Study of Safety of Molybdenum and Iron-Molybdenum Nanoclaster Polyoxometalates Intended for Targeted Delivery of Drugs

doi:10.4236/jbnb.2011.225066

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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.

E-mail: #[email protected]

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.

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