Journal of Biomaterials and Nanobiotechnology, 2011, 2, 557-560
doi:10.4236/jbnb.2011.225066 Published Online December 2011 (http://www.scirp.org/journal/jbnb)
557
Study of Safety of Molybdenum and
Iron-Molybdenum Nanoclaster Polyoxometalates
Intended for Targeted Delivery of Drugs*
Alexander A. Ostroushko1, Irina G. Danilova2#, Irina F. Gette2, Svetlana Yu. Medvedeva2,
Margarita O. Tonkushina1, Anna V. Prokofieva1, Maria V. Morozova1
1Ural State University Named after A. M. Gorky, Ekaterinburg, Russia; 2Institute of Immunology and Physiology, Ural Branch, Rus-
sian Academy of Sciences, Ekaterinburg, Russia.
Received September 16th, 2011; revised October 29th, 2011; accepted November 20th, 2011.
ABSTRACT
Nanocluster polyoxometalates with a structure of buckyball (fullerene) are a promising means of targeted delivery of
drugs in the body. In this paper, based on an analysis of histological sections of liver and kidney and peripheral blood,
showed a significant reduction of toxicity of buckyballs, containing ions of molybdenum (VI), the substitution of Mo (V)
ions to Fe (III), in contrast to buckyballs based on Mo (V). The absence of accumulation of molybdenum in rats with a
daily intramuscular injection of aqueous solutions of both drugs within a month was confirmed.
Keywords: Buckyball, Polyoxometalates, Direct Delivery, Toxicity
1. Introduction
The search for effective and safe means of targeted de-
livery of drugs is one of the most pressing issues of nano-
biotechnological studies [1-3]. Candidates for the role
nanocontainers or nanokernel is a group of compounds,
united by the name of polyoxometalates (POM) with the
structure of keplerates (buckyballs or fullerenes) [4,5].
There is a number of prerequisites for success when us-
ing this type of compounds: water solubility, an ability to
reversibly absorb various organic compounds [6], to
form complexes [7], an ability to decompose within a
certain time [6,7] and move under the influence of weak
electric fields [6].
One of the most typical buckyball representatives is
molybdenum buckyball Мо132 (1). Polyoxometalates
with a structure of keplerate were first synthesized about
12 years ago under the guidance of Professor A. Müller
(Bielefeld University, Germany). These compounds
(Мо132) consist of coordination oxygen polyhedra sur-
rounding ions of molybdenum, as well as stabilizing
ligands (acetate ion, etc.), water molecules. Coordination
polyhedra by self-assembly in aqueous solutions form up
the structure close to spherical, containing a whole 132
molybdenum ion with a charge of 6+ and 5+. In water
the resulting compounds form polyanions of about 2.5
nm. A related compound is an iron-molybdenum bucky-
ball Mo72Fe30 [5] (2), in which Mo(V) ions are replaced
by Fe(III). Researchers’ attention was attracted by the
internal cavity in keplerates structure and “windows”
through which the exchange of water molecules and
various substances is possible. Depending on the acidity
of the solutions and their concentrations kepleratys de-
compose into simpler compounds of molybdenum, simi-
lar to other known polyoxometalates.
VI V
4 427260372330272
342
(NH)[MoMoO(H CCOO)(HO)]
30H CCOONH250H O (1);
7230 2523122 722
2282 2912
[MoFeO(CH COO){MoO(H O)}
{H Mo O (HO)}(H O)]150H O (2).
Molybdenum and iron are essential micronutrients.
Biological function of molybdenum is its participation in
redox reactions as a cofactor oxidases, including hy-
poxanthine oxidase, xanthine oxidase, catalyzing the
formation of uric acid from purine bases [8,9]. Daily re-
quirement for molybdenum for the man is 0.15 - 0.5 mg,
its soluble compounds are easily absorbed in the intes-
*This work was financially supported by the Russian Foundation for
Basic Research (Grant 10-03-00799).

Study of Safety of Molybdenum and Iron-Molybdenum Nanoclaster Polyoxometalates Intended 558
for Targeted Delivery of Drugs
tines and does not accumulate in the body. The biological
role of iron is related to its participation in the transport
and binding of oxygen (hemoglobin, myoglobin) and in
redox reactions: heme iron is a coenzyme of cytochromes,
catalase, peroxidase, and non-heme iron Fe2+ is a cofac-
tor required for oxidation of amino acids and cholesterol.
Excess of iron in the body is accompanied by the visceral
injury (hemochromatosis), activation of free radical oxi-
dation. Need for iron is 10 - 20 mg/day, but since only
5% - 10% of iron consumed is absorbed [8,9].
Exchange of buckyballs based on molybdenum and
iron may be different from the usual exchange of com-
pounds of these elements in the body due to the unique
features of nanoflakes, so it is necessary to study the
toxicity of buckyballs. Preliminary researches show of
complexes formation possibility for buckyballs with a
known enough immunomodulator aminophtalhidraside
or its derivatives. Aminophtalhidraside promotes regen-
erative processes in an organism. Therefore we assume
further to investigate transport of aminophtalhidraside by
intramuscular introduction with buckyballs. Other of
possible method of introduction of a complex is also the
electrophoresis method.
The purpose of this study: the study of toxicity of
buckyballs based on molybdenum and iron, designed as
containers or cores for transport of drugs.
2. Materials and Methods
Mo132 synthesis was carried out published in [4], and
Mo72Fe30—by the method [5]. The initial reagents were
ammonium heptamolybdate (NH4)6Mo7O24·4H2O (che-
mically pure), hydrazine sulfate N2H4·H2SO4, sodium
hydroxide NaOH and sodium chloride NaCl qualification
(pure for analysis), ammonium chloride NH4Cl and hy-
drochloric acid HCl (purity), ferric chloride (III)
FeCl3·6H2O (Panreac, the content of main substance 97 -
102 wt%), acetic acid (chemically pure) CH3COOH.
Accumulation of buckyballs was investigated during
the experiment on 27 rats of both sexes weighing 200 -
230 g, contained on a standard diet of the vivarium. Con-
ditions of housing and treatment to animals corresponded
to the EU Council Directive of November, 24 1986 “On
the approximation of laws, regulations and administrative
statutes of the EU on the protection of animals used for
experimental and other scientific purposes “(86/609EES).
Animals were divided into 3 groups: 1—intact rats, 2—
introduction of molybdenum buckyballs, 3—introduction
of iron-molybdenum buckyballs. Previously, it was
found that the area of acceptable over time stability of
buckyballs is a weak acidic or neutral environment, and
the best method of buckyballs introducing—intramus-
cular injections was chosen [10,11]. Injections were
made in the area of the gastrocnemius muscle in a con-
centration of 10−2 mol/L daily for a month. The dose of
buckyballs injected corresponded to the upper limit of
the normal daily intake of molybdenum and was 21.5
times less than the daily dose of iron. Animals were with-
drawn from the experiment by an overdose of ether. Mo-
lybdenum content in organs was determined by atomic
emissive spectrometer with inductively coupled plasma
iCAP-6500 Duo (Thermo Scientific) after mineralization
of samples.
Preparation of tissue samples of liver and kidney for
histological examination was performed on an automated
processor Leica EG 1160, followed by filling in paraffin.
Slices of thickness 3 - 5 μm were stained with hematoxy-
lineosin, pirofuchsin by Van Gieson and Weigert. Mi-
croscopic examination was performed on a microscope
Leica DM 2500, image analysis was performed in Video-
TesT “Morphology” 5.0. Photomicrographs of histologi-
cal specimens were obtained using a digital color camera
“CAM 2800”.
Analysis of peripheral blood was performed on an
automated hematology analyzer Celly 70 Biocode Hycel.
Statistical analysis of the material was carried out us-
ing the programs Statistica 6.0 (Stat. Soft.Inc.) and Mi-
crosoft Exel 2003. During the statistical hypotheses test-
ing a significance level of 5% (P < 0.05) was used.
3. Results and Discussion
In the study of molybdenum content in organs of rats
receiving molybdenum buckyballs (group 2), no accu-
mulation of this element in the liver and bones was re-
vealed. In kidneys a sharp decrease in its content by 80%
(P < 0.05) was revealed. Experiments allowed us to es-
tablish the lack of accumulation of molybdenum (Table
1) in the liver, kidneys, bones, skin of animals receiving
iron-molybdenum buckyballs (group 3). Lack of molyb-
denum accumulation may be associated with the decom-
position of buckyballs into simpler forms that can be easily
Table 1. The molybdenum content (mkg/g in a correspond-
ing part) in rats bodies.
Mean content of Мо
Organ
Experimental animals Intact animals
Liver 5.3 7.2
Kidney 8.3 12.0
Ossa 1.2 1.8
Cutaneous covering
with hair side 3.0 2.9

Study of Safety of Molybdenum and Iron-Molybdenum Nanoclaster Polyoxometalates Intended 559
for Targeted Delivery of Drugs
removed from the body.
Histological analysis of kidney sections of animals
from group 2 no structural changes in the glomeruli were
detected, but in the part of tubules lumen the eosinophilic
mass (Figure 1) and focal hyperemia of sinusoidal blood
vessels a “sludge-complex” formation were determined.
Histological analysis of kidney sections of animals
from group 3, in contrast to group 2, only focal hypere-
mia of sinusoidal vessels (Figure 2) revealed, the tubules
epithelium was not changed. The presence of eosino-
philic masses and hyperemia of vessels showed sensiti-
zation and inflammation in the kidney tubules. These
changes are largely present at the animals receiving mo-
lybdenum buckyballs.
In the liver of animals from group 2 diffuse hyperemia
of central venous and focal hyperemia of the veins of
portal tracts were revealed. In the parenchyma of the
Figure 1. The kidneys of animals of the 2nd group: in the
tubules lumen there are eosinophilic masses. Hematoxylin
and eosin stain. Magnification ×200.
Figure 2. The kidneys of animals of the 3rd group: focal
hyperemia of sinusoidal vessels. Hematoxylin and eosin
stain. Magnification ×100.
organ focal hyperemia of sinusoids with the formation of
“sludge complex” was noted (Figure 3).
In a part of the periportal tracts periportal leukocytic
infiltration was found. Increase in the number of Kupfer
cells in sinusoids was visually marked. Part of the hepa-
tocytes showed signs of granular dystrophy.
In the liver of animals from group 3 (Figure 4), unlike
in the liver of animals from group 2, the structural
changes of hepatocytes were not observed. Central vein
and the vein of portal tracts were plethoric. In some of
them the “sludge complex” formation was determined
that may be associated with increased blood clotting due
to increasing number of platelets and hematocrit index.
Comparing the results of morphometry it was estab-
lished that index of alteration of the animals from group
2 increased in comparison with the index of intact ani-
mals by 13 times, while the figure in group 3 was 3.9
Figure 3. The liver of animals from the 2nd groups: the
central vein hyperemia, the “sludge-complex” formation in
sinusoids. Hematoxylin and eosin stain. Magnification ×400.
Figure 4. The liver of animals from the 3rd groups: hy-
peremia of the central vein and the “sludge complex” for-
mation. Hematoxylin and eosin stain. Magnification ×100.

Study of Safety of Molybdenum and Iron-Molybdenum Nanoclaster Polyoxometalates Intended
for Targeted Delivery of Drugs
560
times higher than the control level (P < 0.05). Alteration
processes were accompanied with activation of the or-
gan’s intracellular regeneration, since the number of bi-
nucleated cells in the liver of animals from groups 2 and
3, respectively, increased by 1.6 and 1.4 times. Perhaps
this is due to an increase in activity and number of sinu-
soidal cells in the liver. Sinusoidal cells (Kupffer cells,
Ito cells) secrete growth factors, cytokines that promote
regeneration. An increase in number of sinusoidal cells
by 1.5 times in comparison with control was registered
for the animals from group 2 only.
Analysis of peripheral blood led to the conclusion that
there is no characteristic feature of many chronic intoxi-
cations—anemia of animals receiving injections of both
buckyballs, as far as hemoglobin in erythrocytes of ex-
perimental rats was not reduced. The total number of
leukocytes of experimental animals from groups 2 and 3
did not increase, indicating the absence of an inflamma-
tory process of the whole organism.
More pronounced toxic effect of molybdenum bucky-
balls compared to the iron-molybdenum may occur due
to the presence of molybdenum in the oxidation state 5+,
involved in renewable redox reactions, and accelerated
formation of uric acid and urate intoxication in the renal
tubules.
4. Conclusions
1) The toxicity of iron-molybdenum nanocluster poly-
oxometallates significantly reduced in comparison with
molybdenum, which is confirmed by histological studies
and analysis of the peripheral blood of animals.
2) The selective cumulativity of buckyballs in the
studied tissues was not found, which allows to suggest
reversibility of detected changes.
REFERENCES
[1] S. D. Caruthers, S. A. Wickline and G. M. Lanza,
“Nanotechnological Applications in Medicine,” Current
Opinion in Biotechnology, Vol. 18, No. 1, 2007, pp. 26-
30. doi:10.1016/j.copbio.2007.01.006
[2] R. C. Dutta, “Drug Carriers in Pharmaceutical Design:
Promises and Progress,” Current Pharmaceutical Design,
Vol. 13, No. 7, 2007, pp. 76-79.
doi:10.2174/138161207780249119
[3] T. Yamase, “Polyoxometalates for Molecular Devices:
Antitumor Activity and Luminescence,” Molecular En-
gineering, Vol. 3, 1996, pp. 365-373.
[4] A. Müller, E. Krickemeyer, H. Bögge, M. Schidtmann
and F. Peters, “Organizational Forms of Matter: An Inor-
ganic Superfullerene and Keplerate Based on Molybde-
num Oxide,” Angewandte Chemie International Edition,
Vol. 37, No. 24, 1998, pp. 3360-3363.
[5] A. Müller, S. Sarkar, Q. N. Shah, H. Bögge, M.
Schmidtmann, S. Sarkar, P. Kögerler, B. Hauptfleisch, A.
X. Trautwein and V. Schünemann, “Archimedian Synthe-
sis and Magic Numbers: ‘Sizing’ Giant Molybdenum—
Oxide Based Molecular Spheres of the Keplerate Type,”
Angewandte Chemie International Edition, Vol. 38, No.
21, 1999, pp. 3238-3241.
doi:10.1002/(SICI)1521-3773(19991102)38:21<3238::AI
D-ANIE3238>3.0.CO;2-6
[6] A. A. Ostroushko, M. O. Tonkushina and N. A. Mar-
tynova, “Mass and Charge Transfer in Systems Contain-
ing Nanocluster Molybdenum Polyoxometallates with a
Fullerene Structure,” Russian Journal of Physical Chem-
istry A, Vol. 84, No. 6, 2010, pp. 1022-1027.
[7] A. A. Ostroushko, M. Yu. Sennikov and M. O. Tonku-
shina, “Interaction of Polyoxometalate Mo132 with Poly
(Vinyl Alcohol),” Russian Journal of Inorganic Chemis-
try, Vol. 54, No. 4, 2009, pp. 611-617.
doi:10.1134/S0036023609040184
[8] Yu. V. Chistyakov, “Fundamentals of Bioinorganic
Chemistry,” ColosS, Moscow, 2007, pp. 400-488.
[9] A. Sh. Zaychik and L. P. Churilov, “Pathophysiology.
Vol. 2: Fundamentals of Pathochemistry,” Elby-SPb,
St.-Petersburg, 2001, pp. 403-423.
[10] A. A. Ostroushko, I. G. Danilova, S. J. Medvedeva, I. F.
Gette and M. O. Tonkushina, “Studying of Safety of Mo-
lybden Nanocluster Polyoxometalates Intended for Ad-
dress Delivery of Medicinal Substances,” Ural medical
Journal, Vol. 9, No. 74, 2010, pp. 114-117.
[11] A. A. Ostroushko, I. G. Danilova, S. J. Medvedeva, I. F.
Gette, A. V. Prokofieva and M. V. Morozova, “Studying
of Polyoxometalates with Fullerene Structure as Potential
Agents of Address Delivery of Substances,” European
Symposium on Biomaterials and Related Areas, B.56,
Jena, 13-14 April 2011.