Post on 25-Feb-2020
Nano Res
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*().
Nano Res., Just Accepted Manuscript • DOI: 10.1007/s12274-014-0597-6
http://www.thenanoresearch.com on October 8, 2014
© Tsinghua University Press 2014
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Nano Research
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 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 Korea
3Department of Chemistry, Korea University, Seoul
136-701, Republic of Korea
†These authors contributed equally to this work.
Simple colourimetric redox-polyaniline nanoindicator; Silica-coated
polyaniline nanoparticles with adsorbed fluorophores Cy3 and Cy7
(FPSNICy3 and FPSNICy7) were fabricated as proton-sensitive
nanoindicators.
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
Revised: day month year
Accepted: day month year
(automatically inserted by
the publisher)
© Tsinghua University Press
and Springer-Verlag Berlin
Heidelberg 2014
KEYWORDS
Redox, pH, intracellular
compartments, organic
quencher, conducting
polymer, nanoindicator
ABSTRACT 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.
Address correspondence to Yong-Min Huh, ymhuh@yuhs.ac; Seungjoo Haam, haam@yonsei.ac.kr
Nano Research
DOI (automatically inserted by the publisher)
Research Article
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2 Nano Res.
1. Introduction
Real-time tunable ratiometric fluorescent proton
organic sensors that can efficiently measure
optical-fluorescence-based ratiometric signals in
living cells have attracted much interest in the quest
to understand diverse cellular processes.[1] The
scope offered by trafficking vesicular transport of
living cells is revealing the science behind various
cellular processes and allowing researchers to better
understand physiological and pathological
processes.[2] Vesicular transport plays a significant
part in the formation and maintenance of various
compartments as well as in the communication
between cells and the environment.[3] Thus, for the
comprehensive understanding of native cellular
processes, the vesicle should be considered essential
to maintaining homeostasis of every vesicular
transport so that the functions of specific
intracellular regions or organelles are not
disturbed.[4,5,6] As cellular dysfunction is often
associated with an abnormal proton level in
organelles, the vesicular proton plays a particularly
crucial role in cell biology by staying generally
between 6.8 and 7.4 in the cytosol and between 4.5
and 6.0 in the cell’s acidic organelles since proteins
depend on the proton level to maintain their
structures and functions.[7,8] Therefore, extensive
research efforts have been directed toward the
development of simple nanoprobes which can
provide real-time time-resolved pH information
rather than simple fragmental changes because the
cellular redox environment is not static and
fluctuates through different stages of the cell
cycle.[9] In addition, there is significant interest in
the scientific community to better understand and
track the progression of vesicular transport for cell
cycle and apoptosis.[10] A number of
nanoparticle-based proton sensors have attracted
more and more attention owing to their remarkable
advantages, the most important of these being that
it is easy to simultaneously assemble diverse dyes
on the same nanoparticle and continuously monitor
concentrations of target species in a simple and
reliable manner.[11] Optically addressed biosensors
of this type often use fluorescence
resonance-energy-transfer (FRET) in signal
transduction.[12] The challenge in the development
of any fluorescent sensor is the induced signal
change, which converts the recognition event to an
optical signal. Owing to their operational simplicity
and high sensitivity in comparison with
proton-permeable microelectrodes nuclear
magnetic resonance (NMR) and absorbance
spectroscopy,[2,13,14,15,16] FRET, a mechanism
describing the non-radiative and
distance-dependent energy transfer between two
chromophores, has been mostly used in various
sensing systems for proteins, peptides, nucleic acids,
and small molecules.[17,18] However, single or
dual fluorophore-labeled nanoprobes using FRET
exhibit proton-level detection ranges that are too
limited, with a maximum range of two pH units, to
perform intracellular measurements for the
endolysosomal pathway.[19,20] The actual pH
would fall outside the range of the latest generation
of developed nanosensors since the pH differs by
more than two pH units between the early
endosomes and lysosomes. Therefore, a nanoprobe
employing the quenching effect is a more powerful
tool to obtaining useful information in cell biology.
Among nanoquenchers, Au nanoparticles have
been widely utilised because of their successful
confinement of the electric field near a metal surface
and their stability against the surroundings.[21]
However, the detection of pH changes using
Au-based particles requires conjugation of
pH-sensitive polymers such as poly(lysine),
poly(acrylic acid), and chitosan.[22,23] Furthermore,
the structural change of the functional group to the
‘fluorescence-on’ state is irreversible in these probes.
In other words, once these probes become strongly
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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 FPSNICy7 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.
2. Results and Discussions
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
can be converted into soluble Mn2+ in an acidic
environment, the presence of the iron-oxide phase
enabled the polymer synthesis under much milder
acidic condition at room temperature.[25] We
synthesised two partially reversible oxidised forms
of PANI, the deprotonated EB and protonated ES
states, which exhibit distinct absorbance peaks at
750 and 650 nm, respectively.[26,27] Subsequently,
two pH-insensitive fluorophores, Cy3 and Cy7,
which exhibit efficient quenching performance with
PANI, were further adsorbed onto the PANI surface
to generate dual fluorescent signals. The
optical-absorbance peak of polyaniline was
red-shifted as a result of its transition from the EB
state to the ES state in the entire physiologically
relevant range of the endosome–lysosome pathway,
as shown in Figure. 1. To assess the feasibility of
using a pH-insensitive fluorophore-adsorbed
silica-coated polyaniline nanoindicator (FPSNI) as
an organic nanoquencher, we investigated the
quenching effect of FPSNICy3 and FPSNICy7 (with
Cy3 and Cy7, respectively, as the fluorophore
adsorbed on the nanoindicators) on fluorophores,
biocompatibility, and in vitro ratiometric
fluorophores intensities. Island-shape nanoparticles
(IsNPs) with an average size of approximately 63±
5.32 nm, each consisting of the core iron oxide
(Fe3O4) nanoparticle and exterior MnO
nanoparticles, were synthesised via heteroepitaxial
growth.[28,29] Silica-coated IsNPs (SIsNPs) were
then obtained using the Stöber method through
ammonia-catalysed hydrolysis of
tetraethylorthosilicate (TEOS) in an aqueous basic
solution.[30] This mesoporous silica layer provided
monodispersity based on framed structures where
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).
2.2 Characterization of FPSNIs.
The TEM image in Figure. 2a,b reveals that for each
IsNP and SIsNP, the IsNP was completely
encapsulated by a mesoporous silica shell with a
uniform layer thickness (~26 nm). Subsequently, the
polyaniline–mesosilica-shell nanoindicator (PSNI)
was synthesised by introducing a dilute sulphuric
acid solution and the aniline monomer into the
mesopores (as a nanoreactor) of the SIsNPs. The
polymerisation was initiated by oxidation with
heterostructured nanoparticles (Fe3O4–MnO). The
absorption spectra of PSNIs, obtained using
Fe3O4–MnO, at various pH values were compared
to the ones polymerised with MnO (Figure. S1). The
use of heterostructured nanoparticles led to the
upward shift of the doping level by approximately
one order of magnitude. This is due to the presence
of the interface between the iron oxide and MnO
because Mn ions diffused toward the iron oxide,
forming a new metallic interface.[31] Mn-doped
iron ions were then located in the interface of the
two different metals, which assisted the change in
pH to switch on the absorption peak of PANI for
distinction over acidic cellular compartments. The
transmission electron microscopy (TEM) and
atomic force microscopy (AFM) images in Figure.
2c,d verify that the mesoporous silica shell
successfully provided a space for polymerisation
(Figure. S2). The distinctive chemical structures of
the PSNI were verified by Fourier-transform
infrared (FT-IR) spectroscopy with the characteristic
bands of PANI: C=C and C=N stretchings of the
quinone ring at 1565 cm-1; aromatic amine vibration
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5 Nano Res.
Figure 2. Morphologic characterization of FPSNIs. Transmission
electron microscopy (TEM) images of (a) IsNP, (b) SIsNP, and (c)
PSNI. (d) Atomic force microscopy (AFM) image of PSNI. Scale
bar: 50 nm. The red circles in inset figure 2a indicate Fe3O4 is
embedded in MnO.
at 1305 cm-1 in the emeraldine base state of PANI;
Si–O–Si stretching at 1100 cm-1 owing to the
presence of the silane bond for both PSNI and
SIsNPs (Figure. S4a).[32] The X-ray diffraction
pattern (XRD) of the IsNPs revealed peaks at 2θ
values of 35.02˚, 40.68˚, 58.86˚, 70.22˚, and 74.06˚
(Figure. S4b, orange line) owing to the presence of
MnO nanoparticles, and the peaks corresponded to
the (111), (200), (220), (311), and (222) reflections,
respectively (JCPDS 07-0230). The collapse of the
MnO crystallinity indicates the change from MnO
to Mn2+ in an acidic environment (Figure. S4b, green
line). Furthermore, X-ray photoelectron
spectroscopy (XPS) of the PSNI detected the peaks
of carbon, nitrogen, oxygen, and silicon because of
the presence of aromatic amine and the quinoid
ring of PANI as well as the silane bonds on the silica
shell, indicating that PANI was formed in the
mesosilica pores (Figure. S4c).
To assess the conversion ratio of MnO to Mn2+ ions,
the Mn2+ ions in the supernatant were quantified by
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.
The UV spectra were analysed through 6 reversible
cycles of switching between the oxidised ES state
and the reduced EB state by alternately adding
solutions of 1 M HCl and 1 M NaOH. The results
demonstrate the robust and reversible pH sensing
performance of the PSNI. The response of the PSNI
in the biogenic pH range (pH 3–8) was analysed
using UV–vis spectroscopy (Figure. 3b). In the
range of pH 6–8, the polaron bands (420 nm and
750–900 nm) in PANI of the PSNI disappeared and
a strong absorption band (~600 nm) emerged as a
result of the excitation from the highest occupied
molecular orbital (HOMO) of the three-ring benzoid
part of the PANI to the lowest unoccupied
molecular orbital (LUMO) of the localised quinoid
ring and the two surrounding imine nitrogen
atoms.[33,34] Since the pH difference between the
endosome (pH ~6.5) and lysosome (pH ~5.5) is
approximately 1, a nanoindicator that can switch on
an absorption peak with a range that is narrower
than 1 pH unit is required.[35] As seen in Figure. 3c,
the PSNI successfully distinguished a pH interval
as small as 0.4 in the biological range with pH
3.73–6.67. Therefore, the PSNI demonstrated the
feasibility of using the shift in the absorbance peak
to differentiate the intracellular proton level: it
exhibited remarkable performance for sensitive
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6 Nano Res.
intercellular pH trafficking with the advantageous
feature of the ability to sense finer pH variations in
a wider detectable range compared to previously
report FRET-based trafficking agents.
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
substantial overlap of the emission spectra of Cy3
and Cy7 with the absorption spectra of the EB and
ES transitional states of PANI. To synthesise
FPSNICy3 and FPSNICy7, Cy3 and Cy7 were adsorbed,
respectively, on the surface of PSNI by vortexing for
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 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)
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7 Nano Res.
2.5 In vitro evaluation of cytotoxicity and
trafficking vesicular transport.
The cytotoxicity of the PSNIs was evaluated by
measuring the inhibition of cell growth using the
MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra
zolium bromide] assay against HT1080 cells. The
result indicates the negligible cytotoxicity of the
PSNIs (Figure. S6). The detection of pH changes
with respect to intracellular compartments
(endosome and lysosome) using FPSNIs was
performed against HT1080 cells (Figure. 5a). The
FPSNIs were treated with HT1080 cells for different
incubation time intervals of 0.5, 1.5, and 4 h. Their
fluorescence images were then obtained using a
confocal laser scanning microscope. The feasibility
of trafficking intracellular compartments by FPSNIs
was evaluated (Figure. 5b), where FPSNIs were
co-localised with the early endosome marker, EEA1,
at 0.5 and 1.5 h; after an incubation period of 4 h,
the FPSNIs overlapped with the lysosome marker,
lysotracker blue DND-22. In the early endosome in
particular, the fluorescence intensity of FPSNICy7
was strong whereas the intensity of FPSNICy3 faded
out. In the endosome, PANI in the PSNI was in the
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 FPSNICy3 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
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8 Nano Res.
EB state since its pH was approximately 6 where its
absorption peak was located at 570 nm. Because of
the EB state of PANI, the emission fluorescence of
FPSNICy3 was quenched; hence, the emission of
FPSNICy7 was apparently observed in the
endosomes. On the other hand, after the 4 h
incubation period, the fluorescence intensity of
FPSNICy3 was restored while the intensity of
FPSNICy7 diminished. In the lysosome, PANI in the
PSNI was in the ES state since the lysosomal pH
was lower than 5, and its absorption peak shifted to
770 nm. Thus, owing to the ES state of PANI, the
emission fluorescence of FPSNICy7 was quenched;
therefore, the emission of FPSNICy3 was clearly
observed in the lysosome. The fluorescence
intensity ratio of FPSNICy3/FPSNICy7 was increased
proportionately as the amount of protons increased
owing to the increase in the absorbance ratio (λ570/
λ770) of PANI in the PSNI. As a result of the selective
quenching according the proton level, the
nanoindicators exhibited gradual changes in
fluorescence intensities. Furthermore, there was
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
serve as efficient nanoindicators in intracellular
compartments.
2.6 Fitting equation of FPSNIs for pH analysis in
single cell.
The fluorescence intensity ratio of
FPSNICy3/FPSNICy7 proportionally decreases as the
amount of protons increases owing to the increment
of the absorbance ratio at 550 nm to 750 nm the
excitation wavelength of Cy3 and Cy7, respectively.
Due to such aspect, the nanoindicators performed
fluctuated fluorescence intensities at distinct acidic
compartments whereas organelle markers which
are EEA1 and lysotracker blue DND-22, the
identification dyes for the early endosomes and
lysosomes, showed monotonous intensities at all
distinct times. This reveals that FPSNIs fulfill the
capability of trafficking within early endosomes to
acidic lysosomes in Figure. 6a Moreover, due to the
equation introduced in Figure. 6b,c, the
quantification of intracellular compartment pH was
enabled from the fluorescence intensity ratio. This
feature is advantageous for FPSNIs over fluorescent
acidotropic probe such as lysotracker or EEA1,
which can only display a color and further it is not
able to distinguish the deviation in surrounding pH
in intracellular organelles. These in vitro results
imply that FPSNI may serve as an efficient
nanoindicator in intracellular component.
Figure 6. Fitting equation of FPSNIs for pH analysis in single cell.
(a) In vitro dual emission fluorescence image of HT1080 cells
treated with FPSNICy3 and FPSNICy7 for distinct durations taken
at 4 h by confocal laser scanning microscope. pH titration curve
of the (b) PSNI obtained from the UV-Vis absorbance ratio
λ570/λ770 and (c) fluorescence intensity ratio of FPSNICy3/FPSNICy7
as a function of pH. As pH decreases the absorbance at 570 nm
decreases while the fluorescence intensity of FPSNICy3 increases.
3. Conclusion
We have 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, FPSNIs were transiently
localised in the endosome 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
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.
Synthesis of island-like nanoparticles (IsNP). First
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
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]
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10 Nano Res.
Synthesis of polyaniline - mesosilica shell
nanoindicators (PSNI). For the preparation of PSNI,
50 mg of SIsNP were dissolved in 0.5 mL of
deionized water. Then 1 mL of 1.83 M sulfuric
acid and aniline (43.88 mmol) were added
simultaneously. The mixture was vortexed for 20
min and centrifugation were done two times with
excess water.
Synthesis of fluorophore adsorbed
polyaniline–mesosilica shell nanoindicator
(PEGylated-FPSNI). 100 mg of PSNI were
dissolved in 3 mL of ethyl alcohol. 0.2 mg of Cy3
or Cy7 were individually added and vortexed for
48 h. After FPSNICy3 and FPSNICy7 were formed,
and Si-PEG-COOH (0.4 μmol) were added then
the sample was vortexed again for overnight. The
PEGylated-FPSNI was centrifuged three times
with excess deionized water and re-suspended in
1 mL of PBS.
Characterization of IsNP, SIsNP, PSNI and
PEGylated-FPSNI. The absorbance spectra of
particles were measured using a spectrometer
(Optizen 2120UV, MECASYS, Korea),
respectively. The morphologies were evaluated
using a high-resolution transmission electron
microscope (HR-TEM, JEM-2100 LAB 6 , JEOL
Ltd., Japan) and atomic force microscopy (model
dimension 3100, Digital Instrument Co., USA),
and characteristic bands were confirmed by
Fourier-transform infrared spectroscopy (FT-IR,
Perkin Elmer, USA). To verify diffraction patterns
and band gap energy of inorganic nanoparticles
X-ray diffraction (Rigaku, X-ray Diffractometer
Ultima3) and X-ray photoelectron spectroscopy
(k-alpha, Thermo Scientific, U.K.) were used. For
quantifying the fluorescence of FPSNICy3 at 550
nm excitation and 570 nm emission, and FPSNICy7
at 750 nm excitation and 770 nm emission using a
hybrid multi-mode microplate reader (Synergy
H4, BioTek, USA). Moreover, stained cells were
observed by laser scanning confocal microscope
(LSM 700, Carl Zeiss, Jena, Germany)
Assessment of in vitro cell viability. Cell viability
was quantified using a colorimetric assay based
ontheMTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphe
nyltetrazolium bromide] assay (Roche, Germany).
The HT1080 was obtained from American Tissue
Type Culture (ATCC, USA), and cells were
plated at a density of 2.5 ⅹ 104 cells/100 μL in a
96-well plate and were incubated at 37 ℃ in a
5% CO2 atmosphere. The cells were incubated for
24 h with 100 μL of PSNI re-suspended in MEM
supplemented with 3% FBS and were then rinsed
with 100 μL of PBS (pH 7.4, 1mM). The cells were
then added to 100 μL of MEM supplemented
with 3% FBS, 1% antibiotic anti-mycotic and
non-essential amino acid and were treated with
10 μL of freshly-prepared tetraolium salt. After 2
h, the plate was assayed using an enzyme-linked
immunosorbent assay (ELISA, Spetra MAX 340,
Molecular device USA) at an absorbance
wavelength of 450 nm and a reference
wavelength of 650 nm.
Treatment for intracellular compartment trafficking.
For the seeding of HT1080 cells onto the confocal
dishes, 1x105 cells/mL were seeded and settled for
24 h for well attachment to the dish. HT1080 cells
were rinsed with PBS (pH 7.4, 1 mM) two times
and 0.1 mg of FPSIsNICy3 and 0.2 mg of
FPSIsNICy7 were dispersed in minimum essential
media (MEM) supplemented with 3% fetal
bovine serum (FBS), 1% antibiotic anti-mycotic
and non-essential amino acid (Gibco® , Invitrogen,
USA) After incubation for different hours which
were 30 min, 1 h and 30 min and 4 h were
incubated under 37 ℃ and 5% of CO2 condition.
Immunocytochemistry stains. For staining of
lysosome after incubation for different hours at
37 ℃ during incubation lysotracker (7 μM)
should be treated for 2 h before fixation. At
predetermined time intervals, the cells were
washed with PBS (pH 7.4, 1 mM) two times then,
fixed in 4% paraformaldehyde in PBS for 10 min.
The fixed cells were permeabilized with 0.1%
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11 Nano Res.
Triton X-100 in PBS for 10 min, blocked with 1%
bovine serum albumin (BSA) in PBS for 1 hour,
and stained with rabbit polyclonal anti-EEA1
which is a marker for early endosome, was
diluted in PBS containing 1% BSA (1:200) for 1
hour. After being washed three times with PBS to
remove excess antibodies, the cells were
incubated with secondary antibody of rabbit IgG
conjugated with Alexa Fluor488 (Invitrogen,
USA) diluted in PBS containing 1% BSA (1:300)
for 1 hour. The stained cells were examined using
a laser scanning confocal microscope. All cell
staining procedure were performed at room
temperature.
Acknowledgements
“This work was supported by BioNano
Health-Guard Research Center funded by the
Ministry of Science, ICT & Future Planning
(MSIP) of Korea as Global Frontier
Project" (H-GUARD_2013-11-2072) and “This
work was supported by the national research
foundation of Korea (NRF) grant funded by the
Korea government (MEST)” (2010-0019923)
Electronic Supplementary Material: Supplementary
material (Absorption spectra (Figure S1), TEM images
(Figure 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
online version of this article at
http://dx.doi.org/10.1007/s12274-***-****-* (automatically
inserted by the publisher).
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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, ymhuh@yuhs.ac; Seungjoo Haam, haam@yonsei.ac.kr
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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
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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.
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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
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Nano Res.
Supernant SIsNP_after
0
20
40
60
80
100
120
Mn2+
Fe2+
C
/Cto
tal (%
)
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.
Concentration (g/mL)
10-6 10-5 10-4 10-3 10-2 10-1 100 101
Cell
via
bil
ity (
%)
0
20
40
60
80
100
120
Figure S6. Assessment of cytotoxicity. Growth inhibition assay of HT1080 cells treated with PSNI.