Tumor cell-specific prodrugs using arsonic acid-presenting iron oxide nanoparticles with high sensitivity

Article abstract of DOI:10.1016/j.bmc.2012.06.012

We report the tumor cell-selective prodrugs based on the arsonic acid-presenting iron oxide nanoparticles. We synthesized the well-dispersed nanoparticles having arsonoacetic acid which is composed of the low toxic As(V) form. From the analyses of the reaction products, it is suggested that the reduction by dithiothreitol with arsonoacetic acid and the modified nanoparticles could generate the highly-toxic As(III) species. In the MTT assays, it was found that the cell viabilities of HeLaS3 and especially HepG2 were reduced in the presence of the modified nanoparticles. In contrast, a slight effect on viability was observed with primary mouse hepatocytes. The viabilities showed good agreements with the amounts of intracellular reduced glutathione concentrations. Furthermore, the valid concentrations of the modified nanoparticles for tumor-specific cytotoxicity were similar level in MRI measurements. These results indicate that arsonic acid-presenting nanoparticles should be a good platform for developing highly-sensitive tumor-specific prodrugs.

Full text of DOI:10.1016/j.bmc.2012.06.012

Bioorganic & Medicinal Chemistry 20 (2012) 4675–4679
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Tumor cell-specific prodrugs using arsonic acid-presenting iron oxide
nanoparticles with high sensitivity
a
a
b
a
c
Hiroki Minehara , Asako Narita , Kensuke Naka , Kazuo Tanaka , Moeko Chujo ,
c
a,
⇑
Masaya Nagao , Yoshiki Chujo
Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
Department of Chemistry and Materials Technology, Graduate School of Science and Technology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585, Japan
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the tumor cell-selective prodrugs based on the arsonic acid-presenting iron oxide nanoparti-
cles. We synthesized the well-dispersed nanoparticles having arsonoacetic acid which is composed of the
low toxic As(V) form. From the analyses of the reaction products, it is suggested that the reduction by
dithiothreitol with arsonoacetic acid and the modified nanoparticles could generate the highly-toxic
As(III) species. In the MTT assays, it was found that the cell viabilities of HeLaS3 and especially HepG2
were reduced in the presence of the modified nanoparticles. In contrast, a slight effect on viability was
observed with primary mouse hepatocytes. The viabilities showed good agreements with the amounts
of intracellular reduced glutathione concentrations. Furthermore, the valid concentrations of the modi-
fied nanoparticles for tumor-specific cytotoxicity were similar level in MRI measurements. These results
indicate that arsonic acid-presenting nanoparticles should be a good platform for developing highly-sen-
sitive tumor-specific prodrugs.
Received 3 April 2012
Revised 6 June 2012
Accepted 6 June 2012
Available online 15 June 2012
Keywords:
Arsonic acid
Iron oxide
Nanoparticle
Prodrug
Anticancer drug
Ó 2012 Elsevier Ltd. All rights reserved.
1
. Introduction
The biodistributions of iron oxide nanoparticles with approxi-
The differences of the environmental factors between tumor
and normal cells are used as a trigger for activating prodrugs which
must undergo chemical conversion by metabolic processes before
becoming an active pharmacological agent, highly-specific anti-
cancer drugs are developed, leading to the suppression of the
mately 10 nm of diameter can be readily tuned by the surface
modifications. For example, human serum albumin-tethered
nanoparticles are accumulated at the tumor regions via the
enhanced permeability and retention effect in which nanometer-
sized macromolecules are specifically delivered to the tumor
1
6
administration amounts and undesired adverse effects. Reductive
environments in the tumor cells were focused on the characteris-
7
–9
tics on tumor regions.
Intracellular reductive environments are
2
region. Thereby, because of the superior effect to accelerate the
mainly controlled by modulating the amount of thiol groups in
1
0–12
transverse relaxation of water tissue in NMR measurements, they
are applied as a contrast agent for cancer imaging and diagnosis.
In addition, surface-modified iron oxide nanoparticles were also
applied as vesicles for drug delivery and site-selective drug release.
Various kinds of molecules are directly tethered to the surface and
sustained at the surface modifications.3 By the target reactions or
the micro-environmental changes, the sustained bio-active or
the reduced glutathione (GSH).
Therefore, the thiol-responsive
functional groups or molecules can be a candidate for the designs
of the reduction-responsive prodrugs. Indeed, reductive environ-
ment-responsive prodrugs were developed not only for the imag-
ing of cancer regions but also for anticancer drugs.
Organic arsenic compounds containing the As(V)–C bond are
regarded as a reduction-responsive prodrug. In the presence of
the reducing agent such as GSH, the transformation from As(V)
,4
5
probe molecules can be released. In particular, since the location
of the nanoparticles is monitored by MRI, the releasing of the
loaded drugs is readily confirmed. Hence, the protocols of the sur-
to As(III) occurs with the bond cleavage of As–C, resulting in the
5
13,14
generation of highly-toxic arsenic species.
Since the concentra-
face modification for functionalizing iron oxide nanoparticles are
strongly desired for developing new series of the tumor-specific
drug delivery and releasing system.
tions of GSH in tumor cells are higher than those in normal cells,
the transformation from As(V) to As(III) could be accelerated. Con-
sequently, the tumor-selective cell death would be induced.1
However, there is still room to improve the specificity for biodistri-
bution. In particular, effective accumulation of arsonic compounds
should be necessary to suppress unexpectedly transformation to
As(III) species before the localization at the target sites. Therefore,
5,16
⇑
Corresponding author. Tel.: +81 75 383 2604; fax: +81 75 383 2605.
E-mail address: chujo@chujo.synchem.kyoto-u.ac.jp (Y. Chujo).
0
968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.06.012

4
676
H. Minehara et al. / Bioorg. Med. Chem. 20 (2012) 4675–4679
it should be of great significance to demonstrate the conjugation
systems using arsonic compounds with other biocompatible
materials.
2.4. The evaluation of the number of arsonoacetic acid on the
surface of particles
Herein, we report the highly-sensitive anticancer prodrugs
based on the arsonic acid-presenting iron oxide nanoparticles.
We prepared arsonoacetic acid-tethered nanoparticles and investi-
gated the reactivity with the thiol compound. It was found that the
bond cleavage was induced between the As–C bond, leading to the
generation of highly-toxic As(III) species. From the MTT assays, it
was found that the significant decreases of cell viability were in-
duced in the tumor cell lines in the presence of the modified nano-
particles with similar concentrations for MRI measurements. In
addition, the degree of the toxicity showed good agreements with
the intracellular GSH concentrations. Our findings propose that the
arsonic acid can be a key functional group for developing a new
series of prodrugs as anticancer agents with high sensitivity.
The standard samples with various concentrations were pre-
pared by mixing naked SPIOs and arsonoacetic acid. Then, from
the signal intensity of Fe, As and S elements in the XRF spectra,
the standard curve was made. The ratios between Fe, As and S con-
tents of the samples were calculated by fitting the observed values
on the standard curves. From these procedure, we obtained 4.56
wt % of an As element as an average from the three samples. There-
fore, 1 g of the sample contains 0.0456 g of an As element (=0.112 g
of arsonoacetic acid and 0.888 g of Fe
3
O
4
). According to the density
3
19
of Fe O (5.18 g/cm ) and the average radii of SPIO (ca. 4 nm), the
3 4
molecular weight of the single SPIO particle is determined as
0.84 MDa. Then, it can be estimated that 1 g of the sample involves
1.06 lmol (0.888/0.84 MDa) of SPIO and 610 lmol (0.112/183.98)
of arsonoacetic acid. Finally, the number of arsonoacetic acid per
the single particle was approximately determined as 570 ± 40.
2
2
. Experimental section
2
.5. The reaction of arsonoacetic acid with dithiothreitol (DTT)
.1. General
1H NMR spectra were obtained with a JEOL EX-400 spectrome-
To the solution containing arsonoacetic acid (0.2 mmol) in D
2
O
(
2
1.0 mL), DTT (0.2 or 0.4 mmol) was added and stirred at 40 °C for
ter (400 MHz). Transmission electron microscopy (TEM) was per-
formed using a JEOL JEM-100SX operated at 100 kV electron
beam acceleration voltage. One drop of the sample solution was
deposited onto a copper grid and the excess of the droplet was
blotted off the grids with filter paper. Subsequently the sample
was dried under ambient conditions. Powder X-ray diffraction
1
h. From the H NMR spectra, conversions of arsonoacetic acid
were determined from integral ratios between the methylene pro-
tons in arsonoacetic acid and the methyl protons in acetic acid.
2
.6. The reaction of the modified nanoparticles with DTT
(
XRD) patterns were recorded on a SHIMADZU X-ray diffractome-
The modified nanoparticles (4 mg) were dispersed in water
10 mL) containing 10 mM or 100 mM DTT and stirred at 40 °C
ter-6000 with high-intensity Cu K
0
a
radiation at a scanning rate of
À1
(
.02° S in 2h ranging from 2° to 90°. FT-IR spectra were recorded
for 2 h. The nanoparticles were collected by the magnets and
washed with 20 mL of water five times. Then, the samples were
dried in vacuo, and XRF analyses were executed to estimate the ar-
sine content.
on a Perkin Elmer 1600 infrared spectrophotometer using a KBr
disk dispersed with the powder sample. X-ray fluorescence (XRF)
spectra were recorded on a Rigaku Primini with PdK
k = 0.05859 nm). The powder samples were used for XRD, FT-IR,
XRF, and SQUID measurements. Dynamic light scattering (DLS)
a radiation
(
2
.7. Cell viability assay
was measured to determine the hydrodynamic radii (r
H
) of the
samples on an FPAR-1000, Otsuka electronics Co., Ltd.
Primary mouse hepatocytes, HepG2, and HeLaS3 cells were
used to test the toxic effects of various samples as assessed in
the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
2
.2. Materials
Iron(II) chloride hexahydrate, dithiothreitol and sodium hydride
(
MTT) assay. Cells were grown in Dulbecco’s modified Eagle’s med-
ium (DMEM) containing 10% fetal bovine serum, 100 units/mL
penicillin, 100 g/mL streptomycin and incubated at 37 °C in
humidified 5% CO . Primary mouse hepatocytes were prepared
were purchased from Wako Pure Chemical Industries, Ltd (Osaka,
Japan). Iron(III) chloride tetrahydrate was purchased from Kanto
Chemicals Co., Ltd (Tokyo, Japan). All reagents were used as sup-
plied, unless stated otherwise. Arsonoacetic acid was synthesized
according to the previous work.17
l
2
2
0
by the method of collagenase perfusion from 8 week-old male
ICR mice. Briefly, livers were perfused at 37 °C for 5 min with
SC-1 solution consisting of 8000 mg/L NaCl, 400 mg/L KCl,
8
8.17 mg/L NaH
HEPES, 350 mg/L NaHCO
.25, followed by digestion at 37 °C for 6 min with 0.05% collage-
nase dissolved in SC-2 solution consisting of 8000 mg/L NaCl,
2
PO
4
Á2H
2
O, 120.45 mg/L Na
, 190 mg/L EGTA, 900 mg/L glucose, pH
2 4
HPO , 2380 mg/L
2
.3. Synthesis of the iron oxide nanoparticles
3
7
Preparation of the nanoparticles was according to our previous
1
8
report. Iron(III) chloride hexahydrate (1.081 g, 4 mmol) and iro-
n(II) chloride tetrahydrate (0.3976 g, 2 mmol) were dissolved in
water (120 mL). After the addition of oleic acid (0.2 mL) with
mechanically stirring at 1000 rpm, 15 mL of aqueous ammonium
hydroxide (28%) was added to the solution all at once. Undecanoic
acid (0.2 mL) was continuously added to the solution in four addi-
tions every 5 min with stirring at 1000 rpm at 80 °C. After stirring
for 30 min, the resulting dark brown suspension was cooled to
room temperature. Undecanoic acid-coated nanoparticles (10 mg)
were dispersed in toluene (2 mL), and arsonoacetic acid (27 mg)
as a suspension in methanol (1 mL) was added under sonication.
The dispersion was continuously sonicated overnight. The arsonic
acid-presenting iron oxide nanoparticles were collected and
washed with water five times using a magnet.
4
2
7
00 mg/L KCl, 88.17 mg/L NaH
380 mg/L HEPES, 350 mg/L NaHCO
.25. The digested liver was excised, cut into small pieces and dis-
2
PO
4
Á2H
2
O, 120.45 mg/L Na
2
HPO
O, pH
4
,
3
, 560 mg/L CaCl
ÁH
2 2
persed by pipetting. The resulting suspension was filtered through
a 70 m mesh and hepatocytes were collected by centrifugation at
l
5
0Âg for 1 min. The cells were washed with DMEM 4 times and
cultured in DMEM containing 10% fetal bovine serum and 10 nM
dexamethasone. One day before the nanoparticle addition, HepG2
cells or Hela S3 cells were seeded at 1500 cells/100
well plate, while primary hepatocytes were seeded at 15,000 cells/
00 l/well. Three days after the cells were incubated with the
modified nanoparticles, 10 l of 5 mg/mL MTT in phosphate buf-
fered saline was added to each well, and the plates were kept in
ll/well in a 96
1
l
l

H. Minehara et al. / Bioorg. Med. Chem. 20 (2012) 4675–4679
4677
a CO
removed, the cells were lysed by adding 100
.01 M NH Cl and were incubated at 37 °C overnight. The degree
of MTT reduction (i.e., cell viability) in each sample was subse-
quently assessed by measuring absorption at 600 nm using a plate
reader. The absorbance values measured from the three wells were
averaged, and the percentage MTT reduction was calculated by
dividing this average by the absorbance measured from a control
sample lacking the modified particles.
2
incubator for an additional 4 h. After MTT solution was
water. In addition, non-specific aggregation of the particles was
hardly observed during the analyses.
ll of 10% SDS,
0
4
Initially, the reaction products from arsonoacetic acid with the
thiol compound were investigated. Ioannou et al. reported that thi-
ols were able to break the C–As bond resulting in production of
1
3,14
As(III) compounds.
Correspondingly, the arsonic acid-contain-
ing liposomes were induced to show the strong toxicity triggered
by the reduction leading to the arsenic acid releasing in the cells.
Based on these facts, we evaluated the reaction products from
the reductive degradation of arsonoacetic acid. Figure 1 represents
1
the H NMR spectra of the reaction solutions containing arsonoace-
3
. Results and discussion
tic acid in the presence of the same equivalent of DTT before and
after 2 h incubation. The characteristic peak appeared at 1.9 ppm
which could be assigned as the methyl group of acetic acid. In addi-
tion, the time-courses of the amounts of arsonoacetic acid were
We designed the arsonoacetic acid-presenting iron oxide nano-
particles for highly-sensitive prodrugs. Iron oxide nanoparticles are
a suitable platform to introduce the drugs into the cells by anchor-
ing at the surfaces. In addition, the stable coordination of arsono-
acetic acid to the surface of iron oxide can be formed using the
carboxyl group and maintained under the biological conditions.
Thereby, the arsonic acid moiety should be effectively accumulated
monitored in the presence of various concentrations of DTT (Table
1
1
). The reaction yields were estimated with the H NMR spectros-
copy by calculating the conversion of arsonoacetic acid. The reac-
tions completed by 2 h, and the observed conversion increased
from 49% to 70% corresponded to the increase of the molar ratio
of DTT against arsonoacetic acid. These results indicate that the
reductive bond cleavage at the C–As bond, resulting in the produc-
tion of highly-toxic arsenic compounds.
Same reactions were executed with the modified nanoparticles.
The reactions were monitored with the XRF spectroscopy by eval-
uating the existence ratio of the C–As bond against the Fe–O bond.
Figure 2 shows the XRF spectra of the modified nanoparticles be-
2 3 2
into the cells. By transforming As(V) in –CH AsO H group to As(III)
by thiol groups, the highly-toxic arsenic species would be gener-
1
3,14
ated.
Moreover, the minimum length of the aliphatic arsonic
acid should hardly disturb the magnetic interaction between iron
oxide and water tissue. Indeed, we have observed the clear con-
trast in T
of the commercial contrast agent.
2
-weighted MR images with higher sensitivity than that
21,22
Hence, arsonoacetic acid is
the simple but highly-sophisticated molecules as a candidate for
the prodrug which can be traced in MRI.
fore and after the treatment with DTT. The peak arising from AsK
a
was observed at 2h = 34° before the reaction. On the other hand,
the peak decreased after the reaction with 1 mM DTT. These data
clearly indicate the bond cleavage between the C–As bond in
arsonoacetic acid and the release of the arsenic element from the
surface of the nanoparticles. In addition, from the measurements
of the time-courses of the changes of arsenic element in the mod-
ified nanoparticles, the residual arsine content at the modified
nanoparticles decreased to 33% by reacting with 1 mM DTT
The arsonic acid-presenting nanoparticles were synthesized via
the ligand exchange from undecanoic acid to arsonoacetic acid
according to our previous report.18 The chemical structure of the
modified nanoparticles is shown in Scheme 1. The identifications
of the modified nanoparticles were executed with TEM and DLS
for measuring the diameters (7.6 nm ± 1.4 nm from TEM,
9
.4 ± 2.4 nm from DLS), and XRD. Elemental analysis and XRF spec-
troscopy suggested that the number of the arsonic acid presented
from the single SPIO was approximately 600. It is reported that
(Fig. 3). In the presence of 10 mM DTT, the residual arsine content
further decreased to 22% after 2 h reactions. These data support the
reductive bond cleavage between the C–As bond by DTT, leading to
the generation of inorganic arsenic compounds. In particular, it is
said that the intracellular concentrations of thiol groups are be-
the interfacial areas of carboxylate were between 30 and
4
0 Å2.
23,24
From these facts, it is roughly estimated that the surface
of the particles could be fully covered by arsonoacetic acid. It
should be mentioned that by FT-IR measurements, arsonoacetic
acid is confirmed to coordinate to the surfaces of the iron oxide
particles at the carboxylic unit, but not at the arsonic acid unit. This
means that arsonoacetic acid is immobilized onto the surface of the
particles. The modified nanoparticles showed good dispersibility in
1
0–12
tween 1 mM and 10 mM.
Therefore, it can be presumed that
the inorganic arsenic species were generated and showed the cyto-
toxicity in our experiments. To examine the coordination at the
surfaces, the FT-IR spectra were measured (Fig. 4). The strong band
À1
at 579 cm assigned as the Fe–O stretching vibrations related to
the magnetic core was observed. The significant bands observed
À1
at 1100 cm corresponding to As–O and As@O stretching vibra-
2
5
tions disappeared after the reaction. These results suggest the
Scheme 1. Synthesis and the chemical structure of the ligand used in this study.
Reagents and conditions; (a) As
2
O
3
, water, 25 °C, 1 h; (b) oleic acid, undecanoic acid,
Figure 1. 1H NMR spectra of the solutions (run 3) containing arsonoacetic acid and
4
NH OH, water, 80 °C, 30 min; (c) 1, methanol, toluene, ultrasonic, rt, 16 h.
2
DTT in D O before and after 2 h incubation at 40 °C.

4
678
H. Minehara et al. / Bioorg. Med. Chem. 20 (2012) 4675–4679
Table 1
Results of conversion of arsonoacetic acid to acetic acid in the presence of DTT
Run
DTT/1 (mol/mol)
Time (h)
Conversion (%)
1
2
3
4
5
6
1
1
1
2
2
2
1
2
5
1
2
5
27
47
49
49
68
70
Scheme 2. Proposed reaction scheme of the reduction of arsonoacetic acid with
DTT.
tive conditions in the tumor cells could enhance the generation
of inorganic arsenic acid species. The bands corresponding to
À1
C@O stretching vibration were observed around 1400 cm and
À1
1
600 cm from both samples. This fact suggests that the coordina-
tion of carboxyl groups could be maintained during the reductive
decomposition of arsonoacetic acid. These results are summarized
as that the arsonoacetic acid at the surface of the iron oxide nano-
particles should be reduced by thiol groups and converted to acetic
acid (Scheme 2).
Figure 2. XRF spectra of the modified nanoparticles before and after reaction with
mM DTT.
1
The cytotoxicity of the modified nanoparticles was examined
with three kinds of cells (HepG2, HeLaS3, and primary mouse
hepatocytes). The MTT assays were executed in the presence of
2 3
the modified nanoparticles, arsenic trioxide (As O ) as a positive
control, and the water-dispersive naked nanoparticles as a nega-
tive control with various concentrations. Figure 5 represents the
cell viabilities after 72 h incubation with the samples. The strong
toxicity was confirmed from As O in all cells. The naked nanopar-
2 3
ticles hardly affected the cell viability through the entire concen-
tration range. Interestingly, the viabilities of the cancer cell lines
(
HepG2 and HeLaS3) significantly decreased in the presence of
the modified nanoparticles. These data clearly indicate that
arsonoacetic acid tethered to the nanoparticles should be responsi-
ble for cancer cell-selective cytotoxicity. Moreover, the viabilities
were corresponded to the GSH concentrations of each cell line as
7
–9
previously reported (Table 2).
It is strongly suggested that the
Figure 3. Time-courses of the changes of the residual arsine contents with the
modified nanoparticles determined with XRF measurements after the reaction with
DTT.
reduction of As(V) to As(III) should occur and yield highly-toxic
species. The cytotoxicity in HepG2 was obviously observed with
1 lg/mL of the modified nanoparticles. On the other hand, less sig-
nificant effect on those of normal cells was observed even in the
presence of 100-fold higher concentration than active one for
HepG2. This concentration was a similar level to the detection limit
of the conventional MRI contrast agents based on the iron oxide
nanoparticles. These facts suggest that enough concentrations of
the modified nanoparticles over the active amounts were obtained
by the same administration manner with iron oxide nanoparticles
used for an MRI contrast agent.
4
. Conclusion
We demonstrate that the arsonic acid-presenting iron oxide
nanoparticles have anti-cancer activity with high selectivity. The
viabilities of the tumor cell lines as HepG2 and HeLaS3 were signif-
icantly reduced by adding the modified nanoparticles. On the other
hand, the primary mouse hepatocytes were less affected even in
the presence of 10-times. Such tumor cell-selective cytotoxicity
could be explained by the intracellular concentrations of each cell
line. The amount of GSH and the activities of GSH-relating enzymes
are significant biomarkers such for apoptosis or responses of the
vital cells toward stressful materials. Thus, the arsonic acid-based
Figure 4. FT-IR spectra of the modified nanoparticles before and after reduction
with DTT.
cleavage of the C–As bond and the generation of toxic inorganic
arsenic acid species. The tumor-specific cytotoxicity could be trig-
gered by reducing arsonoacetic acid. The relatively-higher reduc-

H. Minehara et al. / Bioorg. Med. Chem. 20 (2012) 4675–4679
4679
Figure 5. Effect of As
2 3
O , naked iron oxide nanoparticles, and the modified nanoparticles on the viability of normal cells ((a) primary mouse hepatocytes) and tumor cell lines
(
(b) HepG2 and (c) HeLaS3). Cells were incubated with various concentrations of the samples for 72 h. Results are expressed as viability (% viable cells in comparison with the
control) versus the arsenic content for upper three graphs.
Table 2
7. Murakami, C.; Hirakawa, Y.; Inui, H.; Nakano, Y.; Yoshida, H. Biosci. Biotechnol.
Biochem. 2002, 66, 1559.
GSH concentrations of three cell lines
8
.
Toyoda, H.; Mizushima, T.; Satoh, M.; Iizuka, N.; Nomoto, A.; Chiba, H.; Mita,
M.; Naganuma, A.; Himeno, S.; Imura, N. Jpn. J. Cancer Res. 2000, 91, 91.
Ueno, S.; Susa, N.; Furukawa, Y.; Aikawa, K.; Itagaki, I.; Komiyama, T.;
Takashima, Y. Jpn. J. Vet. Sci. 1988, 50, 45.
Cell type
GSH concentration (nmol/mg)
35.4a
9.
HepG2
HeLa
Mouse primary hepatocyte
b
13.4
7
1
1
0. Deneke, S. M.; Fanburg, B. L. Am. J. Physiol. 1989, 257, L163.
1. Carmel-Harel, O.; Storz, G. Annu. Rev. Microbiol. 2000, 54, 439.
c
a
b
c
12. Nordberg, J.; Arnér, E. S. J. Free Radical Bio. Med. 2001, 31, 1287.
13. Ioannou, P. V.; Lala, M. A. Appl. Org. Met. Chem. 2000, 14, 261.
Ref. 7.
Ref. 8.
Ref. 9.
1
4. Lala, M. A.; Ioannou, P. V. J. Inorg. Biochem. 2003, 97, 331.
5. Gortzi, O.; Antimisiaris, S. G.; Klepetsanis, P.; Papadimitriou, E.; Ioannou, P. V.
Eur. J. Pharm. Sci. 2003, 18, 175.
1
16. Gortzi, O.; Papadimitriou, E.; Kontoyannis, C. G.; Antimisiaris, S. G.; Ioannou, P.
V. Pharm. Res. 2002, 19, 79.
7. Palmer, C. S. J. Am. Chem. Soc. 1923, 45, 3023.
materials could be applied not only for monitoring these signifi-
cant biological events but also for modulating cell activities using
the reaction from As(V) to As(III) as a trigger.
1
1
8. Minehara, H.; Naka, K.; Tanaka, K.; Narita, A.; Chujo, Y. Bioorg. Med. Chem. 2011,
19, 2282–2286.
9. (a) Linde, D. R.; Frederikse, H. P. R. Handbook of Chemistry and Physics, 75th ed.;
CRC: Boca Ratan, FL, 1995; (b) Amstad, E.; Gillich, T.; Bilecka, I.; Textor, M.;
Reimhult, E. Nano Lett. 2009, 9, 4042.
1
References and notes
20. Seglen, P. O. Methods in Cell Biology, In 2nd ed.; Preseon, D. M., Eds.; Academic
Press: New York, 1976; Vol. 13, Chapter 4, pp 29–83.
1
2
.
.
Xie, J.; Liu, G.; Eden, H. S.; Al, H.; Chen, X. Acc. Chem. Res. 2011, 44, 883.
Xie, J.; Chen, K.; Huang, J.; Lee, S.; Wang, J.; Gao, J.; Li, X.; Chen, X. Biomaterials
21. Tanaka, K.; Narita, A.; Kitamura, N.; Uchiyama, W.; Morita, M.; Inubushi, T.;
Chujo, Y. Langmuir 2010, 26, 11759.
2
010, 31, 3016.
3
.
.
Hirata, N.; Tanabe, K.; Narita, A.; Tanaka, K.; Naka, K.; Chujo, Y.; Nishimoto, S.
Bioorg. Med. Chem. 2009, 17, 3775.
Naka, K.; Narita, A.; Tanaka, Y.; Chujo, Y.; Morita, M.; Inubushi, T.; Nishimura, I.;
Hiruta, J.; Shibayama, H.; Koga, M.; Ishibashi, S.; Seki, J.; Kizaka-Kondoh, S.;
Hiraoka, M. Polym. Adv. Technol. 2008, 19, 1241.
22. Tanaka, K.; Kitamura, N.; Morita, M.; Inubushi, T.; Chujo, Y. Bioorg. Med. Chem.
Lett. 2008, 18, 5463.
23. Imae, T.; Takeshita, T.; Kato, M. Langmuir 2000, 16, 612.
24. Naito, K.; Iwakiri, T.; Miura, A.; Azuma, M. Langmuir 1990, 6, 1309.
25. Gründler, H.-V.; Schumann, H.-D.; Steger, E. J. Mol. Struct. 1974, 21, 149.
4
5
6
.
.
Mornet, S.; Vasseur, S.; Grasset, F.; Dugnet, E. J. Mater. Chem. 2004, 14, 2161.
Tanabe, K.; Harada, H.; Narazaki, M.; Tanaka, K.; Inafuku, K.; Komatsu, H.; Ito,
T.; Yamada, H.; Chujo, Y.; Matsuda, T.; Hiraoka, M.; Nishimoto, S. J. Am. Chem.
Soc. 2009, 131, 1598.