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Toxicology Research
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This article can be cited before page numbers have been issued, to do this please use: A. Ruiz, L. Ali, P. Cáceres-Velez, R. Cornudella, M. Gutiérrez, J. A. Moreno, R. Piñol, F. Palacio, M. L. Fascineli, R. Bentes de Azevedo, M. P. Morales and A. Millán, Toxicol. Res., 2015, DOI: 10.1039/C4TX00241E.
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Toxicology Research View Article Online
DOI: 10.1039/C4TX00241E
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ARTICLE Hematotoxicity of magnetite nanoparticles coated with polyethylene glycol: In vitro and in vivo study
Received Accepted DOI: 10.1039/x0xx00000x www.rsc.org/
A. Ruiza,b§, L. M. A. Alic§, P. R. Cáceres-Vélezd, R. Cornudellae, M. Gutiérreze, J. A. Morenoe, R. Piñolc, F. Palacioc, M. L. Fascinelid, R. B. de Azevedod, M. P. Moralesa* and A. Millánc* Hematotoxicity of magnetite nanoparticles coated with dimercaptosuccinic acid (DMSA) and polyethylene glycol (PEG) has been evaluated by determining their safety in vitro and in vivo in a rat model up to 30 days after administration of a single dose. The in vitro analysis consists on global plasma coagulation (PT, aPTT, Fibrinogen) and platelet aggregation tests while the hematotoxicity studies in vivo include a complete blood count and the possible genotoxic effects analysis in the bone marrow hematopoietic function. Prolonged aPTT values indicate higher anticoagulant effect for NP-DMSA compared with PEG-coated nanoparticles as a consequence of the higher surface charge of the former. The in vivo tests showed that these bioferrofluids do not cause genotoxic effects, affect erythropoiesis or increase the number of immature erythrocytes in the bone marrow at the analyzed dose. However, nanoparticles administration showed a significant effect on the leukocytes counts in animals treated with DMSA coated nanoparticles 24 h after injection. This response is not observed in animals treated with PEG modified nanoparticles which justifies the use of this polymer in biomasking strategies. Keywords: magnetic nanoparticles, toxicity, PEG coated nanoparticles, blood, hematological parameters
Interactions of nanoparticles with blood usually start with protein
Introduction
adsorption3, and subsequently derive in coagulation problems and Magnetic nanoparticles based on iron oxides are being used as contrast agents in magnetic resonance imaging (MRI).
Clinical
applications of these materials in drug delivery, hyperthermia and
other disturbances. Several studies have reported that environmental nanoparticles significantly increase the risk and worsen the prognosis of cardiovascular diseases due to the induction of
1
diagnosis are also being considered seriously . However, a first requisite for in vivo applications is blood compatibility2, otherwise these materials would be immediately discarded for intravenous administration.
thrombotic complications4. Protein adsorption is favored by surface charges, especially positive charges, and can be minimized with an appropriate coating. Carbohydrates and other biological polymers have been considered to be effective against protein adsorption, although polyethylene glycol is the preferred coating for these
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Journal Name DOI: 10.1039/C4TX00241E
purposes5, 6. However there are only few studies investigating the
(Dimercaptosuccinic acid (DMSA) and Polyethylene glycol (PEG))
hematological effects of nanoparticles in vivo in spite of the fact that
could lead to blood coagulation disorders and affect hematological
the majority of nanoparticle formulations are intended for systemic
parameters in vitro and in vivo. These bioferrofluids have previously
administration in clinical applications.
In addition, there are no
been used in biodistribution and pharmacokinetics studies in
standard methodologies for the in vitro assessment of the blood
different animal models. PEG coated nanoparticles have shown a
compatibility of these nanoproducts.
good performance as MRI long circulating agents due to the effect of incorporation of PEG into the nanoparticle surface which is known
The in vitro biocompatibility studies should also include evidence of to provide stealth coating to camouflage them, and thus, temporarily hemolysis.
The unique physicochemical properties of the bypass recognition by macrophages9.
As with any device or
nanoparticles could cause hemolysis by acting on the membrane of pharmaceutical product, DMSA or PEG coated nanoparticles red blood cells. Several studies have revealed the effect of the intended for biomedical application must also be subjected to nanoparticles on the blood by measuring their hemolytic action in vitro7.
extensive blood biocompatibility testing before regulatory approval However, direct results interpretation of these studies is for administration to patients.
To our knowledge, the blood
complicated due to variability of the experimental conditions such as biocompatibility of these products has not been established till date. incubation time of blood with the nanoparticles, the wavelength at Previous hematological studies on iron oxide nanoparticles coated which the hemoglobin is quantified, the centrifugation forces, blood storage time and conditions and blood source (human or rabbit)8.
with poly(4-vinyl pyridine)-polyethylene glycol (PVP-PEG) brush copolymers indicated a non-specific anticoagulant effect of the
Platelets are very sensitive to contact with biological substances,
bioferrofluid10.
such as collagen, ADP and epinephrine, and non-biological ones, We have carried out an extensive in vitro biocompatibility study of including ristocetin, that are commonly used in the hematology our bioferrofluids with blood including coagulation studies, laboratory to detect functional defects of platelets. Foreign materials hemolysis and quantification of leukocytes, erythrocytes and and high shear stress can also induce platelet aggregation. When this platelets, to rule out immediate cytotoxicity of nanoparticles or occurs they tend to produce micro aggregates that are easily detected contact spontaneous platelet aggregation11-13.
Moreover, blood
by modern blood cell counters because they decrease the number of toxicity of these nanomaterials has been studied in terms of global platelets and alter the size distribution curve. Nanoparticles can tests (Prothrombin time (PT), activated partial thromboplastin time potentially damage the cell membrane and even the cytoplasm since (aPTT), Fibrinogen), thrombus-elastography, platelet aggregation they can penetrate inside cells. Blood cell counters not only quantify and degradation products of fibrinogen and fibrin.
Recently, a
blood cells but also can detect abnormalities in shape, size and detailed description of methods has been published, which focuses homogeneity. on the global plasma coagulation tests (PT, aPTT, Fibrinogen) and This study was designed specifically to investigate whether iron
platelet aggregation14. The doses used in this study are higher than
oxide magnetite nanoparticles with a narrow size distribution (Mean
the therapeutically relevant concentration but describe the range
size=7
where we observed an effect on hematological parameters. The
nm;
SD
