was poured into water (200 mL) and
extracted with dichloromethane (3 × 50 mL). The extracts were
combined and washed with water and saturate sodium
bicarbonate solution then dried over anhydrous magnesium
sulfate. After filtration, filtrate was concentrated to 100 mL under
reduced pressure. Petroleum ether (200 mL) was added to the
mixture and stirred. Resulting triphenylphosphine oxide
precipitate was filtered off and filtrate was concentrated to 100
Experimental Section
This article is protected by copyright. All rights reserved.

10.1002/chem.201604741
Chemistry - A European Journal
FULL PAPER
ml. Added another 100 mL petroleum ether and filtered.
Repeated this operation till no precipitate observed, and the
filtrate was evaporated to dryness. The product 2-octyl-1-
dodecyl bromide was obtained in a yield of 92% and used in
subsequent reactions without further purification.
7.12 (4H, d), 7.01 (2H, t), 3.97 (4H, d), 1.84 (2H, m), 1.25-1.50
(64H, broad), 0.89 (12H, m).
Synthesis of Compound 5
Compound 4 (25 mg, 0.02 mmol) and N,N-diisopropylethylamine
(0.2 mL, 1.2 mmol) were dissolved in dichloromethane (2 ml).
Trifluoroborane etherate (0.2 mL, 1.6 mmol) was added and the
mixture was stirred at room temperature for 2 hours. The
reaction mixture was washed with water and dried over
anhydrous sodium sulfate. After removing the solvent, the crude
product was purified by column chromatography, eluting with
dichloromethane to give Compound 5 as a green solid (26 mg,
quantitative yield). 1H NMR (300 MHz, CDCl3): 8.36 (2H, d), 7.88
(2H, t), 7.65 (6H, m), 7.22 (2H, t), 7.08 (4H, d), 3.95 (4H, d), 1.83
(2H, m), 1.25-1.50 (64H, broad), 0.91 (12H, m).
Synthesis of Compound 2
4-Cyanophenol (1.19 g, 10 mmol) and potassium carbonate
(5.52 g, 40 mmol) were dissolved in 60 ml acetone. After being
o
purged for 15 min, the mixture was heated to 100 C for half an
hour. Then, 2-octyl-1-dodecyl bromide (4 g, 11 mmol) was
added to the mixture by syringe. The reaction mixture was
o
stirred at 100 C for 30 h. After cooled to room temperature, the
reaction mixture was added to 100 mL of water and extracted
with ethyl acetate. The extracts were combined and washed with
water then dried over anhydrous magnesium sulfate. The crude
product was purified by flash chromatography (petroleum
ether/dichloromethane from 10: 0 to 10:1) to yield target product
Synthesis of Compound 7
5-bromo-2-cyanomethylpyridine was used to replace 2-
cyanomethylpyridine as the raw materials. The procedure to
synthesize 8 was similar with that of 2 and the similar yield was
achieved. 1H NMR (300 MHz, CDCl3): 8.43 (2H, d), 7.91 (2H, d),
7.64 (4H,d), 7.55 (2H, d), 7.07 (4H, d), 3.95 (4H, d), 1.83 (2H, m),
1.25-1.50 (64H, broad), 0.91 (12H, m).
1
as a light yellow viscous liquid in a yield of 90%. H NMR (300
MHz, CDCl3): 7.58 (2H, d), 6.97 (2H, d), 3.88 (2H, d), 1.81 (1H,
m), 1.25-1.50 (32H, broad), 0.90 (6H, t).
Synthesis of Compound 3
Potassium tert-butoxide (1.68 g, 15 mmol) was added to 2-
methyl-2-butanol 15 mL, and the mixture was heated to reflux.
When the alkali is dissolved, Compound 2 (4 g, 10 mmol) was
added in one portion. Then diisopropyl succinate (1.01 g, 5
mmol) was added over three hours with a dropping funnel. After
heated for another three hours at 110 oC, the mixture was
cooled down and slowly added to a mixture of 100 mL ethanol
(containing 2 mL concentrated hydrochloric acid). The red
precipitate is filtered and washed with ethanol. The solid is
digested in boiling ethanol, filtered and washed with ethanol.
This procedure is repeated until the filtrate is clear. Drying in
vacuum yields 2.1 g (24 %) orange solid. 1H NMR (300 MHz,
CDCl3): 8.42 (2H,s), 8.26 (4H, d), 7.08 (4H, d), 3.96 (4H, d), 1.84
(2H,m), 1.25-1.50 (64H, broad), 0.90 (12H, m).
Synthesis of Compound 8
Compound 8 (133.5 mg, 0.1 mmol), phenylboronic acid (36.6
mg, 0.3 mmol) and K2CO3 (1.38g) was dissolved in degassed
mixture of toluene (10 mL), water (2 mL) and ethanol (2 mL)
under nitrogen atmosphere. It is followed by addition of a
catalytic amount (1% m/m) of Pd(PPh3)4. The resulting mixture
was heated at 100 °C for 6 h, then cooled down to room
temperature. The organic layer was washed by water for three
times and concentrated under reduced pressure. The product
was purified by column chromatography on silica using
dichloromethane/hexane (1/1) as the solvent and dried under
vacuum at 80 °C overnight. Finally, we obtained 121.4 mg dark
green powder (yield 91.3%). 1H NMR (300 MHz, CDCl3): 8.61
(2H, s), 8.09 (2H, d), 7.70 (6H, m), 7.49 (10H, m), 7.10 (4H, d),
3.96 (4H, d), 1.84 (2H, m), 1.25-1.50 (64H, broad), 0.90, (12H,
m).
Synthesis of Compound 4
Compound 3 (0.88 g, 1 mmol) and 2-cyanomethylpyridine or 2-
cyanomethylpyridine (0.295 g, 2.5 mmol) or were heated to
reflux in absolute toluene (20 mL) under nitrogen. Phosphoryl
chloride (0.75 ml, 8 mmol) was then added. The reaction was
monitored by thin-layer chromatography. As soon as 3 was used
up, the reaction mixture was cooled down, quenched by water
and alkalize by sodium bicarbonate solution. Water was
separated and extracted with chloroform. The combined organic
layer was dried over anhydrous sodium sulfate. After filtration,
the volatiles were removed under reduced pressure. The crude
product was purified by column chromatography on silica,
eluting with dichloromethane/petroleum ether (from 1:5 to 1:1) to
give Compound 4 as a bluish green solid (173 mg, 16%). 1H
NMR (300 MHz, CDCl3): 13.6 (2H, s), 8.45 (2H, d), 7.65 (8H, m),
Preparation of nanoparticles in the presence of surfactant
as stabilizer
Taking JAGG NPs with 0.4 mM as representative example,
PCBF (200 µL×0.01M THF solution) and amphiphilic copolymer
PCL-b-POEGMA (100 µL×6.67 g/L THF solution) were mixed in
200 µL of THF. This mixture was rapidly injected into 5 mL of
deionized water under ultrasonication. THF was evaporated by
exposure in open air overnight. Similarly, JAGG dispersions with
concentration of 0.24, 0.16, 0.08 and 0.04 mM were prepared by
using 120, 80, 40 and 20µLof 0.01M THF solution of PCBF,
respectively, while the amount of PCL-b-POEGMA was kept
unchanged. HAGG dispersions with various concentrations were
obtained by the similar procedure.
This article is protected by copyright. All rights reserved.

10.1002/chem.201604741
Chemistry - A European Journal
FULL PAPER
acknowledges the financial support from NTU-Northwestern
Institute for Nanomedicine (M4081502.F40). We thank Dr.
Hongwei Duan for access to the fluorometer. We also thank Dr.
Quan Liu and Mr. Xiang Li for the assistance in the use of the
770-nm laser.
Monitoring the aggregation of PCBF in mixture of THF/water
in the absence of surfactant as stabilizer
An aliquot of PCBF (100 μL, 1 mg/mL in THF) was mixed with
different amounts of THF (x mL), followed by a quick addition of
a known volume of water ((3-x) mL) and mixed with gentle
shaking. Through this process, a series of PCBF dispersions
were obtained with different water fractions (fw = 0%, 10%, 20%,
30%, 40%, 50%, 60% by volume).
Keywords: J-aggregates • Near-infrared • Fluorescence •
Pyrrolopyrrole Cyanines • Bioimaging
[1] (a) E. M. Sevick-Muraca, J. P. Houston, M. Gurfinkel, Curr. Opin. Chem.
Biol. 2002, 6, 642-650; (b) J. V. Frangioni, Curr. Opin. Chem. Biol. 2003,
7, 626-634.
[2] A. L. Vahrmeijer, M. Hutteman, J. R. van der Vorst, C. J. van de Velde, J.
V. Frangioni, Nat. Rev. Clin. Oncol. 2013, 10, 507-518.
Cell culture and cytotoxicity study
L929 mouse fibroblast cells were cultured in folate-free
Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10%
o
[3] H. Maeda, Bioconjugate Chem. 2010, 21, 797-802.
fetal bovine serum and 1% penicillin streptomycin at 37 C in a
[4] (a) M. J. Pittet, F. K. Swirski, F. Reynolds, L. Josephson, R. Weissleder,
Nat. Protoc. 2006, 1, 73; (b) S. Huang, S. Liu, K. Wang, C. Yang, Y. Luo,
Y. Zhang, B. Cao, Y. Kang, M. Wang, Nanoscale 2015, 7, 889-895.
[5] (a) H. Cabral, Y. Matsumoto, K. Mizuno, Q. Chen, M. Murakami, M.
Kimura, Y. Terada, M. R. Kano, K. Miyazono, M. Uesaka, N. Nishiyama, K.
Kataoka, Nat. Nanotech. 2011, 6, 815-823; (b) J. Wang, W. Mao, L. L.
Lock, J. Tang, M. Sui, W. Sun, H. Cui, D. Xu, Y. Shen, ACS Nano 2015, 9,
7195-7206.
humidified environment containing 5% CO2. Before experiments,
the cells were precultured until confluence was reached. The
cytotoxicity of JAGG NPs against L929 cancer cells was
evaluated by PrestoBlue (PB) assay. Briefly, L929 cells were
seeded in 96-well plates (Costar, IL, USA) at an intensity of
5×104 cells mL-1. After 12 h incubation, the cells were exposed to
[6] J. D. Luo, Z. L. Xie, J. W. Y. Lam, L. Cheng, H. Y. Chen, C. F. Qiu, H. S.
Kwok, X. W. Zhan, Y. Q. Liu, D. B. Zhu, B. Z. Tang, Chem. Commun.
2001, 1740-1741.
o
a series of doses of JAGG NPs at 37 C. After the designated
[7] (a) S. Huang, K. Wang, S. Wang, Y. Wang, M. Wang, Adv. Mater. Interfac.
2016; (b) K. Wang, Y. Luo, S. Huang, H. Yang, B. Liu, M. Wang, J. Polym.
Sci., Part A: Polym. Chem. 2015, 53, 1032-1042; (c) C. Yang, H. Liu, Y.
Zhang, Z. Xu, X. Wang, B. Cao, M. Wang, Biomacromolecules 2016, 17,
1673-1683.
[8] (a) E. E. Jelley, Nature 1936, 138, 1009-1010; (b) G. Scheibe, Angew.
Chem 1936, 49, 563; (c) E. E. Jelley, Nature 1937, 139, 631-632; (d) G.
Scheibe, Angew. Chem. Int. Ed. Eng. 1937, 50, 212-219.
[9] (a) A. Herz, Adv. Colloid Interface Sci. 1977, 8, 237-298; (b) D. Möbius,
Adv. Mater. 1995, 7, 437-444; (c) F. Würthner, T. E. Kaiser, C. R. Saha‐
Möller, Angew. Chem. Int. Ed. Eng. 2011, 50, 3376-3410; (d) S.
Sengupta, F. Würthner, Acc. Chem. Res. 2013, 46, 2498-2512.
[10] F. Rotermund, R. Weigand, A. Penzkofer, Chem. Phys. 1997, 220, 385-
392.
time intervals, the wells were washed twice with 1×PBS buffer
and 100 µL of freshly prepared PB solution in culture medium
was added into each well. The PB medium solution was carefully
removed after 1 h incubation in the incubator. The absorbance of
PB at 570 nm and 600 nm was monitored by the microplate
reader (Genios Tecan). Cell viability was expressed as the ratio
of the percent PB reduction of the cells incubated with JAGG
NPs suspension to that of the cells incubated with culture
medium only.
[11] (a) J. F. Lovell, C. S. Jin, E. Huynh, H. Jin, C. Kim, J. L. Rubinstein, W. C.
Chan, W. Cao, L. V. Wang, G. Zheng, Nat. Mater. 2011, 10, 324-332; (b)
M. Shakiba, K. K. Ng, E. Huynh, H. Chan, D. M. Charron, J. Chen, N.
Muhanna, F. S. Foster, B. C. Wilson, G. Zheng, Nanoscale 2016, 8,
12618-12625; (c) K. K. Ng, M. Takada, C. C. Jin, G. Zheng, Bioconjugate
Chem. 2015, 26, 345-351.
[12] T. E. Kaiser, H. Wang, V. Stepanenko, F. Würthner, Angew. Chem. Int.
Ed. Eng. 2007, 46, 5541-5544.
[13](a) G. M. Fischer, A. P. Ehlers, A. Zumbusch, E. Daltrozzo, Angew. Chem.
Int. Ed. Eng. 2007, 46, 3750-3753; (b) G. M. Fischer, M. Isomäki‐
In vivo fluorescence imaging
The care and use of laboratory animals were performed
according to the approved protocols of the Institutional Animal
Care and Use Committee (IACUC) at Nanyang Technological
University, Singapore. 50 µL of JAGG solution of PCBF and
HAGG solution of PCBFB (1 mg/ml) were subcutaneously
injected to the left or right side of flanks of 8-week-old NCr nude
mice (Invivos Pte Ltd), respectively. Next, 50 µL of the same
JAGG dispersion of PCBF and HAGG dispersion of PCBFB
were intramuscularly injected to left or right side of rear legs,
respectively. The efficacy of dye as imaging agents was then
examined using an IVIS SpectrumCT in vivo imaging system
(PerkinElmer). The wavelengths of excitation and emission
filters used were 605-640 nm, and 740-800 nm, respectively.
The auto-fluorescence of the skin was removed using a spectral
unmixing software.
Krondahl, I. Göttker‐Schnetmann, E. Daltrozzo, A. Zumbusch, Chem.
Eur. J. 2009, 15, 4857-4864; (c) S. Wiktorowski, C. Rosazza, M. J.
Winterhalder, E. Daltrozzo, A. Zumbusch, Chem. Commun. 2014, 50,
4755-4758; (d) G. M. Fischer, E. Daltrozzo, A. Zumbusch, Angew. Chem.
Int. Ed. Eng. 2011, 50, 1406-1409; (e) G. M. Fischer, C. Jüngst, M.
Isomäki-Krondahl, D. Gauss, H. M. Möller, E. Daltrozzo, A. Zumbusch,
Chem. Commun. 2010, 46, 5289-5291.
[14] K. K. Ng, M. Shakiba, E. Huynh, R. A. Weersink, A. Roxin, B. C. Wilson,
G. Zheng, ACS Nano 2014, 8, 8363-8373.
[15] (a) W. West, S. Pearce, J. Phy. Chem. 1965, 69, 1894-1903; (b) F.
Würthner, Y. Sheng, B. Uwe, Angew. Chem. Int. Ed. Eng. 2003, 42, 3247-
3250; (c) C. Shao, M. Grüne, M. Stolte, F. Würthner, Chem. Eur. J. 2012,
18, 13665-13677; (d) F. Würthner, Acc. Chem. Res. 2016, 49, 868-876.
[16] E. Knapp, Chem. Phys. 1984, 85, 73-82.
[17] S. A. Soper, Q. L. Mattingly, J. Am. Chem. Soc. 1994, 116, 3744-3752.
[18] (a) H. S. Muddana, T. T. Morgan, J. H. Adair, P. J. Butler, Nano Lett.
2009, 9, 1559-1566; (b) E. I. Altınoğlu, T. J. Russin, J. M. Kaiser, B. M.
Barth, P. C. Eklund, M. Kester, J. H. Adair, ACS Nano 2008, 2, 2075-
2084.
Acknowledgements
[19] J. Devoisselle, S. Soulie-Begu, S. Mordon, T. Desmettre, H. Maillols,
Laser. Med. Sci. 1998, 13, 279-282.
[20] (a) W. Cooper, Chem. Phys. Lett. 1970, 7, 73-77; (b) D. Eisele, C. Cone,
E. Bloemsma, S. Vlaming, C. van der Kwaak, R. J. Silbey, M. G. Bawendi,
J. Knoester, J. Rabe, D. V. Bout, Nat. Chem. 2012, 4, 655-662.
MW is grateful to the funding support by a start-up grant
(M4080992.120) of Nanyang Assistant Professorship from
Nanyang Technological University, AcRF Tier 2 (ARC 36/13)
from the Ministry of Education, Singapore. XCJ also
This article is protected by copyright. All rights reserved.

10.1002/chem.201604741
Chemistry - A European Journal
FULL PAPER
[21] (a) X. Q. Li, X. Zhang, S. Ghosh, F. Würthner, Chem. Eur. J. 2008, 14,
8074-8078; (b) V. Huber, M. Katterle, M. Lysetska, F. Würthner, Angew.
Chem. Int. Ed. Eng. 2005, 117, 3208-3212.
[22] C. Yang, Q. T. Trinh, X. Wang, Y. Tang, K. Wang, S. Huang, X. Chen, S.
H. Mushrif, M. Wang, Chem. Commun. 2015, 51, 3375-3378.
[23] (a) S. Huang, P. K. Upputuri, H. Liu, M. Pramanik, M. Wang, J. Mater.
Chem. B 2016, 4, 1696-1703; (b) S. Huang, R. K. Kannadorai, Y. Chen, Q.
Liu, M. Wang, Chem. Commun. 2015, 51, 4223-4226.
This article is protected by copyright. All rights reserved.

10.1002/chem.201604741
Chemistry - A European Journal
FULL PAPER
Entry for the Table of Contents (Please choose one layout)
Layout 1:
FULL PAPER
A novel class of colloidal
Cangjie Yang, Xiaochen Wang,
nanoparticles containing near-infrared
fluorescent J-aggregates of
Mingfeng Wang*, Keming Xu, Chenjie
Xu
pyrrolopyrrole cyanines (PPcys)
exhibit a narrow emission band at 773
nm, a fluorescence quantum yield
comparable to that of indocyanine
green, and significantly enhanced
photostability that is ideal for long-
term bioimaging.
Page No. – Page No.
Robust Colloidal Nanoparticles of
Pyrrolopyrrole Cyanines J-
Aggregates with Bright Near Infrared
Fluorescence in Aqueous Media:
From Spectral Tailoring to
Bioimaging Application
This article is protected by copyright. All rights reserved.