Colourimetric redox-polyaniline nanoindicator for in situ vesicular … · 2014-10-08 ·...

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, [email protected]; Seungjoo Haam, [email protected]

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

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

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


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


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)


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


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]


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%


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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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]


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


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.


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


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.

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