Nanomedicine: Nanotechnology, Biology, and Medicine 2 (2006) 217 – 221
www.nanomedjournal.com
Toxicology
Cerium doping and stoichiometry control for biomedical
use of La0.7Sr0.3MnO3 nanoparticles: microwave
absorption and cytotoxicity study
Sangeeta N. Kale, PhD,a,1 Sumit Arora, MSc,a Kavita R. Bhayani, MSc,a
Kishore M. Paknikar, PhD,a,* Mona Jani, MSc,b Ulhas V. Wagh, PhD,c
Shailaja D. Kulkarni, PhD,d Satish B. Ogale, PhD,e
a
Nanobiotechnology Group, Agharkar Research Institute, Pune, India
b
Computer Science Unit, Fergusson College, Pune, India
c
Interactive Research School for Health Affairs, Pune, India
d
Centre for Materials Characterization, National Chemical Laboratory, Pune, India
e
Physical and Materials Chemistry Division, National Chemical Laboratory, Pune, India
Received 4 September 2006; accepted 14 October 2006
Abstract
La0.7Sr0.3MnO3 nanoparticles doped with cerium (La0.7–x Cex Sr0.3MnO3 where 0 V x V 0.7) as well
as the La1–y Sry MnO3 nanoparticles with different values of y (La/Sr ratio) are evaluated for
cytotoxicity and heating application. Considering hyperthermia as one of the possible application
domains of such materials, the cytotoxicity studies were done on human skin carcinoma and human
fibrosarcoma cell lines. All the samples showed the desired heating effect when subjected to highfrequency exposure at 2.45 GHz. Cytotoxicity studies revealed extremely low cytotoxicity in
Ce-doped samples as well as in samples with a reduced La/Sr ratio. A maximum percentage cell
viability on exposure to these nanoparticles was 95% and 85% for the two groups of samples,
respectively, with a dose of 20 Ag/mL for the x = 0.4 sample. The issues of dopant solubility and
nonstoichiometry are discussed.
D 2006 Elsevier Inc. All rights reserved.
Key words:
Manganite; Nanoparticles; Hyperthermia; Microwave absorption
Nanoscience has witnessed spectacular progress in the
past few years [1,2]. Among the various nanosystems under
active investigation in the biological and pharmaceutical
sector, magnetic nanoparticles are possibly at the forefront
because of the range of their potential applications: drug
delivery systems, hyperthermia applications, magnetic
resonance imaging contrast imaging, magnetic separation,
and magneto-fluids [3 - 6]. A large body of medical evidence
shows that when hyperthermia is used in combination
This work was supported by Nano Cutting Edge Technology Pvt. Ltd.,
Mumbai, India.
4 Corresponding author.
1
On leave from Fergusson College, Pune 411 004, India.
E-mail address: [email protected] (K.M. Paknikar).
1549-9634/$ – see front matter D 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.nano.2006.10.001
with radiation therapy or chemotherapy there is a dramatic
improvement in response rates. Therefore, there is increasing interest in the development of novel and safe materials
that exhibit the desired heating effects.
A review of related research publications reveals the
potential of super-paramagnetic iron oxide nanoparticles in
the context of hyperthermia [7 - 10]. High-frequency alternating fields have been reported to vary between a few
kilohertz to the gigahertz, range and the heating mechanism
is yet to be conclusively determined. A large community of
researchers attributes this heat generation to multiple tracing
of hysteresis curves by the sample in the varying magnetic
fields. The temperature rise is closely related to the shape
and size of these magnetic nanoparticles. Very recently there
have been reports on the evaluations of La0.7Sr0.3MnO3
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S.N. Kale et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 2 (2006) 217–221
(LSMO) nanoparticles for hyperthermia applications [11].
However, there have been no reports on the study of
cytotoxicity in such mixed-valence manganites, and this is
critically important for real-world applications.
We recently examined LSMO nanoparticles as yet
another candidate for heating applications and cytotoxicity.
Because the basic LSMO system has a Curie temperature of
~365 K, it promises self-control over rise in temperature,
which is a highly desirable property. Although these
nanoparticles effectively serve this particular purpose, the
corresponding cytotoxicity is clinically acceptable only
at very low dose values (~20 Ag/mL). In this report we
show that Ce doping in LSMO leads to dramatic improvement in cytotoxicity tolerance without compromising
microwave absorption effectiveness and the related heating
effects relevant to hyperthermia. The choice of Ce was
guided by the following considerations: (1) Ce is reported
to be an agent that improves clinical efficacy, (2) Ce
compounds [12] have pharmacological properties to prevent
burn sepsis and systematic response by fixing burn toxins,
(3) CeNO3 is reported to be a new antiseptic for extensive
burns [13]. Thus, in our context Ce could provide two
advantages: first, it could reduce toxicity of the LSMO
system and second, it would operate as a post-burn healing
agent, which is needed after microwave exposure at a tumor
site. In a separate set of experiments, we also examined the
effect of La/Sr stoichiometry on toxicity and hyperthermia
applicability. This study was motivated by some of our
observations on the Ce-doped samples at high concentrations, as discussed later. We find that this rare earth
stoichiometry also affects cytotoxicity.
Materials and methods
The undoped and Ce-doped LSMO nanopowders were
prepared by the citrate gel route [14]. The samples studied
and discussed here are: La0.7–x Cex Sr0.3MnO3, where x = 0
(LSMO), x = 0.1 (LCSMO10), x = 0.4 (LCSMO40), and
x = 0.7 (CSMO); and La1–y Sry MnO3 ( y = 0.5, y = 0.7)
without Ce doping. For evaluation in the context of the
hyperthermia application, 5 mg of Ce-doped LSMO nanoparticles were suspended in 10 mL of toluene. Although the
nanoparticles were sparsely suspended in toluene, this was
done to ensure the temperature measurement uniformity and
also because of toluene’s lower sensitivity to microwave
absorption. The sample was kept in the center of the
microwave chamber. A standard 2.45-GHz, 900-W microwave radiation was used. The exposure time was varied
from 0 to 300 seconds. After each exposure time, the
temperature of the sample was noted. The entire trial was
repeated five times to ensure repeatability of results.
Corresponding results for dry powder were also recorded
for confirmation.
For the cytotoxicity studies the A-431 (human skin
carcinoma) and HT-1080 (human fibrosarcoma) cell lines
were used. The cells were maintained at 378C in a CO2
Fig 1. XRD spectra of (1) LSMO, (2) LCSMO10, (3) LCSMO40, (4)
CSMO, and (5) CeO2 samples. See text for abbreviations. The LSMO peak
at 32.7 degrees (corresponding to the 112-degree plane) is seen to be shifted
and broadened, indicating Ce incorporation into the lattice; but with higher
Ce doping (x z 0.2), we can observe extra signatures in the spectrum
(indicated by arrows) which correspond to the CeO2 phase.
incubator in a saturated-humidity atmosphere containing 5%
CO2. For in vitro cytotoxicity, the cell viability was assayed
using the tetrazolium salt XTT [15 - 17]. The cells were
seeded (1 104 cells/200 AL per well) in 96-well microtiter
plates in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. After overnight
incubation, the medium was aspirated, and fresh medium
containing LSMO nanoparticles was added. The concentrations of various manganite nanoparticles used were
5–100 Ag/mL. After 24 hours of incubation, XTT reagent
(70 AL) was added to each well after removing the medium.
The individual plates were read after 5 hours at specific
wavelengths (415 nm for soluble dye and 630 nm for cells).
Results and discussion
Figure 1 shows the x-ray diffraction (XRD) spectra of
LSMO and Ce-doped LSMO samples. At low Ce concentration a shift and broadening is noted in the primary LSMO
peak at 32.7 degrees (corresponding to the 112-degree
plane), indicating Ce incorporation into the lattice; yet with
higher Ce doping (x = 0.4) we can observe extra signatures
in the spectrum (indicated by arrows) that correspond to the
CeO2 phase. Also, we do not find signatures of the CSMO
phase, confirming the precipitation of only the CeO2 phase.
The XRD results imply that only a limited amount of Ce is
incorporated into the LSMO perovskite structure, the result
being consistent with the earlier reports [18-20]. Alejandro
et al [18] report 3.6% of Ce out of the perovskite system
in La0.67–x Cex Ca0.33MnO3 (with x = 0.2). CeO2 nanoclusters are observed by Yanagida et al [19,20] in their
Nd0.7Ce0.3MnO3 system.
Figure 2, A shows the hysteresis loops for different
samples taken at room temperature (25-278C). The data for
S.N. Kale et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 2 (2006) 217–221
219
Fig 3. Microwave absorption data for (1) LSMO, (2) LCSMO10, and (3)
LCSMO40 samples. It can be seen that for the LSMO sample, the
temperature between 43 and 458C is reached in only 60 seconds; this
increases to a maximum of 528C in 200 seconds and does not rise further
thereafter. Similar trends are shown in (2) and (3).
Fig 2. A, Hysteresis loops for (1) LSMO (2) LCSMO10 (3) LCSMO40, and
(4) CSMO samples taken at room temperature. The data for pure LSMO
show saturation magnetization of ~36 emu/g. The saturation moment is
seen to decrease progressively with increasing Ce concentration. B,
Dependence of magnetic moment on temperature for (1) LSMO, (2)
LCSMO10, and (3) LCSMO40 samples. It can be seen that the moment
reduces and the transition broadens with increasing Ce in the composition.
pure LSMO show saturation magnetization of ~36 emu/g.
The saturation moment is seen to decrease progressively with
increasing Ce concentration. Many earlier findings [18 -20]
do show magnetic frustrations with Ce doping, resulting in
the reduced magnetic moment. Moreover, at higher Ce
concentrations the dopant comes out of the system, but its
inclusion in mixing chemistry results in a different residual
La/Sr stoichiometry than the La0.7Sr0.3MnO3 in the nanoparticles, causing reduced magnetic moment. Figure 2, B
shows the dependence of magnetic moment on temperature
for different samples. It can be clearly seen that the moment
reduces with increasing Ce in the composition. A broad
nature of transition can be attributed to stoichiometry
imbalance and chaotic magnetic structure.
Figure 3 shows the microwave absorption data (averaged
over five trials) for all the samples. It can be seen that for the
LSMO sample, the temperature between 43 and 458C is
reached in only 60 seconds, then increases to a maximum of
528C in 120 seconds, and does not rise further thereafter.
With Ce doping, the heating results do not seem to be
appreciably affected. As is well expected, the CSMO sample
shows a poor response to the microwave exposure.
The most encouraging results are of cytotoxicity studies,
which were done on human fibrosarcoma (HT-1080) and
human skin carcinoma (A-431) cell lines and are shown in
Figures 4, A and 5, A, respectively. Both the figures
distinctly show extremely reduced toxicity with Ce doping.
This gives a very clear indication of significantly improved
biocompatibility and treatment tolerance of Ce-doped
LSMO magnetic nanoparticles. For a dose of 20 Ag/mL,
the maximum percentage cell viability on exposure to
these nanoparticles is seen to be 90% (A-431) and 68%
(HT-1080) for the x = 0.4 sample. However, to clarify the
role of Ce in reducing toxicity, especially at higher Ce
concentrations, we carefully and separately evaluated the
toxicity issue pertaining to La/Sr stoichiometry, because
addition of Ce in the mixing chemistry implies effective
reduction in the La/Sr ratio, because CeO2 comes out of the
LSMO matrix at higher concentration. Therefore, we
synthesized two additional samples, namely La0.3Sr0.7MnO3
and La0.5Sr0.5MnO3—the former with the reduced La
concentration (similar to the composition of LCSMO40)
and the latter with La/Sr ratio equal to 1 (similar to the ratio
of La/Sr in LCSMO40). It is important to mention here that
these two samples did not give us encouraging microwave
absorption heating effects, probably because of their low
magnetic transition temperature. Yet one more sample was
synthesized with the stoichiometry La0.67Ce0.03Sr0.3MnO3.
This sample exhibited an XRD pattern without any
signatures of CeO2 (hence data not shown). We expect that
this sample will have complete incorporation of Ce in the
LSMO matrix, which was subjected to toxicity studies. Very
interestingly, we saw that toxicity of La0.3Sr0.7MnO3
and La 0.5 Sr 0.5 MnO 3 was also very similar to the
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Fig 4. A, Cytotoxicity studies on human fibrosarcoma (HT-1080) cell lines
subjected to (1) LSMO, (2) LCSMO10, and (3) LCSMO40. The graph
shows significant reduction in toxicity because of Ce incorporation. With
the dose of 20 Ag/mL, for LSMO the cell viability is 40%; cell viability
increases to 68% for identical doses of LCSMO10 and LCSMO40. B,
Cytotoxicity studies on human fibrosarcoma (HT-1080) cell lines subjected
to (4) La0.67Ce0.03Sr0.3MnO3, (5) La0.5Sr0.5MnO3, and (6) La0.3Sr0.7MnO3.
The results are shown for comparison with pure LSMO sample.
Fig 5. A, Cytotoxicity studies on human skin carcinoma (A-431) cell lines
subjected to (1) LSMO, (2) LCSMO10, and (3) LCSMO40. The graph
shows significant reduction in toxicity due to Ce incorporation. With the
dose of 20 Ag/mL for LSMO, the cell viability is 72%; cell viability
increases to 90% for identical doses of LCSMO10 and LCSMO40. B,
Cytotoxicity studies on human skin carcinoma (A-431) cell lines subjected
to (4) La0.67Ce0.03Sr0.3MnO3, (5) La0.5Sr0.5MnO3, and (6) La0.3Sr0.7MnO3.
The results are shown in comparison with pure LSMO sample.
La0.67Ce0.03Sr0.3MnO3 system, which in turn is comparable
with the LSCMO10 and LSCMO40 samples (Figures 4, B
and 5, B). This suggests that with limited Ce incorporation
in the sample (~3%) the toxicity reduces significantly as
compared with pure LSMO. With additional increase in Ce
concentration the toxicity changes marginally. Also, the
change in La/Sr ratio results in increased cell viability.
Ce-doped manganite is not a new system, but all the
work on this system is largely restricted to La-Ce-Mn-O and
has discussed the insolubility property of Ce in the bulk
phases [18,20,21]. Literature review does point us to the fact
that CeO2, recognized as bimpuritiesQ in the La-Ce-Mn-O
matrix, does make the system structurally chaotic causing
competition between the intrinsic and extrinsic ferromagnetism. Furthermore, other investigators also claim that if
thin films are synthesized from these bulk systems, they are
able to achieve limited solubility of Ce as is seen in our
samples. Because Ce doping will change the valence of Mn
to Mn2+, there would be a combination of Mn2+, Mn3+, and
Mn4+ states in the system. There is a competition between
nearest-neighbor interaction due to these multiple coexisting
Mn states. The gradual decrease in saturation moment at
room temperature, with increase in Ce doping, can be
attributed to reduction in magnetic transition temperature
due to the strain involved and mixed phases formed.
Because the ionic radius of Mn2+ is 1.5 times that of
Mn4+, the marginal substitution of Ce on Mn site results in
an appreciably large change in the tolerance factor, thereby
making the system structurally and magnetically chaotic.
Srinivasu et al [22] have reported microwave absorption in
the manganite systems, which decreases near the magnetic
transition temperature. Because our systems show magnetic
S.N. Kale et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 2 (2006) 217–221
transition above the temperature required for hyperthermia
treatments, the microwave absorption, and hence the heating
mechanism can be justified.
In conclusion, we have shown that Ce-doped and
stoichiometry-controlled LSMO nanoparticles are viable
heating agents with extremely low cytotoxicity. Although
the results provide strong evidence on microwave heating of
these nanoparticles, their hyperthermia application domain
is yet to be carefully evaluated —especially in light of the
high frequency and power regime of commercial microwave
chambers. Furthermore, although the cytotoxicity findings
are encouraging, proper biocompatible coatings must be
incorporated on these manganite nanoparticles to make them
good candidates in biomedical applications. This work is
currently in progress.
Acknowledgment
The authors at Agharkar Research Institute acknowledge
the funding by Nano Cutting Edge Technology, Ltd. S.B.O.
would like to thank the Department of Science and
Technology (Government of India) for the award of a
Ramanujan Fellowship and grant. The authors also
thank Dr. Darshan C. Kundaliya of the University of
Maryland (College Park, Maryland, USA) for his help in
magnetic measurements.
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