Fluorescent nanogel of arsenic sulfide nanoclusters

Full text of DOI:10.1002/anie.200900586

Communications
DOI: 10.1002/anie.200900586
Nanotechnology
Fluorescent Nanogel of Arsenic Sulfide Nanoclusters**
Junzhong Wang, Kian Ping Loh,* Zhe Wang, Yongli Yan, Yulin Zhong, Qing-Hua Xu, and
Paul C. Ho
[
4]
Hydrogels are smart and environmentally sensitive materials
that can be chemically tailored to exhibit responses to various
stimuli. The three dimensional networks of hydrophilic
polymers imbibing large amounts of water make hydrogels
soft and elastic and offer excellent properties for many
leukemia. Recently it had been demonstrated that the
nanosized form of realgar shows greater cytotoxic effects for
[
5,6]
cancer cells.
The release of arsenic sulfide nanocluster
from the nanogel thus affords a vehicle for releasing a
“molecular drug.” Indeed, we show herein that such compo-
site nanogels combine the useful properties of fluorescent
labeling and good in vitro cytotoxicity towards cancer cells.
Bulk arsenic sulfide is soluble in liquid ammonia,
anhydrous amines,
[
1]
applications.
Stimuli-sensitive hydrogels responsive to
[
1a]
[1b]
[1b]
[1c]
[1d]
pH, temperature, light, biomoleclules, and drugs
[
7a]
hold great promise as intelligent materials for application in
the biomedical fields. Currently, the primary focus for hydro-
gel research is on their biodegradability and drug-delivery
[
7b]
[7c]
and ethylenediamine, owing to the
chemical bonding of arsenic with amine groups. For example,
the two nitrogen atoms in ethylenediamine can donate their
[
1e,2]
[7c]
kinetics.
The hydrogel network can be held together by
covalent, ionic, and noncovalent forces, and this variety
provides the flexibility for stimuli-responsive assembly and
lone pairs to the empty d orbitals of arsenic to form chelate
bonds. A wide range of amines with different pK values can
a
[3]
disassembly, as well as easy biodegradation. The idea of
integrating semiconductor nanoclusters within the three-
dimensional hydrogel network to form functional hybrids
has not been considered. Such hybrid material could poten-
tially display stimuli-responsive optical and electronic proper-
ties. Herein, we describe a route to synthesize nanosized
hydrogel (nanogel) containing arsenic sulfide nanoclusters as
an integral part of its bonding framework. The design is
motivated by the good chelation properties of arsenic atoms
with the amine groups in small organic molecules and amino
acids; the latter undergoes nanogel formation with hydrogen
bonding. As a result, highly hydrophilic nanogel containing
arsenic sulfide nanoclusters can be produced. Interestingly,
such a composite nanogel was found to retain the quantum-
confined optical properties of the arsenic sulfide nanoclusters,
whereby the specific optical properties can be controlled by
adjusting the pH value of the material. Moreover, the release
of arsenic sulfide nanocluster from the nanogel can be
influenced by changes in pH. This feature is promising for
pharmaceutical applications because the bulk form of arsen-
ic(II) sulfide, realgar, has been traditionally used as a drug for
its antipyretic, antiulcer, anticonvulsive, and anti-schistoso-
miasis actions as well as a treatment for promyelocytic
chelate to the arsenic atoms, thus allowing the tuning of the
zeta potential of the complex. Scheme 1 illustrates the two
stages in the formation of the assemblies: first molecular
complex or nanocluster formation and then gel formation.
Depending on the molar ratio of ethylenediamine to arsenic
sulfide, arsenic sulfide chelates of the amine solvent with
[
5,7c]
different degree of complexation can be formed.
Upon the
addition of carboxylic acid, ethylenediamine is converted into
an amide (Figure S1 in the Supporting Information) or a
secondary amine (Scheme S1 in the Supporting Information),
[*] Dr. J. Z. Wang, Prof. K. P. Loh, Y. L. Yan, Y. L. Zhong, Prof. Q.-H. Xu
Department of Chemistry, National University of Singapore
3
Science Drive 3, Singapore 117543 (Singapore)
Fax: (+65)6779-1691
E-mail: chmlohkp@nus.edu.sg
Z. Wang, Prof. P. C. Ho
Department of Pharmacy, National University of Singapore
3
Science Drive 3, Singapore 117543 (Singapore)
[
**] The work was funded by a National University of Singapore
academic grant. We thank the support of MOE-ARF grant “Nano-
cluster Beam Deposition of Nanostructured Films” R-143-000-265-
Scheme 1. Formation of nanogel assemblies: Stage 1: Formation of
arsenic sulfide (AsS) nanocluster/ethylenediamine solution from bulk
As S mineral. Stage 2: Formation of a AsS nanogel after the addition
of carboxylic acid. The AsS nanocluster can be released from the
nanogel at low pH, and the AsS nanocluster is coordinated by
ethylenediamine to regenerate the nanogel at high pH.
4
4
112.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200900586.
6
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Angewandte
Chemie
which acts as a stabilizer of the arsenic sulfide complex in the
aqueous environment. These ethylenediamine derivatives
have rich hydrogen-bonding properties (Figure S2 in the
Supporting Information) and form a three-dimensional (3D)
hydrogen-bonded cross-linked hydrogel network with arsenic
sulfide as the hydrophobic core and the ethylenediamine
derivative as the hydrophilic linker; this constitutes the
second level of supramolecular bonding and gives rise to
the hydrogel formation.
The ethylendiamine derivatives in the hydrogel can
convert between neutral or anionic state (high pH) and
cationic stage (low pH) by deprotonation and protonation.
Changing the pH value of the hydrogel converts it from a
jelly-like semisolid state at high pH into a semiliquid state at
low pH (Figure 1a). The chelation between nitrogen and
arsenic is readily disrupted by protonation of the nitrogen
The nanogel is made up of several units of AsS nanocluster
coordinated to ethylenediamine derivatives. The size of the
nanogel is related to the concentration of AsS in water. When
the concentration of AsS and ethylenediamine increases, the
small colloidal units cross-link together (aggregate) into 3D
hydrogel through hydrogen-bonding among ethylenediamine
derivatives. As seen in the TEM image in Figure 1, AsS
nanogels consist of 3–4 nm arsenic sulfide nanocluster colloi-
dal units (highlighted in Figure 1c). These colloidal units
appear to be interlinked to form shaped features with
constant interparticle distance. In contrast, Figure 1e shows
that after the release of nanoclusters from the nanogel in
acidic conditions, the arrangement of the nanoclusters
becomes more random. Elemental analysis of released nano-
clusters confirmed the presence of arsenic and sulfur in a ratio
close to 1 (Figure S4 in the Supporting Information). In the
nanogel-encapsulated state, the solubility of arsenic sulfide
(
amine) atom in acidic environment, thus causing the arsenic
À1
sulfide nanocluster to be released from the hydrogel. The
addition of base deprotonates the nitrogen (amine) atom and
causes ethylenediamine to coordinate to the arsenic sulfide
nanocluster and revert to the semisolid hydrogel state. In
humid environments, the hydrogel adsorbs water and swells
nanocluster can be improved to several mgmL of arsenic
sulfide in water (bulk realgar is generally thought to be
insoluble in water at pH 7). We make a distinction between
arsenic sulfide nanoclusters and nanocrystals in this study.
The high-resolution TEM image in Figure 1d shows that these
nanolcusters resemble molecular with sizes less than 1 nm.
Neither electron diffraction pattern (EDX) nor X-ray dif-
fraction (XRD) analysis reveal evidence of crystallinity in the
nanogel. In fact, it can be demonstrated that these arsenic
sulfide–ethylenediamine cluster solutions are the precursor
state to the AsS nanocrystal, and the transformation can be
brought about by thermal treatment (Scheme S2 in the
Supporting Information).
(
TEM images in Figure S3 in the Supporting Information).
The jelly-like 3D network of the hydrogel readily
dissolves and forms colloidal dispersion of nanogel in water.
Taking a cue from the nanogel formation between ethyl-
enediamine, arsenic sulfide, and acrylic acid, we hypothesize
that the use of ethylenediamine-N,N’-bis(3-propionic acid)
À
+
+
À
dihydrate (EPH;
·
OOCC H NH C H NH C H COO
2 4 2 2 4 2 2 4
[
8]
2H O) as the chelating agent with As S should provide a
2 4 4
one-step route to hydrogel formation, as shown in Scheme 1.
Indeed, a transparent gel with similar properties can be
obtained by treating EPH with As S . Alternatively, when two
4
4
molar equivalents of CH =CHÀCOOH and As S –
2
4 4
À1
NH C H NH cluster solution (ca. 5 mgmL ) were mixed,
2
2
4
2
phase separation of As S (confirmed by EDX and XPS) and
4
4
highly crystalline EPH occurred (see the Supporting Infor-
mation). The two phases of As4S4 and EPH could be
combined again to form a half-transparent gel when the pH
value of the mixture was increased to 10 with mild heating
(708C). The resulting gel was similar to that prepared by the
one-step process under basic conditions.
Interestingly, the AsS nanogel was observed to be highly
fluorescent (quantum yield greater than 5% in water) in the
visible region. The fluorescence can be switched on or off by
adjusting the pH value (Figure 2). The fluorescence of the
nanogel decreased significantly upon release of AsS nano-
clusters at acidic conditions and transformation into a semi-
liquid. As discussed above, under acidic conditions, the
coordination of ethylenediamine derivatives to AsS is dis-
rupted following protonation of the amine. As a result,
unpassivated AsS clusters are released. The fluorescence
could be recovered when base is added and ethylenediamine
again coordinates to AsS (semisolid state). The need for
Figure 1. a) Reversible semisolid to semiliquid transition of the AsS
nanogel in response to high or low pH conditions, respectively. 1: The
semisolid state is transparent and strongly fluorescent under UV
illumination; 2: fluorescence is quenched in the semiliquid state with
the release of AsS nanoclusters; 3: recovery of transparent and
fluorescent semisolid state at pH>8.0. b) Scanning transmission
electron microscopic image of the nanogel. c) TEM image showing the
assembled nanolcusters inside the nanogel; the orange line traces the
outline of one nanogel particle. d) High-resolution TEM image of the
nanoclusters. e) TEM image showing AsS nanoclusters that were
released from the nanogel at pH 5, after re-dispersion in ethanol.
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Communications
edge and emission peak are red-shifted with increasing
concentration of the nanogel in water. Resonant energy
transfer through an optical near-field interaction between
[
11]
excited states of quantum dots has been reported. Such
energy transfer can occur when the excitation energy transfer
length is in the nanometer range, which agrees with the
physical dimensions of the nanogel matrix and the trend in
concentration dependence.
Remarkably, upconversion fluorescence was also
observed from the AsS nanogel (Figure 2c). The upconverted
emission peak appears at 510 nm when the nanogel is excited
at 800 nm by a laser. Similar to the one-photon fluorescence,
the upconverted fluorescence spectra are also red-shifted with
increasing concentration of the nanogel. This can be seen in
the photograph of the fluorescence lines from different
concentrations of nanogel in water (Figure 2d). The quadratic
power dependence of the intensity of the fluorescence
suggests that this is a two-photon absorption process, which
[
12]
may originate from intermediate surface states.
The versatility of the AsS nanogel framework is such that
small amino acids can be added to tune its biocompatibility.
For example, instead of ethylenediamine, arginine or argi-
nine/lactic complex can be used as the chelating agent. The
distal end of arginine is capped by a complex guanidinium
Figure 2. Optical properties of the AsS nanogel. a) The photolumines-
cence (PL) spectra of the nanogels before (black line) and after (red
line) the release of AsS nanoclusters. The emission peak from a
[
5]
ca. 3 nm As S nanocrystal (gray peak) is plotted for comparison.
4
4
b) Concentration-dependent absorption and PL spectra of the AsS
nanogel in water (inset: photograph of the nanogel under UV
illumination). There is a red shift of the spectra as the concentration
group with a high pK value of 12, thus ensuring that the
a
group is positively charged in neutral, acidic, and even basic
environments; the positive charge is favorable for cellular
uptake. Because of the conjugation between the double bond
and the nitrogen lone pairs, the positive charge is delocalized,
enabling the formation of multiple hydrogen bonds in the
nanogel. In this case, we designed a AsS nanogel based on the
lactide–ethylenediamine–arginine system, in which arginine
provides the first stage complexation with AsS. We observed
that the nanogel is stable in the Hanksꢀ balanced salt culture
medium, and little aggregation was observed in the cytotox-
icity testing. In vitro stability testing of the nanogels towards
metal ions found no sign of aggregation even in aqueous
À1
of the AsS nanogel is increased from 40 to 200 mgmL . c) Two-
photon fluorescence spectra of the nanogel excited by a laser at
8
00 nm. Inset: quadratic dependence of fluorescence intensity with
power of the laser (slope: 2.02). d) Two-photon fluorescence from AsS
nanogels at three different concentrations in water.
surface passivation for these cluster materials to exhibit
fluorescence suggests that its origin may be related to surface
energy traps that become emissive upon stabilization. These
traps could be related to the charge transfer from the N atoms
in the chelated ethylenediamine ligands to the empty
d orbitals of arsenic, or the recombination of carriers from
À1
2+
À1
3+
solution containing 0.1 mgmL Ca and 50 mgmL Fe or
2
+
Zn .
In vitro cytotoxicity of these AsS nanogels towards certain
[
9]
conduction band and hole surface states.
The emission window (450–600 nm) of the AsS nanogel is
red-shifted significantly from that of arsenic(II) sulfide nano-
crystal (287-450 nm). As shown in Figure 2a, the nanogel has
an emission band centered at 2.8 eV, whereas ca. 3 nm
arsenic(II) sulfide nanocrystal has an emission band centered
human cancer cells was found. Plots of cell survival versus
concentration of As (Figure 3a) show that for selected human
ovarian (OVCAR-3) and cervical (HeLa) cancer cells, the
[5,6]
nanogels show improved cytotoxicity.
For example, the
IC50 value, the concentration of a drug that is required for
50% inhibition in vitro, could be less than 1 mm As for
OVCAR-3 cancer cells after a 72 hour treatment period,
which is much lower than that reported for nanosized
[5]
at 4.3 eV at the same excitation wavelength. Thus, the
optical and electronic properties of the nanocluster is distinct
from that of the nanocrystal. A shift in the absorbance and
emission bands of the AsS nanogel with excitation wave-
length was observed, which reflects not only effects from
nanogel or nanoclusters of different sizes, but also a
distribution of different emissive sites on each nanocluster
[
5,6]
[13]
realgar,
and comparable to that of arsenic trioxide. In
contrast, the nanogels show much less cytotoxicity towards
normal human lung fibroblast cell (MRC-5) and colonic
fibroblast cell (CCD-18Co) and two other cancer cells (MCF-
7 and A549). The in vitro therapeutic efficacy can be
combined with the optical properties of the AsS nanogel;
Figure 3b shows that fluorescence labeling of the cancer cells
could be obtained using the AsS nanogel. Fluorescence
labeling experiments show that cells that were killed exhib-
ited positive fluorescence, corresponding to the uptake of AsS
(Figure S5 in the Supporting Information). Therefore, the
[
10]
surface.
Another interesting effect is the concentration
dependence of the emission, which may be due to partial
overlapping of the absorption edge and the emission band.
Furthermore, the size heterogeneity of the nanocluster in the
nanogel may allow energy transfer to proceed from smaller
clusters to larger ones, leading to enhancement of the red-
shifted emission. Figure 2b show that both the absorption
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Keywords: antitumor agents · arsenic · gels · self-assembly ·
.
toxicology
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6285