Cation-anion interaction-directed formation of functional vesicles and their biological application for nucleus-specific imaging

Article abstract of DOI:10.1039/c8nj01503a

A strategy for construction of counterion-induced vesicles in aqueous media has been described using imidazolium salts with multiple imidazolium moieties and alkyl carboxylate counteranions. The spontaneous formation of vesicles can be achieved by the simple selection of the counteranion to satisfy the requirement of the packing parameter for vesicle formation. Functional fluorescent vesicles can also be formed by the introduction of an aggregation-induced emission (AIE) fluorophore, tetraphenylethene (TPE), as the core of the imidazolium salt TPEI-C8, which also exhibits highly specific nucleus imaging in living cells. Considering the distinct AIE characteristic of TPEI-C8, the aggregation-induced strong fluorescence emission of TPEI-C8 in the cell nucleus may be the main reason for the specific fluorescence images of the cell nucleus. The strategy for the construction of functional vesicles reported in this study has shown great potential for the construction of other functional vesicles.

Full text of DOI:10.1039/c8nj01503a

View Article Online
View Journal
NJC
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: Q. Kong, W.
Zhuang, G. Li, Q. Jiang and Y. Wang, New J. Chem., 2018, DOI: 10.1039/C8NJ01503A.
Volume 40 Number
1
January 2016 Pages 1–846
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
NJC
New Journal of Chemistry
www.rsc.org/njc
A journal for new directions in chemistry
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
rsc.li/njc

Page 1 of 7
Pl Ne ae swe Jd oo u nr no at l ao df j Cu sh te mm ai sr tgr iyn s
View Article Online
DOI: 10.1039/C8NJ01503A
Journal Name
ARTICLE
Cation–anion interaction directed formation of functional vesicles
and their biological application for nucleus-specific imaging
Received 00th January 20xx,
Accepted 00th January 20xx
Qunshou Kong, Weihua Zhuang, Gaocan Li,* Qing Jiang* and Yunbing Wang
DOI: 10.1039/x0xx00000x
A strategy for construction of counterion-induced vesicles in aqueous media has been described by imidazolium salts with
multiple imidazolium moieties and alkyl carboxylate counteranions. The spontaneous formation of vesicles can be
achieved by the simple selection of the counteranion to satisfy the requirement of packing parameter for vesicle
formation. Functional fluorescent vesicles can also be formed by introduction of an aggregation induced emission (AIE)
fluorophore tetraphenylethene (TPE) as the core of the imidazolium salt TPEI-C8, which aslo exhibits highly specific
nucleus imaging in living cells. Considering the distinct AIE characteristic of TPEI-C8, the aggregation induced strong
fluorescence emission of TPEI-C8 in the cell nucleus may be the main reason for the specific fluorescence images of the
cell nucleus. This strategy for construction of functional vesicles reported in this study has shown great potential for
construction of other functional vesicles.
www.rsc.org/
fluorescence quenching caused by the molecular aggregation
in a high concentration may occur for conventional organic
fluorescent molecules, which is known as the aggregation
Introduction
Vesicles are well-organized assemblies composed by
amphiphilic substances containing a bilayer membranous
structure with an aqueous void volume in the interior. In
recent years, vesicles have been widely used in different fields
from drug/gene delivery and artificial bioreactor to modes of
2
2,23
caused quenching (ACQ) effect.
In the year of 2001, Tang
group firstly reported a new kind of fluorescent material and
strong fluorescence can be observed in aggregated state owing
to the restriction of intramolecular rotation, which is called
2
2,24
1
-5
aggregation-induced emission (AIE) effect.
Since then, a
biological membranes and so on.
However, these
large number of AIE molecules have been developed and
different types of AIE-based fluorophores such as
tetraphenylethene (TPE) have been widely used for bioimaging
amphiphilic materials employed in the previous studies usually
suffer from complex molecular structure and multi-step
synthesis. As an important type of vesicle, catanionic vesicles
have attracted more and more attention since the first report
2
5-33
with great cell imaging ability.
6
In the past years, a great deal of work about vesicle
formation induced by amphiphilic counterions has been
by Kaler et al. , due to their facile formation just by a simple
mixing of cationic and anionic surfactants in aqueous solution.
As a universal method, a great deal of work based on cationic–
3
4-41
studied by Kunz et al.
Recently, a strategy of counterion-
7
-
induced vesicle (CIV) formation via simple imidazolium salts
with EDTA as counterion has been reported by us and vesicles
anionic surfactant mixtures has been done in the past years.
5
Nevertheless, some drawbacks remained such as the
1
4
2
can be formed in the aqueous media. In this work, a simple
and efficient method for the formation of vesicles has been
developed by taking advantage of multi-electric charged
cations for vesicle formation. As we known, only micellar
structures rather than vesicle assemblies can be formed for
simple imidazolium halides or sodium alkyl carboxylate in the
requirement of multiple surfactants and the need of fine
regulation of the ratio of the cationic/anionic surfactant.
Otherwise, the precipitation of the surfactants may occur due
to the lack of electrostatic repulsion. Therefore, the
development of a simple and efficient method to construct
catanionic vesicles is in high demand for exploring other
functional catanionic vesicles.
4
3
aqueous media. Based on this vesicle construction method,
vesicles with controllable size can be formed by selection of
cations with multiple imidazolium moieties and simple
counteranions of alkyl carboxylate anions. In this work, there is
no need to regulate the ratio of cations and anions for the
construction of vesicles and the vesicles can be formed
spontaneously in the aqueous solution. Furthermore,
functional fluorescent vesicles with AIE effect have also been
constructed based on this method by adapting the widely used
TPE structure as the emission moiety of the cation. More
In recent years, fluorescent imaging has emerged as a
powerful tool for monitoring the life activities of the organism
1
6-21
with high spatial and temporal resolution
. However, the
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 2 of 7
View Article Online
DOI: 10.1039/C8NJ01503A
ARTICLE
Journal Name
importantly, these molecular assemblies triggered by the alkyl diameter of about 390 nm were also observed for TPAI-C8 (Fig.
carboxylate counteranions can facilitate the TPE-based cations 1d). The size of these nanoparticles determined from TEM is
penetrating the cell membranes and exhibit highly specific relatively smaller than that from DLS, which was mainly
targeting to the nucleus.
attributed to the difference in the hydrodynamic diameter of
the fully hydrated vesicles in solution and the dry samples in
the collapsed state.
Results and discussion
Synthesis and characterization
Et
Br
Et
Br
O
Et
Et
O
N
N
N
N
n
O
O
n
N
N
N
N
O
N
N
N
N
Anion exchange
+
AgO
n
TPAI-Br
n = 3: TPAI-C6
n = 5: TPAI-C8
O
N
Br
Et
N
O
n
Et
Et
Et
Br
O
Et
Et
O
Br
N
N
N
N
5
O
O
5
N
N
N
N
N
N
N
N
O
Anion exchange
+
AgO
5
O
O
Br
Et
N
N
Br
Et
5
O
N
N
O
5
TPEI-Br
TPEI-C8
Et
Et
Scheme 1 Preparation of functional multiple imidazolium salts
with alkyl carboxylate counterions.
These functional multiple imidazolium salts with different alkyl
carboxylate counterions were obtained by a simple anion
exchange process through the reaction of imidazolium
bromide with silver alkyl carboxylate, followed by Fig. 1 Distribution of the hydrodynamic diameters of vesicles
recrystallization from dichloromethane and diethyl ether to formed by (a) TPAI-C6 (20 mM) and (b) TPAI-C8 (10 mM)
get the target compounds (Scheme 1). The chemical structures determined by DLS. (c) TEM micrographs of vesicles formed by
of these compounds were confirmed by NMR spectroscopy TPAI-C6 (40 mM) in aqueous solution on a carbon-coated
and high resolution mass spectrometry. All the target copper grid without being stained with an aqueous solution of
compounds exhibited good solubility in water and slight 2% phosphotungstic acid. (d) TPAI-C8 (10 mM) in aqueous
yellowish solutions could be formed at room temperature.
Self-assemble behaviors of TPAI with different counteranions
With these functional multiple imidazolium salts in hand, the
solution on a carbon-coated copper grid stained with an
aqueous solution of 2% phosphotungstic acid.
As we know, an important characteristic of vesicles different
self-assemble behaviors of TPAI-C6 and TPAI-C8 with relatively from micelles is that vesicles have a hydrophilic cavity, in
simple cationic structures were firstly determined to which water-soluble compounds can be trapped. To gain
investigate the feasibility of our vesicle construction method further confirmation of the vesicle formation in the aqueous
and different measurements such as dynamic light scattering solution, the glucose leakage assay, entrapment experiment of
4
4
(
DLS), transmission electron microscope (TEM) and the glucose water-soluble glucose, was then conducted. The glucose can
leakage assay have been conducted in this study. The critical be trapped into the vesicles in the process of the vesicle
aggregation concentrations (CACs) of TPAI-C6 and TPAI-C8 formation, while the glucose leakage takes place from the
were determined and CAC values about 10 mM and 7 mM disrupted vesicles by the addition of detergent such as Triton
were obtained for TPAI-C6 and TPAI-C8, respectively (Fig. S1). X-100. Subsequently, a series of fast enzyme-catalyzed
To our delight, molecular assemblies of vesicles were formed reactions including phosphorylation and oxidization are
spontaneously for both TPAI-C6 and TPAI-C8 in their aqueous involved for the escaped glucose and the nicotinamide
solutions. As shown in Fig. 1a, DLS measurements indicated adenine dinudeotide phosphate (NADP) is also reduced
that nanoparticles with
a hydrodynamic diameter of quantitatively to the corresponding NADPH, which exhibits a
approximately 237 nm could be observed for TPAI-C6. When a typical absorbance at 340 nm and correlates directly with the
counteranion with a longer alkyl chain was employed, amount of glucose efflux. Given this process is very fast, the
nanoparticles with a larger size of approximately 478 nm could release of entrapped glucose can be monitored by determine
be formed for TPAI-C8 (Fig. 1b).
the increase of the absorbance intensity at 340 nm.
Subsequently, vesicle morphological structures were directly
The vesicles of TPAI-C8 were selected as a mode to carry out
observed through the TEM measurement. As illustrated in Fig. the entrapping experiment. The glucose-loaded vesicle
c, the TEM image acquired from the TPAI-C6 showed the solution with a total concentration of 300.0 mM for glucose
1
clear formation of the spherical aggregates with a diameter of was prepared by adding glucose directly into the tested
about 180 nm. Similar to TPAI-C6, spherical aggregates with a solution of TPAI-C8 at room temperature. After this mixture
2
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 7
Pl Ne ae swe Jd oo u nr no at l ao df j Cu sh te mm ai sr tgr iyn s
View Article Online
DOI: 10.1039/C8NJ01503A
Journal Name
ARTICLE
was vortexed for 3h at room temperature, the free glucose TPE groups were restricted and strong fluorescent emission
was removed through a gel column of sephadex G-50, could be observed due to the AIE effect (Fig. S2).
hexokinase, glucose-6-phosphhate dehydrogenase, NADP, ADP,
and ATP were added into the vesicle solution. As shown in Fig.
2a, a weak absorbance at 340 nm could be observed before
the addition of Triton X-100. As a contrast, the vesicle
structure could be destroyed by the addition of Triton X-100
and the rapid release of encapsulated glucose resulted into a
sharp increase of the absorbance in the 340 nm (Fig. 2a). This
observation of the efficient entrapment of the great amount of
glucose demonstrated clearly the spontaneous formation of
vesicles for TPAI-C8 in its aqueous solution. Furthermore, a
long-term stability of the entrapment of glucose could be
observed and the total release of glucose was less than 45%
even after 7 day (Fig. 2b), which made this vesicle system
would be a promising candidate for high-efficiency and long-
term encapsulation of hydrophilic contents.
Fig. 3 (a) Distribution of the hydrodynamic diameters of CIVs
formed by TPEI-C8 in aqueous solution (2.0 mM); (b) TEM
micrograph of CIVs formed by TPEI-C8 in aqueous solution (2.0
mM) on a carbon-coated copper grid stained with an aqueous
solution of 2% phosphotungstic acid.
Nucleus-specific fluorescent imaging
Fig. 2 (a) Comparison of the absorbance spectra of glucose-
loaded vesicles before and after adding Triton X-100 (5% v/v).
(
b) Percent leakage of glucose from CIVs over time. The
vesicles were lysed at the 7th day upon addition of Triton X-
00.
1
Fig. 4 Confocal images of Hela cells cultured with TPEI-C8 and
TPEI-Br at the concentrations of 50 μM: (a) TPEI-C8 under
irradiation at 405 nm (λem = 420-470 nm), (b) TPEI-Br under
irradiation at 405nm (λem = 420-470 nm). Scale bar: 10 μm.
Construction of functional fluorescent vesicles with AIE effect
Our current method for construction of vesicles is simple, yet
capable of developing other functional fluorescence vesicles.
Tetraphenylethene (TPE), as a classic AIE molecule, exhibits
intense fluorescent emission in the aggregate state. As shown
in Scheme 1, the functional imidazolium salt TPEI-C8 with
octanoate anion as counteranion and TPE as the molecular
core has been developed with a fluorescence quantum yield of
With this fluorescent AIE imidazolium salt of TPEI-C8 in hand,
biological application of this functional imidazolium salt for
bioimaging was investigated. The Hela cells were cultured and
incubated with 50 μM TPEI-C8 and 50 μM TPEI-Br for 4h. Then,
the cells were washed with fresh PBS for 3 times. Afterwards,
the process of the cell imaging was observed by the confocal
laser microscopy. To our delight, this functional imidazolium
salt TPEI-C8 could penetrate the cell membranes and label
Hela cells in the location of the nucleus with a bright blue
emission (Fig. 4a). However, only a very weak blue emission
could be observed for imidazolium salt TPEI-Br with bromide
as counteranion during the cell imaging process (Fig. 4b),
which suggested that the octanoate anion and corresponding
molecular self-assemble structures might play key roles in the
penetration of the cell membranes for these imidazolium salts.
Subsequently, the specific localization in nucleus was
confirmed by the co-staining experiments of Hela cells with
TPEI-C8 and red-fluorescent tracker Reddot1 (Fig. 5 and Fig.
S3). As shown in Fig. 5c, the blue fluorescence images of TPEI-
C8 and the red fluorescence images of Reddot1 collected from
different channels overlapped very well. After calculation using
0.25 and the fluorescence lifetime of 9.76 ns in its aggregate
state. The critical aggregation concentration (CAC) of TPEI-C8
was also determined and CAC value about 0.45 mM was
obtained TPEI-C8 (Fig. S1). As expected, functional fluorescent
vesicles could be formed spontaneously for TPEI-C8 in its
aqueous solution, which was determined by DLS and TEM
measurements. As shown in Fig. 3, a hydrodynamic diameter
of approximately 369 nm and a mean diameter of around 220
nm could be obtained from the analysis of DLS and TEM,
respectively. Similar to TPAI-C6 and TPAI-C8, the relatively
smaller vesicle size determined from TEM for TPEI-C8 was
mainly due to the difference states of these vesicles of the
fully hydrated state and the collapsed state. The AIE active
fluorescence emission of this functional imidazolium salt TPEI-
C8 was investigated subsequently. Once the molecular self-
assemble of TPEI-C8 occurred by adding the poor solution, the
Image-Pro Plus software, Pearson's coefficient (R = 0.83) and
r
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 4 of 7
View Article Online
DOI: 10.1039/C8NJ01503A
ARTICLE
Journal Name
1 2
Manders's coefficients (m = 1.00 and m = 1.00), the by the counteranion to satisfy the requirement of packing
parameters quantifying the staining region overlap for the blue parameter for vesicle formation. This counterion-induced
and red fluorescence images, were obtained respectively, vesicle formation strategy is also capable of developing
which clearly demonstrated the specific targeting to the functional fluorescent vesicles by introduction of an AIEgen
4
5,46
nucleus of living cells similar to the reported literatures.
tetraphenylethene (TPE) as the core of the imidazolium salt
Although the mechanism is not clear, we assumed that TPEI-C8. This functional imidazolium salt TPEI-C8 exhibits
electrophoretic force and suitable lipophilicity triggered by the highly specific nucleus imaging in living cells and the
multiple imidazolium moieties and octanoate anion might be aggregation induced strong fluorescence emission of TPEI-C8
the major driving force for TPEI-C8 to penetrate the cell in the cell nucleus may be the main reason for the specific
membrane and precisely target to the nuclelus.
fluorescence images of cell nucleus. The cation–anion
interaction directed functional vesicle formation strategy
reported herein would provide new guidelines for the
development of other novel functional vesicles.
Experimental section
General remarks
1
NMR spectra were obtained on a Bruker AV II-400. The H
Fig. 5 Co-staining of Hela cells with TPEI-C8 and Reddot1: (a)
TPEI-C8 (50 μM) for 4 h (λex = 405 nm, λem = 420−470 nm); (b)
Reddot1 for 5 min (λex = 633 nm, λem = 650−670 nm); (c) panels
NMR spectroscopy chemical shifts were measured relative to
D
2
3
O-d
2
as the internal reference (D
C NMR spectroscopy chemical shifts were given using CDCl
and CDOD-d as the internal standard (CDCl : δ = 77.16 ppm
Cytotoxicity is another important factor for the cell imaging, and CDOD-d : δ = 49.86 ppm). Glucose leakage assay was
2
O-d
2
: δ = 4.79 ppm). The
1
3
-d
(a) and (b) merged; and (d) bright-field image. Scale bar: 5 μm.
4
3
4
and the cytotoxicity of TPEI-C8 was evaluated using MTT monitored on a Cary 50 Bio UV−vis spectrometer. TEM studies
assays. As shown in the Fig. 6, no obvious toxicity could be were carried out using a HITACHI H-600 instrument, operating
observed for the cultured Hela cells with TPEI-C8 at different at 75 kV. DLS experiments were recorded using a Malvern Zeta
concentration, and up to 85% of the cultured Hela cells could sizer Nano ZS particle analyser instrument. Cell experiment
survive at a concentration even up to 80
µ
M after incubation was observed under confocal laser scanning microscopy (CLSM,
for 24 h, showing better biocompatiability comparing the ZEISS, LSM 780) and flow cytometry (BectoDickinson, USA).
2
6
reported TPE based Luminogen.
Materials and reagents
Chemicals: Unless otherwise noted, all reagents were obtained
from commercial suppliers and used without further
purification. TPAI-Br and TPEI-Br were synthesized according
4
7,48
to published procedures.
Typical Preparation of Counterion-Induced Vesicles: TPAI-
C8 (845.0 mg, 0.1 mmol) was added into 10.0 mL of deionized
water at room temperature. The resulting solution was then
left to stand and the vesicles formed spontaneously within
minutes.
Glucose-leakage assay: Glucose-entrapping vesicles were
prepared by adding TPAI-C8 into the 300.0 mM of glucose in
trisbuffer (pH = 7.5). The mixture was vortexed for 4 h, and
then let them stand for 1 h. The prepared vesicles solution was
passed through the column of sephadex G-50 by using
trisbuffer (pH = 7.5) as the eluent to eliminate the free glucose.
The concentration of TPAI-C8 in the stock solution was 40 mM.
The glucose-entrapping vesicles were collected. 10 μL vesicle
stock solution, 2000 μL tris buffer (pH = 7.5), 500 μL enzyme
solution (10 units per mL of hexokinase/glucose-6-phosphoate
dehydrogenas and 2.0 mM ATP dissolved in the above Tris
buffer) and 500 μL NADP solution (1.0 mM dissolved in the
Fig. 6 Growth of Hela cells in the presence of different
concentrations of TPEI-C8 after 24 h incubation.
Conclusions
In summary, a strategy for construction of counterion-induced above Tris buffer) were added into the cuvette. The
vesicles in aqueous media by imidazolium salts with multiple absorbance of NADPH at 340 nm was measured. Subsequently,
imidazolium moieties and alkyl carboxylate counteranions has 100 µL of Triton X-100 (5% v/v) was added into the cuvette and
been developed. The formation of vesicles is mainly triggered the absorbance at 340 nm was measured. The absorbance of
4
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 7
Pl Ne ae swe Jdoo u nr no at l ao df j Cu sh te mm ai sr tgr iyn s
View Article Online
DOI: 10.1039/C8NJ01503A
Journal Name
ARTICLE
NADPH at 340 nm was monitored. Subsequently, the vesicles
Synthesis of silver hexanoate: Following the general
were lysed by the addition of 100 µL of Triton X-100 (5% v/v) procedure, sodium hexanoate (2.76 g, 20.0 mmol) and AgNO
and the absorbance at 340 nm (Amax) was used to calculate the (3.44 g, 20.2 mmol) were used to obtain the desired product
percent leakage [= (A – A )/(Amax – A ) × 100]. A and A are (4.07 g, 91.4%).
the initial and intermediate absorbance, respectively.
3
t
0
0
0
t
Synthesis of silver octanoate: Following the general
The measurement of the critical vesicle concentration procedure, sodium octanoate (3.32 g, 20.0 mmol) and AgNO
CVC): The measurement of the critical vesicle concentration (3.44 g, 20.2 mmol) were used to obtain the desired product
3
(
4
9
(CVC) was measured according to the reported literature.
(4.69 g, 93.44%).
The measurement of CVC for TPAI-Cn: The CVC of TPAI-Cn
General procedure for the synthesis of the TPAI-C6
，TPAI-
was measured by the fluorescence emission of TPA buried in C8, TPEI-C8: The silver carboxylate (0.1528 g, 0.9 mmol) was
the vesicles. Briefly, the stock solution of TPAI-Cn was diluted added to the aqueous solution of TPAI-Br or TPEI-Br (0.31
to various desired concentrations (from 1 mM to 100 mM). mmol) and the white precipitate immediately changed into a
The fluorescence spectra of all solutions were measured with yellow brown precipitate. After being stirred at room
the excitation wavelength at 380 nm, and the intensity values temperature under dark for 24 h, the reaction mixture was
of all solutions at 450 nm were recorded to determine the CVC. filtered. The filtrate was stirred with 50.0 mg active carbon for
2
h to remove the trace silver precipitate and then
The measurement of CVC for TPEI-C8: The CVC of TPEI-C8
was measured by the fluorescence emission of TPE buried in
the vesicles. Briefly, the stock solution of TPEI-C8 was diluted
to various desired concentrations (from 10 μM to 1000 μM).
The fluorescence spectra of all solutions were measured with
the excitation wavelength at 380 nm, and the intensity values
of all solutions at 480 nm were recorded to determine the CVC.
concentrated under vacuum. The crude product was dissolved
into dichloromethane, and was filtered to remove the
excessive TPAI-Br or TPEI-Br. The filtrate was concentrated
under vacuum, then the crude product was recrystallized with
dichloromethane and diethyl ether to get light yellow solid and
dried under vacuum to get the desired product.
Synthesis of the TPAI-C6: Following the general procedure,
TPAI-Br (0.2358 g, 0.31 mmol), silver hexanoate (0.1528 g, 0.9
Cell culture: Hela cells were incubated in Dublecco’s
Minimum Eagle’s Medium (DMEM, Hyclone) supplemented
with 10% (v/v) fetal bovine serum (FBS, Gibco), 1 units/mL of
penicillin (Sigma-Aldrich), and 0.1 mg/mL of streptomycin
mmol) and de-ionized water (50.0 mL) were used to obtain the
1
desired product (0.1983 g, 75%). H NMR (D
2
O-d
2
, 400 MHz): δ
=
0.77 (t, J = 7.2 Hz, 9H), 1.17 (s, 12H), 1.45 (m, 6H), 1.47 (t, J =
.2 Hz, 9H), 2.03 (t, J = 7.6 Hz, 6H), 4.23 (m, 6H),7.31 (d, J = 9.2
(
Sigma-Aldrich) at 37 °C in a humidified atmosphere containing
% CO
Confocal imaging experiments: For co-staining experiments,
7
5
2
.
Hz, 6H), 7.31(d, J = 9.2 Hz, 6H), 7.49(d, J = 8.8 Hz, 6H), 7.60 (d, J
13
=
3
2.4 Hz, 3H), 7.75 (d, J = 2.0 Hz, 3H). C NMR (CDCl -d, 100
Hela cells was incubated with TPEI-C8 (50 μM) in Dublecco’s
Minimum Eagle’s Medium (DMEM, Hyclone) for 4 h at 37 °C.
After incubation, Hela cells were washed twice with PBS. Then,
MHz): δ = 15.5, 22.4, 26.4, 32.0, 38.5, 45.5, 120.6, 122.1, 123.2,
3+
+
25.4, 130.6, 147.1 ppm. HRMS (ESI ): calcd for C33
1
H
36
N
7
[M-
+
X] 530.3016, found 530.3027.
(500 μL) Reddot1 was added to incubate for 5 min at 37 °C.
Synthesis of the TPAI-C8: Following the general procedure,
TPAI-Br (0.2358 g, 0.31 mmol), silver octanoate (0.2259 g, 0.9
Finally, Hela cells were washed twice with PBS before imaging.
Cytotoxicity assays: The cytotoxicity study of TPEI-C8 was
examined by MTT. Hela cells were seeded in the 96-well plate
and treated for 24 h with different concentrations of TPEI-C8
solutions (5, 10, 20, 40 and 80 μM). After the treatment, 20 μL
PBS containing 5 mg/mL MTT was added to each well and the
plate was incubated at 37 °C for 4 h. The purple formazan
crystals were dissolved in 200 μL of DMSO, and the absorbance
of each sample was measured on the multi-detection
microplate reader (Bio-Rad 550) at 490 nm. The cell viability
was calculated by the following formula: viability (%) = (mean
absorbance value in treated wells/ mean absorbance value in
control wells) × 100.
mmol) and de-ionized water (50.0 mL) were used to obtain the
1
desired product (0.2413 g, 83%). H NMR (D
2
O-d
2
, 400 MHz): δ
=
0.70 (t, J =6.8 Hz, 9H), 1.14 (s, 24H), 1.38 (m, 6H), 1.46 (t, J =
.2 Hz, 9H), 2.00 (t, J = 7.4 Hz, 6H), 4.22 (m, 6H), 7.31 (d, J = 8.8
7
Hz, 6H), 7.50 (d, J = 8.8 Hz, 6H), 7.61 (d, J = 2.0 Hz, 3H), 7.76 (d,
13
J = 2.0 Hz, 3H). C NMR (CDCl
3
-d, 100 MHz): δ = 13.3, 14.8,
1
1
C
6.9, 22.2, 26.1, 29.1, 37.4, 45.5, 57.5, 121.4, 122.6, 123.5,
+
25.5, 130.4, 147.8, 183.3 ppm. HRMS (ESI ): calcd for
3+
+
[M-X] 530.3016, found 530.3049.
33
H
36
N
7
Synthesis of the TPEI-C8: Following the general procedure,
TPEI-Br (0.1781 g, 0.17 mmol), silver octanoate (0.1096 g, 0.66
mmol) and de-ionized water (50.0 mL) were used to obtain the
1
desired product (0.2145, 79%). H NMR (D
General procedure for the synthesis of the silver alkyl
carboxylate: Sodium carboxylate was dissolved in de-ionized
water. To the sodium carboxylate solution (0.2 M, 100 mL), a
2
O-d
2
, 400 MHz): δ =
0
7
8
.74 (t, J = 7.0 Hz, 12H), 1.18(s, 16H), 1.4 (m, 8H), 1.47 (t, J =
.2 Hz, 12H), 2.03 (t, J = 7.6 Hz, 8H), 4.23 (m, 8H), 7.31 (d, J =
3
solution of AgNO (0.405 M, 50 mL) was added dropwise under
.8 Hz, 8H), 7.49 (d, J = 8.8 Hz, 8H), 7.60 (d, J = 2.4 Hz, 4H), 7.75
13
vigorous stirring. The precipitate compound was filtered,
washed with de-ionized water and dried under vacuum. The
desired product silver carboxylate was obtained as a white
powder.
4
(d, J = 2.0 Hz, 4H). C NMR (CDOD-d , 100 MHz): δ = 13.0, 14.1,
2
1
2.4, 26.2, 28.9, 29.3, 31.6, 45.2, 121.2, 121.8, 123.2, 132.6,
3+
+
34.1, 143.5, 181.8 ppm. HRMS (ESI ): calcd for C33
H
36
N
7
[M-
+
X] 712.3890, found 712.3885.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 6 of 7
View Article Online
DOI: 10.1039/C8NJ01503A
ARTICLE
Journal Name
2
4. J. Luo, Z. Xie, J. W. Y. Lam, L. Cheng, H. Chen, C. Qiu, H. S.
Acknowledgements
Kwok, X. Zhan, Y. Liu D. Zhu and B. Z. Tang, Chem. Commun.,
This work was financially supported by grants from the
National NSF of China (No. 21502129), China Postdoctoral
Science Foundation Funded Project (2017M612956), and the
National 111 Project of Introducing Talents of Discipline to
Universities (No. B16033).
2
2
001, 1740-1741.
5. J. Huang, Y. Yu, L. Wang, X. Wang, Z. Gu and S. Zhang, ACS
Appl. Mater. Interfaces, 2017,
6. C. W. T. Leung, Y. Hong, S. Chen, E. Zhao, J. W. Y. Lam and
B. Z. Tang, J. Am. Chem. Soc., 2013, 135, 62-65.
7. J. Li, K. Liu, Y. Han, B. Z. Tang, J. Huang and Y. Yan, ACS
Appl. Mater. Interfaces, 2016, , 27987-27995.
8. K. K. Ng and G. Zheng, Chem. Rev., 2015, 115, 11012-
9, 29030-29037.
2
2
8
Notes and references
2
1
2
3
3
3
1
. C. G. Palivan, R. Goers, A. Najer, X. Zhang, A. Car and W. Meier,
Chem. Soc. Rev., 2016, 45, 377-411.
. D. L. Perrier, L. Rems and P. E. Boukany, Adv. Colloid.
Interfac., 2017, 249, 248-271.
. J. Song, P. Huang, H. Duan and X. Chen, Accounts. Chem.
Res., 2015, 48, 2506-2515.
. P. Vader, E. A. Mol, G. Pasterkamp and R. M. Schiffelers,
Adv. Drug. Deliv. Rev., 2016, 106, 148-156.
1042.
9. G. Fan and D. Yan, Sci. Rep., 2014, 4, 4933.
2
0. G. Fan and D. Yan, Adv. Opt. Mater., 2016,
1. D. Yan and D. G. Evans, Mater. Horiz., 2014,
2. D. Yan, H. Yang, Q. Meng, H. Lin and M. Wei, Adv. Funct.
4
, 2139-2147.
1
, 46-57.
3
Mater., 2014, 24, 587-594.
4
3
2
3
3. S. Li, Y. Lin and D. Yan, J. Mater. Chem. C, 2016, 4, 2527-
534.
5. P. Xing and Y. Zhao, Adv. Mater., 2016, 28, 7304-7339.
4. J. J. Garcia-Celma, L. Hatahet, W. Kunz, and K. Fendler,
6
. E. W. Kaler, A. K. Murthy, B. E. Rodriguez and J. A. N.
Langmuir, 2007, 23, 10074-10080.
5. S. Dengler, G. J. T. Tiddy, L. Zahnweh and W. Kunz, Colloid.
Surface. A, 2014, 457, 414-418.
6. A. Renoncourt, N. Vlachy, P. Bauduin, M. Drechsler, D.
Zasadzinski, Science, 1989, 245, 1371-1374.
. Y. Chang, K. Yang, P. Wei, S. Huang, Y. Pei, W. Zhao and Z.
Pei, Angew. Chem. Int. Ed., 2014, 53, 13126-13130.
3
7
3
8
2
9
1
1
. Y. Chen, F. Qiao, Y. Fan, Y. Han and Y. Wang, Langmuir,
Touraud, J.-M. Verbavatz, M. Dubois, W. Kunz, and B. W.
017, 33, 2760-2769.
Ninham, Langmuir, 2007, 23, 2376-2381.
. M. Gao, X. Zhu, L. Wu and L. Qiu, Biomacromolecules, 2016,
3
7. R. Klein, M. Kellermeier, D. Touraud, E. Müller and W. Kunz,
7, 2199-2209.
J. Colloid Interf. Sci., 2013, 392, 274-280.
0. C. R. Lόpez-Barrόn, D. Li, L. DeRita, M. G. Basavaraj and N.
3
4
3
8. R. Klein, D. Touraud and W. Kunz, Green Chem., 2008, 10,
J. Wagner, J. Am.Chem. Soc., 2012, 134, 20728-20732.
1. M. Dubois, Th. Gulik-Krzywicki, J.-C. Dedieu, C. Vautrin, S.
Désert, E. Perez and T. Zemb, Nature, 2001, 411, 672-675.
2. Th. Zemb, M. Dubois, B. Demé and Th. Gulik-Krzywicki,
Science, 1999, 283, 816-819.
3. S. A. Wheaten, F. D. O. Ablan, B. L. Spaller, J. M. Trieu and
P. F. Almeida, J. Am. Chem. Soc., 2013, 135, 16517-16525.
4. Y. Yang, L. Liu, X. Huang, X. Tan, T. Luo and W. Li, Soft
Matter, 2015, 11, 8848-8855.
33–435.
1
9. N. Vlachy, C. Merle, D. Touraud, J. Schmidt, Y. Talmon, J.
Heilmann and W. Kunz, Langmuir, 2008, 24, 9983-9988.
0. D. Rengstl, B. Kraus, M. V. Vorst, G. D. Elliott and W. Kunz,
Colloid. Surface. B, 2014, 123, 575-581.
1. N. Vlachy, M. Drechsler, J.-M. Verbavatz, D. Touraud and W.
Kunz, J. Colloid Interf. Sci., 2008, 319, 542-548.
2. G. Li, S. Zhang, N. Wu, Y. Cheng and J. You, Adv. Funct.
Mater., 2014, 24, 6204-6209.
1
4
1
4
1
4
1
2
1
5. C. Li, S. Zhang, J. Pang, Y. Wu and Z. Gu, Adv. Funct. Mater,
4
4
3. A. Shioi and T. A. Hatton, Langmuir, 2002, 18, 7341-7348.
015, 25, 3764-3774.
4. S. C. Kinsky, J. A. Haxby, D. A. Zopf, C. R. Alving and C. B.
6. W. W.-W. Hsiao, Y. Y. Hui, P.-C. Tsai and H.-C. Chang, Acc.
Kinsky, Biochemistry, 1969,
5. Y. Cheng, G. Li, Y. Liu, Y. Shi, G. Gao, D. Wu, J. Lan and J.
You, J. Am. Chem. Soc., 2016, 138, 4730-4738.
6. T. Peng, N.-K. Wong, X. Chen, Y.-K. Chan, D. H.-H. Ho, Z.
8, 4149-4158.
Chem. Res., 2016, 49, 400-407.
7. Y. Tang, D. Lee, J. Wang, G. Li, J. Yu, W. Lin and J. Yoon,
Chem. Soc. Rev., 2015, 44, 5003-5015.
8. W. Xu, Z. Zeng, J.-H. Jiang, Y.-T. Chang and L. Yuan, Angew.
Chem. Int. Ed., 2016, 55, 13658-13699.
4
1
4
1
Sun, J. J. Hu, J. Shen, H. EI-Nezami and D. Yang, J. Am. Chem.
Soc., 2014, 136, 11728-11734.
1
2
2
4
2
9. H. Zhu, J. Fan, J. Du and X. Peng, Acc. Chem. Res., 2016, 49,
4
7. N. Sinha, T. T. Y. Tan, E. Peris and F. E. Hahn, Angew. Chem.
Int. Ed., 2017, 56, 7393-7397.
8. N. Sinha, L. Stegemann, T. T. Y. Tan, N. L. Doltsinis, C. A.
Strassert and F. E. Hahn, Angew. Chem. Int. Ed., 2017, 56
115-2126.
0. H.-S. Peng and D. T. Chiu, Chem. Soc. Rev., 2015, 44, 4699-
722.
4
,
1. Y. Xia, R. Zhang, Z. Wang, J. Tian and X. Chen, Chem. Soc.
2
4
785-2789.
9. J. Huang, Y. Yu, L. Wang, X. Wang, Z. Gu and S. Zhang, ACS
, 29030-29037.
Rev., 2017, 46, 2824-2843.
2. M. Huang, R. Yu, K. Xu, S. Ye, S. Kuang, X. Zhu and Y. Wan,
Chem. Sci., 2016, , 4485-4491.
3. J. Mei, Y. Hong, J. W. Y. Lam, A. Qin, Y. Tang and B. Z. Tang,
Adv. Mater., 2014, 26, 5429-5479.
2
Appl. Mater. Interfaces, 2017,
9
7
2
6
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 7 of 7
New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ01503A
Graphic Abstract
----------------------------------------------------------------------------
-
Cation–anion interaction directed formation of functional vesicles and their biological
application for nucleus-specific imaging
Qunshou Kong, Weihua Zhuang, Gaocan Li,* Qing Jiang* and Yunbing Wang
A strategy for construction of counterion-induced vesicles in aqueous media has been described by
imidazolium salts with multiple imidazolium moieties and alkyl carboxylate counteranions.
Furthermore, the imidazolium salt with an aggregation induced emission (AIE) fluorophore
exhibits highly specific nucleus imaging in living cells.