A Cell-Targeted Non-Cytotoxic Fluorescent Nanogel Thermometer Created with an Imidazolium-Containing Cationic Radical Initiator

Article abstract of DOI:10.1002/anie.201801495

A cationic fluorescent nanogel thermometer based on thermo-responsive N-isopropylacrylamide and environment-sensitive benzothiadiazole was developed with a new azo compound bearing imidazolium rings as the first cationic radical initiator. This cationic fluorescent nanogel thermometer showed an excellent ability to enter live mammalian cells in a short incubation period (10 min), a high sensitivity to temperature variations in live cells (temperature resolution of 0.02–0.84 °C in the range 20–40 °C), and remarkable non-cytotoxicity, which permitted ordinary cell proliferation and even differentiation of primary cultured cells.

Full text of DOI:10.1002/anie.201801495

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Accepted Article
Title: A Cell-Targeted Non-Cytotoxic Fluorescent Nanogel
Thermometer Created with an Imidazolium-Containing Cationic
Radical Initiator
Authors: Seiichi Uchiyama, Toshikazu Tsuji, Kyoko Kawamoto,
Kentaro Okano, Eiko Fukatsu, Takahiro Noro, Kumiko Ikado,
Sayuri Yamada, Yuka Shibata, Teruyuki Hayashi, Noriko
Inada, Masaru Kato, Hideki Koizumi, and Hidetoshi Tokuyama
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201801495
Angew. Chem. 10.1002/ange.201801495
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Angewandte Chemie International Edition
10.1002/anie.201801495
COMMUNICATION
A Cell-Targeted Non-Cytotoxic Fluorescent Nanogel Thermometer
Created with an Imidazolium-Containing Cationic Radical Initiator
[a]
[b]
[a]
[c][d]
Eiko Fukatsu,[a]
Seiichi Uchiyama,* Toshikazu Tsuji,* Kyoko Kawamoto, Kentaro Okano,
[c]
[b]
[b]
[a]
[e]
Takahiro Noro, Kumiko Ikado, Sayuri Yamada, Yuka Shibata, Teruyuki Hayashi,
[f]
[a]
[b]
[c]
Noriko Inada, Masaru Kato, Hideki Koizumi, and Hidetoshi Tokuyama*
Abstract: A cationic fluorescent nanogel thermometer based on
thermo-responsive N-isopropylacrylamide and environment-sensitive
benzothiadiazole was developed with a new azo compound bearing
imidazolium rings as the first cationic radical initiator. This cationic
fluorescent nanogel thermometer showed an excellent ability to
enter live mammalian cells in a short incubation period (10 min), a
high sensitivity to temperature variations in live cells (temperature
resolution of 0.020.84C in the range 2040C), and remarkable
non-cytotoxicity, which permitted ordinary cell proliferation and even
differentiation of primary cultured cells.
over 40 luminescent thermometers have been applied in
measuring the temperature of live cells and even in mapping the
intracellular temperature distribution.[
4]
Very recently, the
relationships between the intracellular temperature and
biological events were actively investigated using luminescent
thermometers,[5] and our fluorescent nanogel thermometer[1b]
unveiled the role of apoptosis signal-regulating kinase 1 (ASK1)
in the thermogenesis of brown adipocytes.[6]
For versatile cellular applications of fluorescent nanogel
thermometers, the ability to spontaneously enter live cells is
highly demanded. Although microinjection and electroporation
are established techniques for introducing nanogels into live
cells,[7] these procedures are too cumbersome for the treatment
of a large number of cells and cannot be applied to a tissue or
an individual organism. Generally, natural and artificial cationic
structures (including positively ionizable amino groups) are
known to support the ability of nanoparticles to enter live cells.[
Nevertheless, this concept cannot be easily applied to
Fluorescent nanogel thermometers consisting of a thermo-
responsive poly(N-isopropylacrylamide) (polyNIPAM) unit and
an environment-sensitive fluorescent unit show temperature-
dependent fluorescence characteristics (e.g., fluorescence
intensity and fluorescence lifetime).[
1,2]
Due to the polyNIPAM
8]
structure, these thermometers are some of the most sensitive
luminescent thermometers ever reported[3] (the average change
in the fluorescence intensity in our record case[1a] is 45%C–1)
and are capable of performing the first intracellular temperature
measurements in a single live cell.[1b] This breakthrough has
accelerated the development of intracellular thermometry, and
fluorescent nanogel thermometers because no cationic radical
initiators are available yet.[9] Here, we developed a newly
designed cationic radical initiator, 2,2-azobis-(2-1,3-dimethyl-
4
1
,5-dihydro-1H-imidazol-3-ium-2-yl))propane triflate (ADIP, Fig.
a), and synthesized cationic fluorescent nanogel
a
thermometer (1) with this first cationic radical initiator. Then, we
confirmed the unique functions of 1 in terms of its ability to enter
live mammalian cells under standard incubation conditions, its
sensitive fluorescence responses to temperature variations
when introduced into live cells, and its remarkably low
cytotoxicity in propidium iodide (PI) staining and cell
[
a]
b]
Dr. S. Uchiyama, K. Kawamoto, E. Fukatsu, Y. Shibata, Dr. M. Kato
Graduate School of Pharmaceutical Sciences
The University of Tokyo
7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan
E-mail: seiichi@mol.f.u-tokyo.ac.jp
T. Tsuji, K. Ikado, S. Yamada, Dr. H. Koizumi
Central Laboratories for Key Technologies
KIRIN Company Limited
proliferation/differentiation assays. Notably, 1 was superior to
[
our previous polymeric thermometers[
1,4a,8d,10,11]
in both its ability
to enter live cells and its non-cytotoxicity.
1-13-5 Fukuura Kanazawa-ku, Yokohama Kanagawa 236-0004,
A new cationic radical initiator, ADIP, was synthesized from
a neutral radical initiator, 2,2-azobisisobutyronitrile (AIBN), in
three steps (Fig. 1b). In the first reaction, the cyano groups of
AIBN were converted to imidate groups under acidic conditions.
Then, N-methylenediamine was attached to the imidate groups,
which was followed by cyclization to form the five-membered
imidazoline rings. In the final reaction, methylation was
performed using methyl trifluoromethanesulfonate to afford ADIP.
The overall yield of the three reactions was satisfactorily high
Japan
E-mail: Toshikazu_2_Tsuji@kirin.co.jp
Dr. K. Okano, T. Noro, Prof. H. Tokuyama
Graduate School of Pharmaceutical Sciences
Tohoku University
Sendai Miyagi 980-8578, Japan
E-mail: tokuyama@mail.pharm.tohoku.ac.jp
Dr. K. Okano
[
c]
[d]
[e]
[f]
Department of Chemical Science and Engineering
Kobe University
Kobe Hyogo 657-8501, Japan
Dr. T. Hayashi, Dr. N. Inada
The Graduate School of Biological Sciences
Nara Institute of Science and Technology
Ikoma Nara 630-0192, Japan
Dr. T. Hayashi
(
90%). ADIP could be stored without decomposition in a freezer
at –18 C.
The ability of ADIP to produce radicals was confirmed by
monitoring the reduction of p-alkoxyiodobenzene[12] (Fig. S1).
The radical production rate corresponding to the thermal
Department of Nutrition
Koshien University
decomposition rate (k
determined from the changes in the absorption spectrum in
solution (Fig. S2). Table 1 lists the experimentally determined k
d
) and half-life (t1/2) of ADIP were
10-1 Momijigaoka Takarazuka Hyogo 665-0006, Japan
Supporting information for this article is given via a link at the end of
the document.
d
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Angewandte Chemie International Edition
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COMMUNICATION
values in various solvents at 50 and 70C and the corresponding
contrast to 1, the conventional anionic nanogel thermometer[1c]
prepared with an anionic radical initiator, ammonium persulfate
(APS), could not enter HeLa cells under the same incubation
conditions (Fig. S9). The incorporation efficiency of 1 in HeLa
cells decreased to 26.211.6% when the incubation temperature
was reduced to 4C, indicating that the introduction of 1 into
HeLa cells at 37C occurred through both biological processes
(i.e., endocytosis) and chemical diffusion through the plasma
membrane. As demonstrated in Figs. 2c and S10, 1 was easily
introduced into other mammalian cell lines, including MOLT-4
(human acute lymphoblastic leukemia, 99.10.8%), HEK293T
(human embryonic kidney, 96.44.1%), RAW264.7 (mouse
macrophage, 94.04.7%), and premature brown adipocytes
t
1/2 values. The k
larger than those of AIBN (3.210
,4-dioxane and water (20:80, w/w, pH 7) ) and the anionic
d
values of ADIP were two orders of magnitude
–
5
–1
s
at 70C in a mixture of
[13]
1
radical initiator potassium persulfate (1.110 s–1 at 50C in
water[ ). The usefulness of ADIP as a cationic radical initiator
was also confirmed by preparing nanogels of styrene (Fig. S3),
NIPAM (Fig. S4), and polyethylene glycol-type monomers (Figs.
S5 and S6). The positive zeta potentials of these nanogels
–6
14]
(
shown in Figs. S3S6) clearly indicated that the cationic
imidazolium group of ADIP was successfully incorporated on the
surfaces of the prepared nanogels.
a)
(
99.50.5%). Once introduced into cells, 1 mainly existed in the
cytoplasm rather than in the plasma membrane (Fig. S11), and
no leakage from the cells was observed.
Next, the fluorescence properties of 1 were investigated.
Based on the established functional mechanism,[16] 1 showed
ADIP
stronger fluorescence (Fig. S12) and a longer fluorescence
lifetime with increasing temperature in aqueous solutions. No
changes in the fluorescence response of 1 were observed with
variations in the environmental pH (410) and ionic strength
b)
HCl gas
AIBN
•2HCl
Toluene-MeOH
rt
(
0.20.4) (Fig. S13), which revealed the functional
independence of 1. The fluorescence responses of 1 were then
confirmed in live cells. When HeLa cells containing 1 were
observed with a confocal laser scanning microscope, the mean
fluorescence intensity of 1 in a single cell at 34C was 1.5 times
higher than that at 26C (Fig. 3a, cell number n=9). The
fluorescence spectrum of 1 in MOLT-4 cells was recorded on a
cell suspension using a spectrofluorometer, and a stronger
fluorescence signal was observed at higher temperatures (Fig.
MeOTf
CH Cl
2
rt
99%
ADIP
Toluene-MeOH
rt
2
9
0% (2 steps)
Figure 1. Novel cationic radical initiator, ADIP. a) Chemical structure and b)
synthetic scheme.
3
b). The fluorescence signal shown in Fig. 3b was confirmed to
originate primarily from 1 inside the MOLT-4 cells (Fig. S14). Fig.
c depicts the temperature-dependent fluorescence intensity of
1 in MOLT-4 cells (i.e., the calibration curve for intracellular
3
d
Table 1. Rate constants for radical production (k , means.d.) and the half-
lives (t1/2, mean) of ADIP under various conditions.
thermometry of MOLT-4 cells[17]
corresponding temperature resolution
)
(left axis) and the
[18]
Solvent
T [°C]
k
d
[10−4s−1
1.13 ± 0.01
]
t
1/2 [min]
102
9.2
58
7.7
6.9
determined from
repeated measurements (right axis). Another temperature-
dependent parameter, the fluorescence lifetime of 1 in MOLT-4
cells, was also monitored by a time-correlated single photon
counting (TCSPC) technique on a fluorometer (Fig. 3d). Fig. 3e
illustrates the temperature-dependent changes in the
fluorescence lifetime of 1 in MOLT-4 cells (i.e., the calibration
Water
50
70
50
70
70
Water
12.6 ± 0.2
1.98 ± 0.02
15.0 ± 0.4
16.7 ± 0.1
Methanol
Water-DMSO (4:1, v/v)
Water-DMSO (1:1, v/v)
curve[17]
resolution[
0.020.40C based on fluorescence intensity measurements
)
(left axis) and the corresponding temperature
18]
(right axis). The temperature resolution of
1
(
and 0.110.84C based on fluorescence lifetime measurements
in the range of 2040C) are as high as those of the most
sensitive conventional fluorescent thermometers applied in live
A cationic fluorescent nanogel thermometer, 1, consisting of
thermo-responsive polyNIPAM units and fluorescent DBThD-AA
units was prepared with ADIP (Fig. 2a). Characterization of 1
was performed using TEM (the diameter was 22.3±5.3
[19]
cells, which include a small organic molecule (0.3C),
an
[1b]
anionic thermo-responsive nanogel (0.290.50C),
a cationic
[11]
thermo-responsive linear polymer (0.011C),
a
green
(
(
mean±s.d.) nm, Fig. S7), zeta potential measurements
[20]
fluorescent protein (1.2C),
nanoparticles containing
europium complex (0.3C), a gold nanocluster (0.30.5C),
a
[22]
+29.11.0 mV, Fig. S7), and UV-vis absorption spectroscopy.[
15]
[21]
As shown in Fig. 2b, 1 was easily introduced into HeLa (human
epithelial carcinoma) cells (incorporation efficiency=91.88.7%)
under optimized incubation conditions (0.05 w/v% in a 5%
glucose solution at 37C) within a short period (10 min) (for
optimization of the incubation conditions, see Fig. S8). In
and
a
nitrogen-vacancy center in a diamond nanocrystal
[23]
(
0.044C).
In the present study, the fluorescence intensity
measurements and the fluorescence lifetime measurements
showed comparable temperature resolutions (i.e., sensitivity). In
further applications, the fluorescence intensity measurement at a
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Angewandte Chemie International Edition
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COMMUNICATION
single wavelength will suffer from a reduction in accuracy
because it is affected by experimental conditions (e.g.,
concentration of the fluorescent thermometers and power of the
excitation source). Although the fluorescence lifetime
a)
Fluorescence
DIC
26 C
34 C
2
0 μm
measurement requires
a longer acquisition time (~1 min)
compared to the fluorescence intensity measurement (~1 sec), it
can be straightforwardly applied to intracellular temperature
mapping due to its independency from the fluctuation of
experimental conditions.[4a]
a)
b)
c)
2
2
.4
.2
2
.8
.6
3
2
2
Excitation: 440 nm
.5
/
C
4
3
3
0
6
2
1
1
1.4
1.2
1
1.5
1
Thermo-responsive
polyNIPAM unit
Ionic terminal
28
2
4
0
0
0
.5
2
0
0.8
500 600 700 800
20 25 30 35 40
Wavelength/nm
Temperature/C
d)
e)
CH2 CH
C
N
O
11
10
9
3
103
102
Excitation: 405 nm
2.5
N
/
C
N
N
2
40
S
3
3
5
0
1.5
1
8
SO2NMe2
01
1
25
2
0
7
0.5
0
Fluorescent
DBThD-AA unit
Crosslinking
MBAM unit
00
1
6
20
25 30 35 40
Temperature/C
0
20 40 60 80
Time/ns
b)
DIC
Fluorescence
Merge
Figure 3. Function of cationic fluorescent nanogel thermometer 1. a) DIC
image (left) and confocal fluorescence images (middle: at 26C, right: at 34C)
of HeLa cells treated with 1 (0.05 w/v%). b) Change in the fluorescence
5
0 μm
spectrum of
1 in live MOLT-4 cells with increasing temperature. c)
Fluorescence intensity response to temperature variations (excitation: 440 nm)
and the corresponding temperature resolution of 1 in MOLT-4 cells. d) Change
in the fluorescence decay curve of
1 in MOLT-4 cells with increasing
temperature. The solid lines shown in panel d) are fitting curves, and the
residual errors are indicated in Fig. S15. e) Fluorescence lifetime response to
temperature variations (excitation: 405 nm, emission: 580 nm) and the
corresponding temperature resolution of 1 in MOLT-4 cells. The error bars
(s.d.) for the fluorescence responses in panels c) and e) are too small to be
visualized.
c)
5
0 μm
One of the remarkable advantages of 1 prepared with the
new cationic radical initiator, ADIP, was its low toxicity to live
cells. In PI assays of cells treated with 1, the viabilities of HeLa
and MOLT-4 cells were determined to be 1000.0 and
Figure 2. Cationic fluorescent nanogel thermometer 1 and its spontaneous
entry into live cells. a) Chemical structure of 1. At lower temperatures, 1 swells
by absorbing water into its interior, where the environment-sensitive DBThD-
AA units are quenched by neighboring water molecules. When heated, 1
shrinks with the release of water molecules, resulting in fluorescence from the
DBThD-AA units. b) Differential interference contrast (DIC) image (left),
confocal fluorescence image (middle), and merged image (right) of HeLa cells
treated with 1 (0.05 w/v%), and c) DIC image (left), confocal fluorescence
image (middle), and merged image (right) of MOLT-4 cells treated with 1 (0.05
w/v%).
98.80.9%, respectively. Next, the cytotoxicity of 1 was more
rigorously assessed by monitoring the proliferation of HeLa cells.
For this experiment, we prepared a new linear copolymer,
poly(NIPAM-co-APTDI-co-DBThD-AA) (2), which had the same
cationic structure as 1 (due to the APTDI units synthesized in
this study), and the previously reported linear cationic copolymer,
[10]
poly(NNPAM-co-APTMA-co-DBThD-AA) (3)
(Fig. 4a) as
references with the ability to enter live cells.[
24]
After
characterization of their fluorescence properties (Fid. S16), the
control linear copolymers (2 and 3) were introduced into HeLa
cells with high incorporation efficiencies (1000.0 and
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Angewandte Chemie International Edition
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COMMUNICATION
9
8.70.3%, respectively, Fig. S17). The numbers of HeLa cells
a)
Thermo-
responsive
unit
Fluorescent
DBThD-AA
unit
treated with 13 were counted in intervals (Fig. 4b). The
proliferation rate of HeLa cells containing 1 was statistically
identical to that of non-treated HeLa cells, whereas the rates for
Cationic
unit
b)
x
y
z
300
●
●
●
●
None (control)
CH2 CH
250
200
1
2
3
2
and 3 were significantly reduced (p=0.025 and 0.033 in the t-


C
O
tests, respectively). The results indicated that 1 is non-cytotoxic
to HeLa cells and that its presence does not interfere with their
growth. Fig. 4c shows representative divisions of HeLa cells
containing 1, in which 1 was observed to distribute in both
divided cells. 1 was also observed to be non-cytotoxic during the
differentiation of brown adipocytes; premature brown adipocytes
treated with 1 were finely differentiated to mature brown
adipocyte in three days (Fig. 4d), and the function of 1 was still
retained in the mature brown adipocytes (Fig. S18). In contrast,
NH
1
1
50
00
50
0
N
N
N
N
N
0
10 20 30 40 50
Time/h
APTMA unit
13 h
APTDI unit
0 h
c)
15 h
21 h
2
and 3 introduced in premature brown adipocytes interfered
5
0 μm
with differentiation and induced their death (Fig. S19). Note that,
despite their extreme importance,[25] cytotoxicity assessments
are sometimes omitted from the studies of nanogels; thus, the
disciplined results of 1 were ideal. The spherical morphology of
1
could limit undesired interactions with biomolecules due to its
smaller surface areas than that of the linear morphology. Thus, 1
is innocuous inside live cells and even enables the differentiation
of brown adipocytes. This feature is another noteworthy
advantage of 1 because the innocuous introduction of cationic
molecules into primary cultured cells (e.g., premature brown
adipocytes) is considerably more difficult than the innocuous
introduction into cell lines (e.g., HeLa cells).[
d)
DIC
Fluorescence
Differentiation
days
3
5
0 μm
26]
Figure 4. Non-cytotoxicity of cationic fluorescent nanogel thermometer 1
toward living cells. a) Chemical structures of the cationic units in control
cationic fluorescent polymeric thermometers 2 and 3. b) Cell growth curves of
HeLa cells containing 1 (black), 2 (blue), 3 (green), and none (control, red)
(means.d.). The t-tests were performed for the data at 50 h. *p < 0.05
compared with the control. c) Division of HeLa cells containing 1. The division
of cells is observed in the red and blue boxes in the images. The observation
time is indicated above each image. d) Differentiation of premature brown
adipocytes treated with 1. DIC images (left) and confocal fluorescence images
(right).
In closing, practical, highly sensitive, and long-term
monitoring of the intracellular temperature is now possible with
cationic fluorescent nanogel thermometer 1, which was prepared
using a novel cationic radical initiator, ADIP. High-throughput
screenings for chemical-induced thermogenesis of mammalian
cells[27] using 1 and related polymers are in progress. Long-term
intracellular temperature mapping is worth investigation by either
combining 1 with fluorescence lifetime imaging microscopy[
or incorporating a reference fluorophore into 1 for ratiometric
imaging.[11] Monitoring of the intracellular temperature during cell
division is another possible application of 1 in biological research
fields.
1c,10]
Keywords: Fluorescence spectroscopy • Fluorescent probes •
Imaging agents • Nanoparticles • Sensors
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2
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K. Kimura, C. Gota, K. Okabe, K. Kawamoto, N. Inada, T. Yoshihara, S.
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Acknowledgements
We are grateful for financial support from the Development of
Advanced Measurement and Analysis System by JST, Grant-in-
Aid for Scientific Research (B) (17H03075) by JSPS, Grant-in-
Aid for Scientific Research (A) (26253001) by JSPS, and Grant-
in-Aid Scientific Research on Innovative Areas (16H00999 and
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COMMUNICATION
4
9
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75, 5926-5935. See also the caption of Fig. 2a.
[17] For a detailed protocol of experiments, see the Supporting Information.
[18] Determined by the established procedure indicated in S. N. Baker, T. M.
McCleskey, G. A. Baker in Ionic Liquids IIIB: Fundamentals, Progress,
Challenges and Opportunities, ACS Symposium Series Vol. 902 (Eds.:
R. D. Rogers, K. R. Seddon), American Chemical Society, Washington,
2005, pp. 171-181, as described in the Supporting Information. Note
that the ″temperature resolution″ was differently expressed as
the ″temperature uncertainty″ in C. D. S. Brites, A. Millán, L. D. Carlos
in Handbook on the Physics and Chemistry of Rare Earths, Vol. 49
(Eds.: J.-C. Bünzli, V. K. Pecharsky), Elsevier B. V., Amsterdam, 2016,
pp. 339-427, but the definitions and calculated values are identical.
[19] S. Arai, M. Suzuki, S. J. Park, J. S. Yoo, L. Wang, N.-Y. Kang, H. H. Ha,
Y.-T. Chang, Chem. Commun. 2015, 51, 8044-8047.
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Angewandte Chemie International Edition
10.1002/anie.201801495
COMMUNICATION
Entry for the Table of Contents (Please choose one layout)
Layout 2:
COMMUNICATION
Seiichi Uchiyama,* Toshikazu Tsuji,*
Kyoko Kawamoto, Kentaro Okano, Eiko
Fukatsu, Takahiro Noro, Kumiko Ikado,
Sayuri Yamada, Yuka Shibata, Teruyuki
Hayashi, Noriko Inada, Masaru Kato,
Hideki Koizumi, Hidetoshi Tokuyama*
N
N
N
N
N
N
Intracellular
thermometry
2
CF3SO3
Cationic
radical initiator
gel
preparation
Page No. – Page No.
A Cell-Targeted Non-Cytotoxic
Fluorescent Nanogel Thermometer
Created with an Imidazolium-
1
2
3
. rapid entry into live cells
. high-sensitivity to T variations
. non-cytotoxicity toward proliferation/differentiation
Containing Cationic Radical Initiator
A cationic fluorescent nanogel thermometer was prepared with a novel cationic
radical initiator. This thermometer showed an excellent ability to enter live
mammalian cells in a short incubation period (within 10 min), a high sensitivity to
temperature variations in live cells (temperature resolution of 0.020.84C), and
remarkable non-cytotoxicity, which permitted ordinary cell proliferation and
differentiation of primary cultured cells.
This article is protected by copyright. All rights reserved.