1. science
2. physics
3. nanotechnology

Nano Research
Nano Res
DOI
10.1007/s12274-014-0597-6
Colourimetric redox-polyaniline nanoindicator for in
situ vesicular trafficking of intracellular transport
Eun Bi Choi1†, Jihye Choi1†, Seo Ryung Bae1, Hyun-Ouk Kim1, Eunji Jang1, Byunghoon Kang1,
Myeong-Hoon Kim1, Byeongyoon Kim 3, Jin-Suck Suh2, Kwangyeol Lee3, Yong-Min Huh2*() and Seungjoo
Haam1*().
Nano Res., Just Accepted Manuscript • DOI: 10.1007/s12274-014-0597-6
http://www.thenanoresearch.com on October 8, 2014
© Tsinghua University Press 2014
Just Accepted
This is a “Just Accepted” manuscript, which has been examined by the peer-review process and has been
accepted for publication. A “Just Accepted” manuscript is published online shortly after its acceptance,
which is prior to technical editing and formatting and author proofing. Tsinghua University Press (TUP)
provides “Just Accepted” as an optional and free service which allows authors to make their results available
to the research community as soon as possible after acceptance. After a manuscript has been technically
edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP
article. Please note that technical editing may introduce minor changes to the manuscript text and/or
graphics which may affect the content, and all legal disclaimers that apply to the journal pertain. In no event
shall TUP be held responsible for errors or consequences arising from the use of any information contained
in these “Just Accepted” manuscripts. To cite this manuscript please use its Digital Object Identifier (DOI®),
which is identical for all formats of publication.
1
Colourimetric redox-polyaniline nanoindicator for in
situ vesicular trafficking of intracellular transport
Eun Bi Choi1†, Jihye Choi1†, Seo Ryung Bae1, Hyun-Ouk
Kim1, Eunji Jang1, Byunghoon Kang1, Myeong-Hoon
Kim1, Byeongyoon Kim3, Jin-Suck Suh2, Kwangyeol
Lee3, Yong-Min Huh2* and Seungjoo Haam1*
1Department
of Chemical and Biomolecular Engineering,
College of Engineering,
Yonsei
University,
Seoul
120-749, Republic of Korea
2Department
of Radiology, College of Medicine, Yonsei
University,
Seoul
120-752,
Republic
of
3Department
of Chemistry, Korea University, Seoul
136-701, Republic of Korea
†These
authors contributed equally to this work.
Korea
Simple colourimetric redox-polyaniline nanoindicator; Silica-coated
polyaniline nanoparticles with adsorbed fluorophores Cy3 and Cy7
(FPSNICy3 and FPSNICy7) were fabricated as proton-sensitive
nanoindicators.
Nano Research
DOI (automatically inserted by the publisher)
Research Article
Colourimetric redox-polyaniline nanoindicator for in situ vesicular
trafficking of intracellular transport
Eun Bi Choi1†, Jihye Choi1†, Seo Ryung Bae1, Hyun-Ouk Kim1, Eunji Jang1, Byunghoon Kang1, Myeong-Hoon Kim1,
Byeongyoon Kim3, Jin-Suck Suh2, Kwangyeol Lee3, Yong-Min Huh2*() and Seungjoo Haam1*( ).
.
Received: day month year
ABSTRACT
Revised: day month year
Vesicular pH modulates the function of many organelles and plays a pivotal
role in cell metabolism processes such as proliferation and apoptosis. Here, we
introduce a simple colourimetric redox-polyaniline nanoindicator, which can
detect and quantify a broader biogenic pH range with superior sensitivity
compared to pre-established trafficking agents employing one-dimensional
turn-on of the FRET signal. We fabricated polyaniline-based nanoprobes, which
exhibited convertible transition states according to the proton levels, as an in
situ indicator of vesicular transport pH. Silica-coated Fe3O4–MnO heterometal
nanoparticles were synthesised and utilised as a metal oxidant to polymerise
the aniline monomer. Finally, silica-coated polyaniline nanoparticles with
adsorbed fluorophores Cy3 and Cy7 (FPSNICy3 and FPSNICy7) were fabricated as
proton-sensitive nanoindicators. Owing to the selective quenching induced by
the local pH variations of vesicular transport, FPSNICy3 and FPSNICy7
demonstrated excellent intracellular trafficking and provided sensitive optical
indication of minute proton levels.
Accepted: day month year
(automatically inserted by
the publisher)
© Tsinghua University Press
and Springer-Verlag Berlin
Heidelberg 2014
KEYWORDS
Redox, pH, intracellular
compartments,
quencher,
organic
conducting
polymer, nanoindicator
Address correspondence to Yong-Min Huh, [email protected]; Seungjoo Haam, [email protected]
2
Nano Res.
1. Introduction
Real-time tunable ratiometric fluorescent proton
on the same nanoparticle and continuously monitor
organic
measure
concentrations of target species in a simple and
optical-fluorescence-based ratiometric signals in
reliable manner.[11] Optically addressed biosensors
living cells have attracted much interest in the quest
of
to understand diverse cellular processes.[1] The
resonance-energy-transfer
scope offered by trafficking vesicular transport of
transduction.[12] The challenge in the development
living cells is revealing the science behind various
of any fluorescent sensor is the induced signal
cellular processes and allowing researchers to better
change, which converts the recognition event to an
understand
optical signal. Owing to their operational simplicity
sensors
that
can
physiological
efficiently
and
pathological
this
type
fluorescence
(FRET)
signal
part in the formation and maintenance of various
proton-permeable
compartments as well as in the communication
magnetic
between cells and the environment.[3] Thus, for the
spectroscopy,[2,13,14,15,16] FRET, a mechanism
comprehensive understanding of native cellular
describing
processes, the vesicle should be considered essential
distance-dependent energy transfer between two
to maintaining homeostasis of every vesicular
chromophores, has been mostly used in various
transport
sensing systems for proteins, peptides, nucleic acids,
intracellular
functions
or
of
(NMR)
the
nuclear
and
absorbance
non-radiative
and
disturbed.[4,5,6] As cellular dysfunction is often
dual fluorophore-labeled nanoprobes using FRET
associated with an abnormal proton level in
exhibit proton-level detection ranges that are too
organelles, the vesicular proton plays a particularly
limited, with a maximum range of two pH units, to
crucial role in cell biology by staying generally
perform
between 6.8 and 7.4 in the cytosol and between 4.5
endolysosomal pathway.[19,20] The actual pH
and 6.0 in the cell’s acidic organelles since proteins
would fall outside the range of the latest generation
depend on the proton level to maintain their
of developed nanosensors since the pH differs by
structures and functions.[7,8] Therefore, extensive
more than two pH units between the early
research efforts have been directed toward the
endosomes and lysosomes. Therefore, a nanoprobe
development of simple nanoprobes which can
employing the quenching effect is a more powerful
provide real-time time-resolved pH information
tool to obtaining useful information in cell biology.
rather than simple fragmental changes because the
Among nanoquenchers, Au nanoparticles have
cellular redox environment is not static and
been widely utilised because of their successful
fluctuates through different stages of the cell
confinement of the electric field near a metal surface
cycle.[9] In addition, there is significant interest in
and their stability against the surroundings.[21]
the scientific community to better understand and
However, the detection of pH changes using
track the progression of vesicular transport for cell
Au-based
cycle
pH-sensitive
A
are
microelectrodes
with
and small molecules.[17,18] However, single or
apoptosis.[10]
organelles
specific
comparison
not
and
regions
the
resonance
in
in
and
that
sensitivity
use
processes.[2] Vesicular transport plays a significant
so
high
often
number
of
intracellular
particles
measurements
requires
polymers
such
for
conjugation
as
the
of
poly(lysine),
nanoparticle-based proton sensors have attracted
poly(acrylic acid), and chitosan.[22,23] Furthermore,
more and more attention owing to their remarkable
the structural change of the functional group to the
advantages, the most important of these being that
‘fluorescence-on’ state is irreversible in these probes.
it is easy to simultaneously assemble diverse dyes
In other words, once these probes become strongly
| www.editorialmanager.com/nare/default.asp
3
Nano Res.
fluorescent in an acidic or basic environment, they
remain strongly fluorescent even after the region
returns to the opposite condition.
Consequently, since sensors made of pH-responsive
ratiometric nanoquencher materials can avoid the
influence of several variants such as concentration
and optical path length, they have been proven to
be an effective way to accurately quantify the pH
values in vesicular transport and even in organelles.
For the study reported here, we selected polyaniline
(PANI) as a tunable ratiometric fluorescent
pH-sensor material because of its optical
responsiveness to minute changes in the proton
level. Conventionally synthesised PANI using
organic oxidant exhibits insensitivity to pH changes
in
biological
phenomena
such
that
the
optical-absorbance peak of PANI is red-shifted as a
result of its transition from an emeraldine base (EB)
to an emeralidine salt (ES) at a pH of 3.[24]
Therefore, we used transitional metals to elevate the
sensitivity of sensors for trafficking intracellular
compartments.
Figure 1. Schematic illustration of organic nanoindicator based on polyaniline nanoparticle for the detection of endolysosomal
compartments. Synthesis steps of nanoindicator based on polyaniline in mesosilica template when using heterometal nanoparticle
(IsNP) as oxidant. Emission of FPSNI Cy7 appears at endosomes. While migrating from endosomes to lysosomes, transition state of
polyaniline transferred to emeraldine salt state due to the increment of proton concentration. The emission of FPSNICy3 gradually
appears at lysosomes.
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research
2. Results and Discussions
solution.[30] This mesoporous silica layer provided
monodispersity based on framed structures where
2.1 Synthesis of FPSNIs.
To synthesise monodisperse silica-coated PANI for
trafficking of the intracellular compartment with
varying
proton
gradients,
Fe3O4–MnO
heterostructured nanoparticles were employed as
an oxidant for the polymerisation of aniline in an
aqueous acidic medium. While manganese oxide
polymerisation of polyaniline (PANI) could occur.
Furthermore, the silica shell enabled simple surface
modification
such
as
PEGylation
(covalent
attachment of polyethylene glycol (PEG) polymer
chains to another molecule) and fluorophore
adsorption (e.g. adsorption of Cy3 and Cy7).
can be converted into soluble Mn2+ in an acidic
2.2 Characterization of FPSNIs.
environment, the presence of the iron-oxide phase
The TEM image in Figure. 2a,b reveals that for each
enabled the polymer synthesis under much milder
IsNP
acidic condition at room temperature.[25] We
encapsulated by a mesoporous silica shell with a
synthesised two partially reversible oxidised forms
uniform layer thickness (~26 nm). Subsequently, the
of PANI, the deprotonated EB and protonated ES
polyaniline–mesosilica-shell nanoindicator (PSNI)
states, which exhibit distinct absorbance peaks at
was synthesised by introducing a dilute sulphuric
750 and 650 nm, respectively.[26,27] Subsequently,
acid solution and the aniline monomer into the
two pH-insensitive fluorophores, Cy3 and Cy7,
mesopores (as a nanoreactor) of the SIsNPs. The
which exhibit efficient quenching performance with
polymerisation was initiated by oxidation with
PANI, were further adsorbed onto the PANI surface
heterostructured nanoparticles (Fe3O4–MnO). The
to
generate
dual
SIsNP,
the
IsNP
was
completely
signals.
The
absorption spectra of
polyaniline
was
Fe3O4–MnO, at various pH values were compared
red-shifted as a result of its transition from the EB
to the ones polymerised with MnO (Figure. S1). The
state to the ES state in the entire physiologically
use of heterostructured nanoparticles led to the
relevant range of the endosome–lysosome pathway,
upward shift of the doping level by approximately
as shown in Figure. 1. To assess the feasibility of
one order of magnitude. This is due to the presence
using
fluorophore-adsorbed
of the interface between the iron oxide and MnO
silica-coated polyaniline nanoindicator (FPSNI) as
because Mn ions diffused toward the iron oxide,
an organic nanoquencher, we investigated the
forming a new metallic interface.[31] Mn-doped
quenching effect of FPSNICy3 and FPSNICy7 (with
iron ions were then located in the interface of the
Cy3 and Cy7, respectively, as the fluorophore
two different metals, which assisted the change in
adsorbed on the nanoindicators) on fluorophores,
pH to switch on the absorption peak of PANI for
biocompatibility,
distinction over acidic cellular compartments. The
optical-absorbance
a
fluorescent
and
peak
of
pH-insensitive
and
in
vitro
ratiometric
PSNIs,
obtained
using
fluorophores intensities. Island-shape nanoparticles
transmission
(IsNPs) with an average size of approximately 63±
atomic force microscopy (AFM) images in Figure.
5.32 nm, each consisting of the core iron oxide
2c,d verify that the mesoporous silica shell
(Fe3O4)
MnO
successfully provided a space for polymerisation
nanoparticles, were synthesised via heteroepitaxial
(Figure. S2). The distinctive chemical structures of
growth.[28,29] Silica-coated IsNPs (SIsNPs) were
the PSNI were verified by Fourier-transform
then obtained using the Stöber method through
infrared (FT-IR) spectroscopy with the characteristic
ammonia-catalysed
bands of PANI: C=C and C=N stretchings of the
nanoparticle
and
exterior
hydrolysis
of
tetraethylorthosilicate (TEOS) in an aqueous basic
electron
microscopy
(TEM)
and
quinone ring at 1565 cm-1; aromatic amine vibration
5
Nano Res.
inductively
coupled
plasma-atomic
emission
spectroscopy (ICP-AES). The calculated value of the
ion concentration reveals that nearly 95% of Mn2+
ions were present in the supernatant and the
residual ions were retained in the shell (Figure. S5).
And, the IsNPs contained 88.5 times more Mn2+ ions
than Fe2+ ions, as confirmed by the ICP-AES
analysis.
2.3 Assessment of redox reversibility of FPSNIs.
To examine the redox response of PANI, which
exhibited different absorption peaks at 650 nm in a
basic environment and 810 nm in an acidic
environment, the absorbance responses to pH
variations were obtained, as depicted in Figure. 3a.
Figure 2. Morphologic characterization of FPSNIs. Transmission
The UV spectra were analysed through 6 reversible
electron microscopy (TEM) images of (a) IsNP, (b) SIsNP, and (c)
cycles of switching between the oxidised ES state
PSNI. (d) Atomic force microscopy (AFM) image of PSNI. Scale
and the reduced EB state by alternately adding
bar: 50 nm. The red circles in inset figure 2a indicate Fe3O4 is
solutions of 1 M HCl and 1 M NaOH. The results
embedded in MnO.
demonstrate the robust and reversible pH sensing
performance of the PSNI. The response of the PSNI
at 1305 cm-1 in the emeraldine base state of PANI;
in the biogenic pH range (pH 3–8) was analysed
Si–O–Si stretching at 1100 cm-1 owing to the
using UV–vis spectroscopy (Figure. 3b). In the
presence of the silane bond for both PSNI and
range of pH 6–8, the polaron bands (420 nm and
SIsNPs (Figure. S4a).[32] The X-ray diffraction
750–900 nm) in PANI of the PSNI disappeared and
pattern (XRD) of the IsNPs revealed peaks at 2θ
a strong absorption band (~600 nm) emerged as a
values of 35.02˚, 40.68˚, 58.86˚, 70.22˚, and 74.06˚
result of the excitation from the highest occupied
(Figure. S4b, orange line) owing to the presence of
molecular orbital (HOMO) of the three-ring benzoid
MnO nanoparticles, and the peaks corresponded to
part of the PANI to the lowest unoccupied
the (111), (200), (220), (311), and (222) reflections,
molecular orbital (LUMO) of the localised quinoid
respectively (JCPDS 07-0230). The collapse of the
ring and the two surrounding imine nitrogen
MnO crystallinity indicates the change from MnO
atoms.[33,34] Since the pH difference between the
to Mn2+ in an acidic environment (Figure. S4b, green
endosome (pH ~6.5) and lysosome (pH ~5.5) is
line).
photoelectron
approximately 1, a nanoindicator that can switch on
spectroscopy (XPS) of the PSNI detected the peaks
an absorption peak with a range that is narrower
of carbon, nitrogen, oxygen, and silicon because of
than 1 pH unit is required.[35] As seen in Figure. 3c,
the presence of aromatic amine and the quinoid
the PSNI successfully distinguished a pH interval
ring of PANI as well as the silane bonds on the silica
as small as 0.4 in the biological range with pH
shell, indicating that PANI was formed in the
3.73–6.67. Therefore, the PSNI demonstrated the
mesosilica pores (Figure. S4c).
feasibility of using the shift in the absorbance peak
To assess the conversion ratio of MnO to Mn2+ ions,
to differentiate the intracellular proton level: it
the Mn2+ ions in the supernatant were quantified by
exhibited remarkable performance for sensitive
Furthermore,
X-ray
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research
6
Nano Res.
intercellular pH trafficking with the advantageous
ES transitional states of PANI. To synthesise
feature of the ability to sense finer pH variations in
FPSNICy3 and FPSNICy7, Cy3 and Cy7 were adsorbed,
a wider detectable range compared to previously
respectively, on the surface of PSNI by vortexing for
report FRET-based trafficking agents.
48 h at room temperature. The amounts of Cy3 and
Cy7 adsorbed on the silica shell, quantified by
fluorescence intensity of supernatant after vigorous
mixing for 48 h, were 0.19 mg and 0.17 mg for
FPSNICy3 and FPSNICy7, respectively. The quenching
effect of PANI on Cy3 and Cy7 with varying pH
levels was shown by the fluorescence intensity ratio
of
FPSNICy3
to
Cy3,
and
FPSNICy7
to
Cy7,
respectively, while the absorbance ratio (λ550 of
FPSNICy3 to Cy3 and λ770 of FPSNICy7 to Cy7) was
fixed regardless of pH changes in the buffer
solution (Figure. 4). The graph reveals that as the
amount of protons increased (transition from EB to
ES state) the absorbance peak of PANI moved
toward 750 nm, which induced the swift quenching
of Cy3 emission while the Cy7 emission was
switched on. Therefore, the selective quenching
effect according to pH level was successfully
demonstrated in the biogenic range.
Figure 3. Redox switching property and sensitivity. (a) Redox
reversibility test of pH nanoindicator (PSNI). The pH PSNI was
changed by adding 1M HCl and 1M NaOH repeatedly.
Absorption titration spectra and photographs (inset) of PSNI
from (b) pH 3 to 8 and (c) pH 3.95 to 7.23. The arrows indicate
the movement of peak as pH increases. Moreover, the titration
graph (c) shows that it has keen proton sensitivity as narrow as
pH 0.3.
2.4 Selective quenching effect in response to
biogenic proton range.
Two fluorophores, Cy3 and Cy7, corresponding to
the absorption peaks of the EB and ES states of
PANI were selected. The emission and excitation
peaks of Cy3 and Cy7 (570 nm and 770 nm, 550 nm
and 750 nm, respectively) exhibited excellent
quenching effect with PANI because of the
Figure 4. Selective quenching effect in response to biological
proton range. Fluorescence intensity and absorbance ratio of
FPSNI to dye in aqueous state at various pH conditions from 4 to
8.The orange color represents FPSNICy3 and red color stands for
FPSNICy7. (Control: free Cy3 and Cy7 at the same concentration
of those in the nanoparticles)
substantial overlap of the emission spectra of Cy3
and Cy7 with the absorption spectra of the EB and
| www.editorialmanager.com/nare/default.asp
7
Nano Res.
2.5 In vitro evaluation of cytotoxicity and
incubation time intervals of 0.5, 1.5, and 4 h. Their
trafficking vesicular transport.
fluorescence images were then obtained using a
The cytotoxicity of the PSNIs was evaluated by
confocal laser scanning microscope. The feasibility
measuring the inhibition of cell growth using the
of trafficking intracellular compartments by FPSNIs
MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra
was evaluated (Figure. 5b), where FPSNIs were
zolium bromide] assay against HT1080 cells. The
co-localised with the early endosome marker, EEA1,
result indicates the negligible cytotoxicity of the
at 0.5 and 1.5 h; after an incubation period of 4 h,
PSNIs (Figure. S6). The detection of pH changes
the FPSNIs overlapped with the lysosome marker,
with
compartments
lysotracker blue DND-22. In the early endosome in
(endosome and lysosome) using FPSNIs was
particular, the fluorescence intensity of FPSNICy7
performed against HT1080 cells (Figure. 5a). The
was strong whereas the intensity of FPSNICy3 faded
FPSNIs were treated with HT1080 cells for different
out. In the endosome, PANI in the PSNI was in the
respect
to
intracellular
Figure 5. In vitro evaluation of FPSNIs as trafficking vesicular transport. (a) Schematic illustration of fluctuation of fluorescence
emission of FPSNIs due to quenching effect of PANI overlaid with intracellular compartment markers (b) In vitro dual emission
fluorescence images of HT1080 cells treated with FPSNI Cy3 and FPSNICy7 for distinct durations taken by confocal laser scanning
microscope by irradiating nanoindicators at 550 nm and 750 nm distinctively. Scale bar: 10 µ
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research
8
Nano Res.
EB state since its pH was approximately 6 where its
amount of protons increases owing to the increment
absorption peak was located at 570 nm. Because of
of the absorbance ratio at 550 nm to 750 nm the
the EB state of PANI, the emission fluorescence of
excitation wavelength of Cy3 and Cy7, respectively.
FPSNICy3 was quenched; hence, the emission of
Due to such aspect, the nanoindicators performed
FPSNICy7
the
fluctuated fluorescence intensities at distinct acidic
endosomes. On the other hand, after the 4 h
was
compartments whereas organelle markers which
incubation period, the fluorescence intensity of
are EEA1 and lysotracker blue DND-22, the
FPSNICy3 was restored while the intensity of
identification dyes for the early endosomes and
FPSNICy7 diminished. In the lysosome, PANI in the
lysosomes, showed monotonous intensities at all
PSNI was in the ES state since the lysosomal pH
distinct times. This reveals that FPSNIs fulfill the
was lower than 5, and its absorption peak shifted to
capability of trafficking within early endosomes to
770 nm. Thus, owing to the ES state of PANI, the
acidic lysosomes in Figure. 6a Moreover, due to the
emission fluorescence of FPSNICy7 was quenched;
equation
therefore, the emission of FPSNICy3 was clearly
quantification of intracellular compartment pH was
observed
in
apparently
the
observed
lysosome.
introduced
in
Figure.
6b,c,
the
fluorescence
enabled from the fluorescence intensity ratio. This
intensity ratio of FPSNICy3/FPSNICy7 was increased
feature is advantageous for FPSNIs over fluorescent
proportionately as the amount of protons increased
acidotropic probe such as lysotracker or EEA1,
owing to the increase in the absorbance ratio (λ570/
which can only display a color and further it is not
λ770) of PANI in the PSNI. As a result of the selective
able to distinguish the deviation in surrounding pH
quenching
according
nanoindicators
the
exhibited
The
in
proton
gradual
level,
the
in intracellular organelles. These in vitro results
changes
in
imply that FPSNI may serve as an efficient
fluorescence intensities. Furthermore, there was
nanoindicator in intracellular component.
colour reversal at distinct acidic compartments
whereas
the
conventional
organelle
markers
showed consistent intensities at all times. This
feature is advantageous for FPSNIs over fluorescent
acidotropic probes such as EEA1 or lysotracker,
which
can
only
display
a
single
colour.
Conventional probes are not able to distinguish the
changes in surrounding pH around intracellular
organelles because they are based on a specific
enzymic
antibody–antigen
or
acquire
the
fluorescence intensity at a considerably low proton
level. These in vitro results imply that FPSNIs could
Figure 6. Fitting equation of FPSNIs for pH analysis in single cell.
serve as efficient nanoindicators in intracellular
(a) In vitro dual emission fluorescence image of HT1080 cells
compartments.
treated with FPSNICy3 and FPSNICy7 for distinct durations taken
at 4 h by confocal laser scanning microscope. pH titration curve
2.6 Fitting equation of FPSNIs for pH analysis in
of
single cell.
λ570/λ770 and (c) fluorescence intensity ratio of FPSNICy3/FPSNICy7
The
fluorescence
intensity
ratio
of
FPSNICy3/FPSNICy7 proportionally decreases as the
the (b) PSNI obtained from the UV-Vis absorbance ratio
as a function of pH. As pH decreases the absorbance at 570 nm
decreases while the fluorescence intensity of FPSNICy3 increases.
| www.editorialmanager.com/nare/default.asp
3. Conclusion
We
have
Synthesis of island-like nanoparticles (IsNP). First
fabricated
novel
PANI-based
nanoindicators to probe the wide range of
intracellular proton levels, which is not feasible
with fluorophores or organic quenchers alone.
After
endocytosis,
localised
in
the
FPSNIs
were
endosome
transiently
where
strong
fluorescence intensity of Cy7 was observed.
Following FPSNI trafficking into the more acidic
organelles, lysosomes, a significant increase in
the fluorescence intensity of Cy3 was observed
owing to the selective quenching effect of FPSNIs
induced by the local pH level. The unique and
robust optical properties of PANI, together with
the pH value in an intracellular environment,
should lead to the development of sensors and
nanostructures with important applications in a
variety
of
areas
including
healthcare,
environment monitoring, and biodefence.
4. Experimental Method Section
of all, 12 nm diameter of Fe3O4 (MNP) were
synthesized
by
the
thermal
decomposition
method.[31] Iron(III) acetylacetonate (2 mmol),
1,2-hexadecanediol (10 mmol), oleic acid (6
mmol), oleylamine (6 mmol), and benzyl ether
(20 mL) were mixed under nitrogen. The mixture
was preheated to 130 °C for 2 h and then heated
to reflux at 300 °C for 30 min. Afterward, the
products were purified by centrifuge with excess
pure ethanol at 6000 rpm for 10 min. Then, 20 mg
of MNP, manganese(II) formate hydrate (0.6
mmol), oleic acid (0.35 mmol), and trioctlyamine
(20 mL) were mixed under nitrogen. The mixture
was preheated to 130 °C for 2 h and then heated
to reflux at 330 °C for 2 h. The products were
purified with excess pure ethanol and were
isolated by centrifugation at 6000 rpm for 10 min.
Synthesis
of
meso
silica
coated
island-like
nanoparticles (SIsNP). To prepare water soluble
SIsNP, IsNP (20 mg) were dissolved in n-hexane
(4 mL). This organic phase was added into the 20
Materials.
Iron(III)
acetylacetonate,
manganese(II)
formate
hydrate,
1,2-hexadecanediol, oleic acid, oleylamine,
trioctlyamine, benzyl ether, polysorbate-80,
tetraethly orthosilicate (TEOS), and aniline were
all purchased from Sigma-Aldrich. Cy3 NHS
ester and Cy7 NHS ester were purchased from
Lumiprobe Corp, FL. Silane-poly(ethylene
glycol)-carboxylic acid (Si-PEG-COOH, Mw
5,000) was purchased from Nanocs, Inc, and
Dulbecco’s phosphate buffered saline (PBS, pH
7.4) was purchased from Hyclone. Lysotracker
blue DND-22 was purchased from invitrogen and
anti-EEA1 was purchased from Abcam (# ab2900).
Dulbecco’s Modified Eagle Medium (DMEM),
fetal bovine serum (FBS), and antibiotic
anti-mycotic and nen essential aminoacid were
purchased from Gibco® , Invitrogen. All other
chemicals and reagents were analytical grade.
Ultrapure deionized (DI) water was used for all
of the synthetic processes.
mL of aqueous phase containing 5 mg of
polysorbate 80. The mixture was emulsified for
20
min
with
an
ultrasonicator
(ULH700S,
Ulssohitech, Korea) at 200 W. After evaporation
of the organic solvent, the products were purified
by centrifugation at 18 000 rpm then the
precipitates were redispersed in deionized water.
The SIsNP were then synthesized by the
modified Stöber method afterward.32 The SIsNP
were synthesized in mixture of alcohol and water
at an ambient temperature using the IsNP as
seeds. IsNP (5mg) were diluted with ethyl
alcohol (3 mL) and 1 mL of 1 M sodium
hydroxide solution. 100 μL of TEOS was added
20 μL for every hour, and after stirring for 12 h, a
meso silica outer shell is formed on the surface of
IsNP through hydrolysis and condensation of
TEOS.[33,34]
10
Synthesis
Nano Res.
shell
Assessment of in vitro cell viability. Cell viability
nanoindicators (PSNI). For the preparation of PSNI,
was quantified using a colorimetric assay based
50 mg of SIsNP were dissolved in 0.5 mL of
ontheMTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphe
deionized water. Then 1 mL of 1.83 M sulfuric
nyltetrazolium bromide] assay (Roche, Germany).
acid and aniline (43.88 mmol) were added
The HT1080 was obtained from American Tissue
simultaneously. The mixture was vortexed for 20
Type Culture (ATCC, USA), and cells were
min and centrifugation were done two times with
plated at a density of 2.5 ⅹ 104 cells/100 μL in a
excess water.
96-well plate and were incubated at 37 ℃ in a
Synthesis
of
polyaniline
of
-
mesosilica
fluorophore
adsorbed
5% CO2 atmosphere. The cells were incubated for
nanoindicator
24 h with 100 μL of PSNI re-suspended in MEM
PSNI
were
supplemented with 3% FBS and were then rinsed
dissolved in 3 mL of ethyl alcohol. 0.2 mg of Cy3
with 100 μL of PBS (pH 7.4, 1mM). The cells were
or Cy7 were individually added and vortexed for
then added to 100 μL of MEM supplemented
48 h. After FPSNICy3 and FPSNICy7 were formed,
with 3% FBS, 1% antibiotic anti-mycotic and
and Si-PEG-COOH (0.4 μmol) were added then
non-essential amino acid and were treated with
the sample was vortexed again for overnight. The
10 μL of freshly-prepared tetraolium salt. After 2
PEGylated-FPSNI was centrifuged three times
h, the plate was assayed using an enzyme-linked
with excess deionized water and re-suspended in
immunosorbent assay (ELISA, Spetra MAX 340,
1 mL of PBS.
Molecular
polyaniline–mesosilica
(PEGylated-FPSNI).
shell
100
mg
of
Characterization of IsNP, SIsNP, PSNI and
PEGylated-FPSNI. The absorbance spectra of
particles were measured using a spectrometer
(Optizen
2120UV,
MECASYS,
wavelength
device
of
USA)
450
nm
at
an
and
absorbance
a
reference
wavelength of 650 nm.
Treatment for intracellular compartment trafficking.
Korea),
For the seeding of HT1080 cells onto the confocal
respectively. The morphologies were evaluated
dishes, 1x105 cells/mL were seeded and settled for
using a high-resolution transmission electron
24 h for well attachment to the dish. HT1080 cells
microscope (HR-TEM, JEM-2100 LAB 6 , JEOL
were rinsed with PBS (pH 7.4, 1 mM) two times
Ltd., Japan) and atomic force microscopy (model
and 0.1 mg of FPSIsNICy3 and 0.2 mg of
dimension 3100, Digital Instrument Co., USA),
FPSIsNICy7 were dispersed in minimum essential
and characteristic bands were confirmed by
media (MEM) supplemented with 3% fetal
Fourier-transform infrared spectroscopy (FT-IR,
bovine serum (FBS), 1% antibiotic anti-mycotic
Perkin Elmer, USA). To verify diffraction patterns
and non-essential amino acid (Gibco® , Invitrogen,
and band gap energy of inorganic nanoparticles
USA) After incubation for different hours which
X-ray diffraction (Rigaku, X-ray Diffractometer
were 30 min, 1 h and 30 min and 4 h were
Ultima3) and X-ray photoelectron spectroscopy
incubated under 37 ℃ and 5% of CO2 condition.
(k-alpha, Thermo Scientific, U.K.) were used. For
Immunocytochemistry stains. For staining of
quantifying the fluorescence of FPSNICy3 at 550
lysosome after incubation for different hours at
nm excitation and 570 nm emission, and FPSNICy7
37 ℃ during incubation lysotracker (7 μM)
at 750 nm excitation and 770 nm emission using a
should be treated for 2 h before fixation. At
hybrid multi-mode microplate reader (Synergy
predetermined time intervals, the cells were
H4, BioTek, USA). Moreover, stained cells were
washed with PBS (pH 7.4, 1 mM) two times then,
observed by laser scanning confocal microscope
fixed in 4% paraformaldehyde in PBS for 10 min.
(LSM 700, Carl Zeiss, Jena, Germany)
The fixed cells were permeabilized with 0.1%
| www.editorialmanager.com/nare/default.asp
11
Nano Res.
Triton X-100 in PBS for 10 min, blocked with 1%
Serra, D.A.; Chichester, C.O.; Engelman, D.M.; Reshetnyak,
bovine serum albumin (BSA) in PBS for 1 hour,
Y.K. Mechanism and uses of a membrane peptide that targets
and stained with rabbit polyclonal anti-EEA1
tumors and other acidic tissues in vivo. Proc. Natl. Acad. Sci.
which is a marker for early endosome, was
U.S.A. 2007, 104, 7893-7898.
diluted in PBS containing 1% BSA (1:200) for 1
[4] Schafer, F. Q.; Buettner, G. R. Redox environment of the
hour. After being washed three times with PBS to
cell as viewed through the redox state of the glutathione
remove
disulfide/glutathione couple. Free Radic. Biol. Med. 2011,
excess
antibodies,
the
cells
were
incubated with secondary antibody of rabbit IgG
30, 1191-1212.
conjugated with Alexa Fluor488 (Invitrogen,
[5] Lewis, J. G.; Lin, K.Y.; Kothavale, A.; Flanagan, W.M.;
USA) diluted in PBS containing 1% BSA (1:300)
Matteucci, M.D.; Deprince, R.B.; Mook, R.A.; Hendren,
for 1 hour. The stained cells were examined using
R.A.; Wagner, R.W. A serum-resistant cytofectin for cellular
a laser scanning confocal microscope. All cell
delivery of antisense oligodeoxynucleotides and plasmid
staining procedure were performed at room
DNA. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 3176-3181.
temperature.
[6] Liu, Y.; Reineke, T. M. Poly(glycoamidoamine)s for gene
delivery. Structural effects on cellular internalization,
Acknowledgements
“This
work
was
buffering capacity, and gene expression. Bioconjugate Chem.
supported
by
BioNano
2007, 18, 19-30.
Health-Guard Research Center funded by the
[7] Busa, W. B.; Nuccitelli, R. Metabolic regulation via
Ministry of Science, ICT & Future Planning
intracellular pH. Am. J. Physiol.
(MSIP)
Frontier
[8] Casey, J. R..; Grinstein, S.; Orlowski, J. Sensors and
“This
regulators of intracellular pH. Nat. Rev. Mol. Cell. Biol. 2010,
of
Korea
as
Global
Project" (H-GUARD_2013-11-2072)
and
1984, 246, R409-438.
work was supported by the national research
11, 50-61.
foundation of Korea (NRF) grant funded by the
[9] Reineke, T. M.; Davis, M. E. Structural effects of
Korea government (MEST)” (2010-0019923)
carbohydrate-containing polycations on gene delivery. 2.
Charge center type. Bioconjugate Chem. 2003 14, 255-261.
Electronic Supplementary Material: Supplementary
[10] Perez-Sala, D.; Collado-Escobar, D.; Mollinedo, F.
material (Absorption spectra (Figure S1), TEM images
Intracellular alkalinization suppresses lovastatin-induced
(Figure
apoptosis in HL-60 cells through the inactivation of a
S2),
Photographs,
absorption
spectra
and
absorption ratio graph (Figure S3), FT-IR spectra, XRD
spectra and XPS spectra(Figure S4), ICP-AES (Figure S5),
Assessment of cytotoxicity (Figure S6)) is available in the
pH-dependent endonuclease. J. Biol. Chem. 1995, 270,
6235-6242.
at
[11] Shi, W.; Li, X.; Ma, H. A tunable ratiometric pH sensor
http://dx.doi.org/10.1007/s12274-***-****-* (automatically
based on carbon nanodots for the quantitative measurement
inserted by the publisher).
of the intracellular pH of whole cells. Angew. Chem. Int. Edit.
online
version
of
this
article
2012, 51, 6432-6435.
References
[12] Peng, H.S.; Stolwijk, J.A.; Sun, L. N.; Wegene,r J.;
[1] Chiu, Y.-L.; Chen, S.-A.; Chen, J.-H.; Chen, K.-J.; Chen,
Wolfbeis, O.S. A nanogel for ratiometric fluorescent sensing
H.-L.; Sung, H.-W. A dual-emission forster resonance energy
of intracellular pH values. Angew. Chem. Int. Edit. 2010, 49,
transfer nanoprobe for sensing/imaging pH changes in the
4246-4249.
biological environment. ACS Nano 2010, 4, 7467-7474.
[13] Davies, T. A.; Fine, R.E.; Johnson, R.J.; Levesque,
[2] Han, J.; Burgess, K. Fluorescent indicators for
C.A.; Rathbun, W.H.; Seetoo, K.F.; Smith, S.J.; Strohmeier,
intracellular pH. Chem. Rev. 2010, 110, 2709-2728.
G.; Volicer, L.; Delva, L.; Simons, E.R. Non-age related
[3] Andreev, O. A.; Dupuy, A.D.; Segala, M.; Sandugu, S.;
differences in thrombin responses by platelets from male
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research
12
Nano Res.
patients with advanced Alzheimer's disease. Biochem.
strong and weak polyelectrolytes. Langmuir 2006, 22,
Biophy. Res. Commun.1993, 194, 537-543.
3864-3869.
[14] Izumi, H.; Torigoe, T.; Ishiguchi, H.; Uramoto, H.;
[26]
Yoshida, Y.; Tanabe, M.; Ise, T.; Murakami, T.; Yoshida, T.;
H.; Suh J.-S.; Lee, K.; Yoo, K.-H.; Kim, E.-K.; Huh, Y.-M.;
Nomoto, M.; Kohno, K. Cellular pH regulators: Potentially
Haam, S. Convertible organic nanoparticles for near-infrared
promising molecular targets for cancer chemotherapy.
photothermal ablation of cancer cells. Angew. Chem. Int.
Cancer. Treat. Rev. 2003, 29, 541-549.
Edit. 2011, 50, 441-444.
[15] Loiselle, F. B.; Casey, J. R. Measurement of cell pH.
[27]
Methods Mol. Biol. 2003, 227, 259-280.
Suh, J.; Huh, Y.; Haam, S.; Yang J. Redox-sensitive
[16]Wray, S. Smooth muscle function and intracellular pH:
colorimetric polyaniline nanoprobes synthesized by a
Measurement, regulation and function. Am. J. Physiol. 1988,
solvent-shift process. Nano. Res. 2013, 6, 356-364.
254, C213-C225.
[28]
[17] Mátyus, L.; Szöllosi, J.; Jenei, A. Steady-state
Thermodynamic
fluorescence quenching applications for studying protein
Experiment and theory. J. Phys. Chem. 1995, 99, 7036-7041.
structure and dynamics. J. Photochem. Photobio.. B: Biol.
[29] Talapin, D. V.; Rogach, A. L.; Haase, M.; Weller, H.
2006, 83, 223-236.
Evolution of an ensemble of nanoparticles in a colloidal
[18] Zhuang, X.; Ha, T.; Kim, H.D.; Centner, T.; Labeit, S.;
solution: Theoretical study. J. Phys. Chem. B 2001, 105,
Chu, S. Fluorescence quenching: A tool for single-molecule
12278-12285.
protein-folding study. Proc. Natl. Acad. Sci. U.S.A. 2000, 97,
[30]
14241-14244.
S.H.
[19] Coupland, P. G.; Briddon, S. J.; Aylott, J. W. Using
formaldehyde resin core/shell nanospheres: Large-scale
fluorescent
synthesis and application for in vivo bioimaging. Adv. Funct.
pH-sensitive
nanosensors
to
report
their
Yang, J.; Choi, J.; Bang, D.; Kim E.; Lim, E.-K.; Park,
Choi, J.; Hong, Y.; Lee, E.; Kim, M.-H.; Yoon, D.S.;
Leff, D. V.; Ohara, P. C.; Heath, J. R.; Gelbart, W. M.
control
of
gold
nanocrystal
size:
Guo, S. R.; Gong, J.-Y.; Jiang, P.; Wu, M.; Lu, Y.; Yu,
Biocompatible,
luminescent
Silver@Phenol
intracellular location after Tat-mediated delivery. Integr. Biol.
Mater. 2008, 18, 872-879.
2009, 1, 318-323.
[31]
[20] Uchiyama, S.; Makino, Y. Digital fluorescent pH
Kim, B.; Lee, M.; Oh, A.; Jin, J.; Chae, Y.; Baik, H.; Suh,
sensors. Chem. Comm. 2009, 2646-2648.
J.-S.; Haam, S.; Huh, Y.-M.; Lee, K. A highly crystalline
[21] Knoll, W.; Interfaces and thin films as seen by bound
manganese-doped
electromagnetic waves. Annu. Rev. Phys. Chem. 1998, 49,
predesigned void volume and shape for theranostic
569-638.
applications. Adv. Mater. 2013, 25, 3202-3208.
[22]
[32]
Febvay, S.; Marini, D. M.; Belcher, A. M.; Clapham,
Phan, V. N.; Lim, E.-K.; Kim, T.; Kim, M.; Choi, Y.;
Kamata,
K.;
iron
oxide
Lu, Y.; Xia,
nanocontainer
with
Y. Synthesis and
D. E. Targeted cytosolic delivery of cell-impermeable
characterization of monodispersed core-shell spherical
compounds
colloids with movable cores. J. Am. Chem. Soc. 2003, 125,
by
nanoparticle-mediated,
light-triggered
endosome disruption. Nano. Lett. 2010, 10, 2211-2219.
2384-2385.
[23] Li, N.; Chang, C.; Pan, W.; Tang, B. A multicolor
[33]
nanoprobe for detection and imaging of tumor-related mrnas
Nanoengineering of inorganic and hybrid hollow spheres by
in living cells. Angew. Chem. Int. Edit. 2012, 51, 7426-7430.
colloidal templating. Science 1998, 282, 1111-1114.
[24]
[34] Jang, J.; Ha, J.; Lim, B. Synthesis and characterization
Gizdavic-Nikolaidis, M.; Travas-Sejdic, J.; Bowmaker,
G. A.; Cooney, R. P.;
Kilmartin, P. A. Conducting polymers
Caruso,
F.;
Caruso,
R.
A.;
Möhwald,
H.
of monodisperse silica-polyaniline core-shell nanoparticles.
as free radical scavengers. Synthetic Metals. 2004, 140,
Chem. Comm. 2006, 1622-1624.
225-232.
[35] Maxfield, F. R.; Yamashiro, D. J. Endosome
[25]
acidification
Nakayama, M.; Tagashira, H.; Electrodeposition of
layered manganese oxide nanocomposites intercalated with
and the pathways of receptor-mediated
endocytosis. Adv. Exp. Med. Biol. 1987, 225, 189-198.
| www.editorialmanager.com/nare/default.asp
Nano Res.
Electronic Supplementary Material
Colourimetric redox-polyaniline nanoindicator for in situ vesicular
trafficking of intracellular transport
Eun Bi Choi1†, Jihye Choi1†, Seo Ryung Bae1, Hyun-Ouk Kim1, Eunji Jang1, Byunghoon Kang1, Myeong-Hoon Kim1,
Byeongyoon Kim3, Jin-Suck Suh2, Kwangyeol Lee3, Yong-Min Huh2*() and Seungjoo Haam1*( ).
.
Supporting information to DOI 10.1007/s12274-****-****-* (automatically inserted by the publisher)
Address correspondence to Yong-Min Huh, [email protected]; Seungjoo Haam, [email protected]
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research
Nano Res.
Figure S1. UV-vis absorption spectra of PSNI. (a) without Fe3O4 (b) with Fe3O4.The result show that in case of
same size of MnO, the pH point where PANI changes its color can be shifted 1 order with Fe3O4.
Figure S2. TEM images of SIsNP and PSNI. showing that location of PANI is influenced by the thickness of silica
shell. Scale bar: 100 nm
| www.editorialmanager.com/nare/default.asp
Nano Res.
Figure S3. Characterization of each nonporous silica and mesoporous silica coated IsNP.
(a) Photographs, (b)
absorption spectra, and (c) absorption ratio ((λ775-λ595)/λ595) graph of IsNP coated with nonporous silica and
mesoporous silica before and after adding monomer stock.
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research
Nano Res.
Figure S4. Structural characterization.
(a) FT-IR spectra of EB state of PANI (black), PSNI (green), and SIsNP
(orange) (ⅰ) C=C and C=N stretching of quinone ring, (ⅱ) aromatic amine vibration, and (ⅲ) Si-O-Si stretching
are represented respectively. (b) X-ray diffraction (XRD) spectra of IsNP (orange), MnO (blue), and PSIsNI
(green). (c) X-ray photoelectron spectroscopy (XPS) spectra of PSNI
| www.editorialmanager.com/nare/default.asp
Nano Res.
120
Mn2+
Fe2+
100
C/Ctotal (%)
80
60
40
20
0
Supernant
SIsNP_after
Figure S5. Relative concentrations (%) of SIsNP and PSNI of transitional metal ions (Fe and Mn) using ICP-AES.
The data reveal that Mn and Fe are dissolve when diluted sulfuric acid is added.
120
Cell viability (%)
100
80
60
40
20
0
10-6
10-5
10-4
10-3
10-2
10-1
100
101
Concentration (g/mL)
Figure S6. Assessment of cytotoxicity. Growth inhibition assay of HT1080 cells treated with PSNI.
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research