Arsonic acid-presenting superparamagnetic iron oxide for pH-responsive aggregation under slightly acidic conditions

Article abstract of DOI:10.1016/j.bmc.2011.02.027

We report the synthesis and characterization of the arsonic acid-presenting superparamagnetic iron oxide (SPIO). We used arsonoacetic acid as the ligand for SPIOs in aqueous media. The surface modification of the SPIOs was accomplished via the ligand exchange from undecanoic acid to the carboxyl moiety of arsonoacetic acid. Consequently, the well-dispersed arsonic acid-presenting SPIOs in water were obtained. We found that the dispersion state of the arsonic acid-presenting SPIOs can be sharply regulated by pH changes in the biological significant region. The well dispersion state of the arsonic acid-presenting SPIOs can be maintained at the neutral pH region. In contrast, the arsonic acid-presenting SPIOs can sensitively form the aggregation below pH 6.1. Moreover, these dispersion states can be controlled reversibly by the pH alteration in the narrow region.

Full text of DOI:10.1016/j.bmc.2011.02.027

Bioorganic & Medicinal Chemistry 19 (2011) 2282–2286
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Arsonic acid-presenting superparamagnetic iron oxide for pH-responsive
aggregation under slightly acidic conditions
a
b
a
a
a,
⇑
Hiroki Minehara , Kensuke Naka , Kazuo Tanaka , Asako Narita , Yoshiki Chujo
a
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
b
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the synthesis and characterization of the arsonic acid-presenting superparamagnetic iron
oxide (SPIO). We used arsonoacetic acid as the ligand for SPIOs in aqueous media. The surface modifica-
tion of the SPIOs was accomplished via the ligand exchange from undecanoic acid to the carboxyl moiety
of arsonoacetic acid. Consequently, the well-dispersed arsonic acid-presenting SPIOs in water were
obtained. We found that the dispersion state of the arsonic acid-presenting SPIOs can be sharply regu-
lated by pH changes in the biological significant region. The well dispersion state of the arsonic acid-pre-
senting SPIOs can be maintained at the neutral pH region. In contrast, the arsonic acid-presenting SPIOs
can sensitively form the aggregation below pH 6.1. Moreover, these dispersion states can be controlled
reversibly by the pH alteration in the narrow region.
Received 5 January 2011
Revised 12 February 2011
Accepted 15 February 2011
Available online 18 February 2011
Keywords:
Arsonic acid
Superparamagnetic iron oxide
pH-responsiveness
Nanoparticle
Ó 2011 Elsevier Ltd. All rights reserved.
1
. Introduction
Since the magnetism of SPIOs (superparamagnetic iron oxides)
with the ability to change the dispersion state under mild acidic
conditions.
Herein, we report the synthesis and characterization of the
arsonic acid-presenting SPIOs. The synthesized SPIOs were well-
dispersed in aqueous media, and the assembly of the arsonic
acid-presenting SPIOs occurs below pH 6.1. In addition, the assem-
bly and dispersion of the arsonic acid-presenting SPIOs can sharply
and reversibly proceed corresponding to the pH changes with bor-
derline at pH 6.1. This is the first report not only about the arsonic
acid-presenting metal nanoparticles but also about the feasibility
of arsonic acid as a pH-responsive unit for pH-sensing probes.
can be enhanced by the assembly, resulting in the creation of neg-
ative contrasts in the image, various kinds of the surface modifica-
tions which can change the dispersibility of SPIOs by the biological
molecules and reactions have been synthesized.1 Indeed, the
monitoring for the significant biological events have been success-
fully accomplished using the surface-modified SPIOs for the appli-
,2
3
cations as biosensors. In particular, the SPIOs which can form the
aggregation under slightly acidic condition are promised to be a
tagging agent for tumor tissues because it is known that the pH
values of the tumor tissues are from 0.4 to 1.0 units lower than
those of the normal tissues (pH 7.4).4
2
2
. Experimental section
The pH-responsiveness of the metal nanoparticles is dominated
.1. General
5
by the dissociation constants of the acid groups of the surfactants.
For example, Toshima and co-workers reported that the carboxylic
acid-modified gold particles can form the aggregation below pH
1H NMR spectra were obtained with a JEOL EX-400 spectrome-
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; then the sample was dried
under ambient conditions. Powder X-ray diffraction (XRD) patterns
were recorded on a SHIMADZU X-ray diffractometer-6000 with
5
a
3
.8. Fiurasek and Reven synthesized phosphonic acid-modified
5b
gold particles which can gather below pH 5.5. Arsonic acid has
higher acid dissociation constant (pKa1 = 4.5, pKa2 = 9.0) than those
a
of carboxylic acid (pK = 4.0) or phosphoric acid (pKa1 = 2.0,
6
pKa2 = 7.5). Hence, arsonic acid derivatives can be expected to be
new series of the surface modifications for providing the particles
°
À1
high-intensity Cu K
a radiation at a scanning rate of 0.02 S in
°
°
2
h ranging from 2 to 90 . FT-IR spectra were recorded on a Perkin
Elmer 1600 infrared spectrophotometer using a KBr disk dispersed
with the powder sample. X-ray fluorescence (XRF) spectra were re-
corded on a Rigaku Primini with PdKa radiation (k = 0.05859 nm).
⇑
Corresponding author. Tel.: +81 75 383 2604; fax: +81 75 383 2605.
E-mail addresses: kazuo123@chujo.synchem.kyoto-u.ac.jp (K. Tanaka), chujo@-
chujo.synchem.kyoto-u.ac.jp (Y. Chujo).
0
968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.027

H. Minehara et al. / Bioorg. Med. Chem. 19 (2011) 2282–2286
2283
Magnetization measurements were performed at room tempera-
ture using MPMS-XL5R SQUID susceptometer, Quantum Design
Co. Ltd. The powder samples were used for XRD, FT-IR, XRF, and
SQUID measurements. Dynamic light scattering (DLS) was mea-
tion times, the samples were added to the same volume of the Aga-
rose XP (Nippon gene) solution at the concentration of 1 wt %. MR
imaging of the samples was carried out using a 7 T Unity Inova MR
Scanner (Varian, Palo Alto, CA). Coronal images of the samples
H
sured to determine the hydrodynamic radii (r ) of the samples
2
were obtained with a T -weighted spin–echo sequence. (Repetition
on an FPAR-1000, Otsuka electronics Co. Ltd.
time (TR) 3000 ms, echo time (TE) 100 ms, the field of view was
4
0 mm, with an image matrix of 256 Â 256. Slice thickness was
2
.2. Materials
5 mm.)
Iron(II) chloride hexahydrate, dithiothreitol and sodium hydride
2.6. Aggregation and dispersion of the SPIOs by changing pH
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 (1) was synthe-
sized according to the previous work.7
To adjust the pH of the samples containing 50 lg/mL of the SPI-
Os, concd. hydrochloric acid (10 N) and 13 M sodium hydroxide
were added. The DLS measurements were carried out at 25 °C.
The CONTIN program was used for data analysis to determine
the average hydrodynamic size.
2
.3. Synthesis of the SPIOs
3
. Results and discussion
Preparation of the SPIOs was according to our previous report.8
Iron(III) chloride hexahydrate (1.081 g, 4 mmol) and iron(II) chlo-
ride tetrahydrate (0.3976 g, 2 mmol) were dissolved in water
As illustrated in Scheme 1, we initially synthesized arsonoacetic
acid (1) as the stabilizer for the SPIOs from chloroacetic acid and
7
(
120 mL). After the addition of oleic acid (0.2 mL) with mechani-
arsenic trioxide in 13 M NaOH aqueous solution. The chemical
structure of the ligand 1 was characterized by 1H NMR, C NMR,
13
cally stirring at 1000 rpm, 15 mL of aqueous ammonium hydroxyl-
ate (28%) was added to the solution all at once. Undecanoic acid
and FT-IR. The undecanoic acid-covered SPIOs were prepared
8
(
0.2 mL) was continuously added to the solution in four additions
every 5 min with stirring at 1000 rpm at 80 °C. After stirring for
0 min, the resulting dark brown suspension was cooled to room
temperature.
according to the previous reports. According to Scheme 2, the 1-
covered SPIO particles were prepared by a ligand exchange from
the undecanoic acid ligand. As shown in Figure 1, the SPIOs before
the ligand exchange were well-dispersed in CHCl . In contrast,
3
3
after the treatment with the ligand 1, the resulting SPIOs showed
good dispersibility only in water. This fact suggests that the ligand
2
.4. Ligand exchange
exchange on the surface of the SPIOs proceeded. The averages of r
H
Undecanoic acid-coated SPIOs (10 mg) were dispersed in tolu-
ene (2 mL), and the ligand 1 (27 mg) as a suspension in methanol
1 mL) was dropwised under sonication. The dispersion was con-
tinuously sonicated overnight. The arsonic acid-presenting SPIOs
were collected and washed with water for five times using a
magnet.
values of the SPIOs before and after the ligand exchange measured
by DLS were determined as 9.1 ± 3.5 nm and 9.4 ± 2.4 nm, respec-
tively. These results indicate that nonspecific aggregation of the
particles should hardly occur.
The ligand 1 has the arsonic and carboxylic acid moieties, which
are potentially able to form the stable bonds to the surface of SPI-
Os. To determine which functional groups between the carboxylic
and arsonic acid are used for the co-ordination to the surface of the
particles, FT-IR measurements were performed. Figure 2 shows the
spectra of the ligands and the SPIOs. The significant peaks assigned
as asymmetrical stretching vibration of C@O were observed around
(
2
.5. Determination of the relaxivities
All samples (100
deionized water were sealed into 5 mm diameter of glass tubes.
For the MR imaging and the calculation of transverse (T ) relaxa-
lL) containing the modified SPIOs in the
À1
9
2
1
600 cm in both the spectra of the ligand 1 and 1-covered SPIOs.
À1
The difference of the peak positions was +11 cm . In contrast,
there was a slight difference in the peak positions of the band de-
rived from As–O and As@O stretching vibration (–1 cm–1). These
results strongly suggest that the ligand 1 should largely co-ordi-
nate to the particles at the carboxylic unit, but not at the arsonic
acid unit. A hard base like carboxylate anion might be favorable
to form higher stable co-ordination with the iron oxide surface.11
Elemental analysis and XRF spectroscopy suggested that the
number of the arsonic acid presented from the single SPIO was
10
Scheme 1. Synthesis and the chemical structure of the ligand used in this study
according to the Ref. 7. Reagents and conditions: (a) As , water, 25 °C, 1 h.
2 3
O
Scheme 2. Synthesis and pH-dependent assembly of the arsonic acid-presenting SPIOs. Reagents and conditions: (a) undecanoic acid, ammonium hydroxide, water, 70 °C,
h; (b) 1, toluene, methanol, sonication for 30 min and then stored at rt for 16 h.
1

2
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H. Minehara et al. / Bioorg. Med. Chem. 19 (2011) 2282–2286
Figure 1. TEM images of the synthetic SPIOs before (left) and after (right) the ligand exchange.
Figure 4. Magnetic hysteresis of the arsonic acid-presenting SPIOs.
plane, using Scherrer formula. The sizes of the undecanoic acid-,
and arsonic acid-presenting SPIOs were estimated as 9.6 nm and
9
.8 nm, respectively. Theses values represent good agreements
Figure 2. FT-IR spectra of the ligands and the SPIOs.
with those of the r values. These results suggest that the ligand
H
exchange occurred only at the surface of the particles and less af-
fected to the magnetic property of the particle cores.
To confirm the superparamagnetism of the particles, the mag-
netization behaviors of the particles were investigated with the
superconducting quantum interference device. As shown in Figure
approximately 600. It has been reported that the interfacial areas
2
12
of carboxylate are between 30 and 40 Å . From these facts, it
can be roughly estimated that the surface of the particles could
be fully covered by the ligand 1. This estimation represents that
the ligand exchange proceeded quantitatively.
Figure 3 shows the results of XRD measurements of SPIOs be-
fore and after the ligand exchange. According to the similar peak
patterns from the samples before and after the ligand exchange,
4, the magnetization curves which represent strong magnetization
(
saturation magnetization is 73.1 memu/g) were obtained from the
arsonic acid-presenting SPIOs. In addition, magnetization can pro-
ceed reversibly without magnetic hysteresis at room temperature.
These facts clearly indicate that the particles have superparamag-
it was confirmed that both SPIOs involve the spinel structure
8
which is typical for magnetite.8 The crystallite size was also esti-
netism. Since SPIOs can be representatively used as a negative
contrast agent in MRI due to the significant ability for enhancing
mated by the half-height width of the strongest reflection (3 1 1)
T
2
relaxation in water tissues, their relaxivities were calculated
from the T -dependency on the concentration. The samples con-
taining the SPIOs dispersed in deionized water with various con-
2
centrations (5, 10, and 25
diameter of glass tubes, and T
2 2
T. The T relaxation time was determined with a T -mapping
lg/mL) were sealed into 5 mm
2
-weighted MRI was performed at
7
mode, and the relaxivity was calculated from the slopes of the lines
according to Figure 5. The arsonic acid-presenting SPIOs provided
À1 À1
approximately 2.5-times larger relaxivty (3.4 mL
lg
s ) than
those of ResovistÒ (1.3 mL
lg
À1 À1 8
s ) which is the commercial con-
trast agent. The thin modification layer on the particles could less
disturb the magnetic interaction of the SPIO core with water
3
d
tissues.
To examine the pH dependency on the assembly of the arsonic
acid-presenting SPIOs, the r values were monitored at variable pH
Fig. 6). The pH titration was performed by adding hydrochloric
acid to the solution. The SPIOs were well-dispersed in the aqueous
media at pH 9. While the r values observed from the dispersions
H
(
Figure 3. X-ray diffraction patterns of the SPIOs before (upper) and after (lower)
the ligand exchange.
H

H. Minehara et al. / Bioorg. Med. Chem. 19 (2011) 2282–2286
2285
of aliphatic arsonic acids are pKa1 = 4.5, pKa2 = 9.0 in the solution.6
The aggregation could be due to protonation of arsonic acid unit
resulting in decrease of static electronic repulsion and increase of
hydrogen bonds between the SPIOs (Fig. 8). Precipitation or color
changes were less observed during the pH titration. Moreover,
average diameters of the particles hardly changed from the pristine
dispersion. These data suggest that the arsonic acid unit can be sta-
bly immobilized on the surface of the SPIOs in this pH region.
4
. Conclusion
In summary, we described the synthesis and characterization of
the arsonic acid-presenting SPIOs. We demonstrated that the
aggregation and dispersion states of the particles can be reversibly
converted corresponding to the pH changes with borderline at pH
6.1. It has been known that the active tumor region tends to be
slightly acidic. The aggregation of SPIOs can generate clear contrast
in MRI. Hence, our materials can be expected to be applied as a
good marker for the detection of the cancer cells with MRI.
Figure 5. T
deionized water, and T
determined by the slope of the linear approximation.
2
dependency on the SPIO concentration. The SPIOs were dispersed in
values were measured at 25 °C. The relaxivities were
2
Acknowledgments
We deeply thank Professor T. Inubushi (Shiga University of
Medical Science) and Dr. M. Morita (Osaka University) for their
kind guidance and significant advices for MRI measurements. We
appreciate Professor S. Kitagawa and Dr. W. Kaneko (Kyoto Univer-
sity) for their help to measure magnetization by superconducting
quantum interference device.
References and notes
Figure 6. The pH-dependent aggregation of the arsonic acid-presenting SPIOs.
1.
(a) Philipse, A. P.; van Bruggen, M. P. B.; Pathmamanoharan, C. Langmuir 1994,
0, 92–94; (b) Srivastava, S.; Samanta, B.; Jordan, B. J.; Hong, R.; Xiao, Q.;
1
Tuominen, M. T.; Rotello, V. M. J. Am. Chem. Soc. 2007, 129, 11776–11780; (c)
Ditsch, A.; Laibinis, P. E.; Wang, D. I. C.; Hatton, T. A. Langmuir 2005, 21, 6006–
6018; (d) Woo, E.; Ponvel, K. M.; Ahn, I.-S.; Lee, C.-H. J. Mater. Chem. 2010, 20,
1511–1515; (e) Matsumoto, Y.; Jasanoff, A. Magn. Reson. Imaging 2008, 26, 994–
998; (f) Qiang, Y.; Antony, J.; Sharma, A.; Nutting, J.; Sikes, D.; Meyer, D. J.
Nanopart. Res. 2006, 8, 489–496; (g) Tanaka, K.; Narita, A.; Kitamura, N.;
Uchiyama, W.; Morita, M.; Inubushi, T.; Chujo, Y. Langmuir 2010, 26, 11759–
11762.
2.
(a) Tromsdorf, U. I.; Bigall, N. C.; Kaul, M. G.; Bruns, O. T.; Nikolic, . M. S.;
Mollwitz, B.; Sperling, R. A.; Reimer, R.; Hohenberg, H.; Parak, . W. J.; Förster, S.;
Beisiegel, U.; Adam, G.; Weller, H. Nano Lett. 2007, 7, 2422–2427; (b) Berret, J.-
F.; Schonbeck, N.; Gazeau, F.; El Kharrat, D.; Sandre, O.; Vacher, A.; Airiau, M. J.
Am. Chem. Soc. 2006, 128, 1755–1761; (c) Zhang, C.; Wängler, B.; Morgenstern,
B.; Zentgraf, H.; Eisenhut, M.; Untenecker, H.; Krüger, R.; Huss, R.; Seliger, C.;
Semmler, W.; Kiessling, F. Langmuir 2007, 23, 1427–1434; (d) Taboada, E.;
Solanas, R.; Rodríguez, E.; Weissleder, R.; Roig, A. Adv. Funct. Mater. 2009, 19,
2319–2324.
3.
(a) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042–6108; (b) Perez, J.
M.; Josephson, L.; Weissleder, R. Chem. BioChem. 2004, 5, 261–264; (c) Yigit, M.
V.; Mazumdar, D.; Kim, H.-K.; Lee, J. H.; Odintsov, B.; Lu, Y. Chem. BioChem.
Figure 7. Reversibility of pH-responsive assembly of the arsonic acid-presenting
SPIOs.
2007, 8, 1675–1678; (d) Tanaka, K.; Kitamura, N.; Morita, M.; Inubushi, T.;
Chujo, Y. Bioorg. Med. Chem. Lett. 2008, 18, 5463–5465; (e) Atanasijevic, T.;
Jasanoff, A. Nat. Protocols 2007, 2, 2582–2589.
(a) Wike-Hooley, J. L.; Haveman, J.; Reinhold, H. S. Radiother. Oncol. 1984, 2,
were below 50 nm in the pH range from 7 to 9, the r
surfactant significantly increased at pH 6.1 with the formation of
the precipitation. Subsequently, the r values reached a plateau be-
low pH 6. These results indicate that the arsonic acid-presenting
SPIOs can assemble with borderline at pH 6.1. In addition, the
assembly/dispersion of the SPIOs can proceed reversibly through
H
value of the
4
.
.
343–366; (b) Gerweck, L. E.; Seetharaman, K. Cancer Res. 1996, 56, 1194–1198;
H
(
c) Gruenberg, J. Nat. Rev. Mol. Cell Biol. 2001, 2, 721–730; (d) Mukheriee, S.;
Ghosh, R. N.; Maxfield, F. R. Physiol. Rev. 1997, 77, 759–803.
(a) Shiraishi, Y.; Arakawa, D.; Toshima, N. Eur. Phys. J. E 2002, 8, 377–383; (b)
Simard, J.; Briggs, C.; Boal, A. K.; Rotello, V. M. Chem. Commun. 2000, 1943–
5
1944; (c) Su, C.-H.; Wu, P.-L.; Yeh, C.-S. Bull. Chem. Soc. Jpn. 2004, 77, 189–193;
a
several pH changes (Fig. 7). The acid dissociation constants (pK )
(d) Selvakannan, PR.; Mandel, S.; Phadtare, S.; Pasricha, R.; Sastry, M. Langmuir
Figure 8. Proposed model of the pH-sensitive dispersion and assembly of the arsonic acid-presenting SPIOs.

2
286
H. Minehara et al. / Bioorg. Med. Chem. 19 (2011) 2282–2286
2
(
003, 19, 3545–3549; (e) Fiurasek, P.; Reven, L. Langmuir 2007, 23, 2857–2866;
f) Naka, K.; Tanaka, H.; Chujo, Y. Polym. J. 2007, 39, 1122–1127.
Fatouros, D. G.; Klepetsanis, P.; Ioannou, P. V.; Antimisiaris, S. G. Int. J. Pharm.
005, 288, 151–156.
9. Lu, Y.; Miller, J. D. J. Colloid Interface Sci. 2002, 256, 41–52.
10. Gründler, H.-V.; Schumann, H.-D.; Steger, E. J. Mol. Struct. 1974, 21, 149–157.
11. Ioannou, P. V. Appl. Organomet. Chem. 2000, 14, 261–272.
12. (a) Imae, T.; Takeshita, T.; Kato, M. Langmuir 2000, 16, 612–621; (b) Naito, K.;
Iwakiri, T.; Miura, A.; Azuma, M. Langmuir 1990, 6, 1309–1315.
6
.
2
7
8
.
.
Palmer, C. S. J. Am. Chem. Soc. 1923, 45, 3023–3028.
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, 1241–1249.