Investigation of hematotoxic effect of nano ZnO, nano Fe3O4 and nano SiO2 in vitro

INTRODUCTION

Nanomaterials are defined as a new class of materials having unique physical and chemical properties. There is great interest in applying nanomaterials in different fields of medicine and industry. The potential benefit of nanomaterials in medicinal devices or drug delivery proposes new methods in diagnosis, medication, and treatment of complicated diseases such as cancers. Any nanomaterials applied as pharmaceuticals or medical devices must be assessed for potential risk for consumers before approval for prescription. Evaluation of nanomaterials interaction with blood ingredients is a part of preclinical risk assessment of newly -synthesized materials, especially for nano-sized pharmaceuticals which are intravenously administrated [1].  Several studies have shown that the unique structure and size of the nanoparticles could lead to oxidative injury and cell toxicity in different cell cultures and also in blood cells [2-3]. 

Formation of reactive oxygen and nitrogen species (RONS) at levels higher  than the body’s antioxidant capacity has been known as the oxidative stress. Reactive oxygen and nitrogen species are generated in the body as the result of normal metabolism and either as the result of exposure to a variety of chemicals, drugs, and pollutants.  Nucleic acids, proteins, lipids are cellular macromolecules that may be harmed by RONS. Oxidative stress also involves in pathogenesis of different diseases including cancer, neurodegenerative diseases, atherosclerosis, kidney and liver damage, rheumatoid arthritis, immunological disorder, and aging [4-5].

Lipid peroxidation of polyunsaturated fatty acids leads to production of malondialdehyde (MDA) that is a useful biomarker of oxidative stress. MDA is commonly measured by the thiobarbituric acid-reactive-substances (TBARS) assay. Small thiols including tripeptide glutathione (GSH) are considered as the protective sulfhydryl antioxidants and radical scavengers. Glutathione protects sensitive thiol groups (-SH)  of proteins against oxidative stress [6]. 

The RBCs are intrinsically susceptible to oxidative stress damage because of exposing to high oxygen pressure and the presence of polyunsaturated fatty acid in their cell membrane.  Furthermore, RBCs do not have endoplasmic reticulum and nucleus to replace damaged proteins [7]. Some studies have investigated the hematotoxic effects of nanoparticles on the blood cells in vivo and in vitro [8-13].

Nano Fe3O4, nano ZnO, and nano SiO2 are well-known nanoparticles that have promising biomedical applications and their oxidative stress effects have been reported in different studies [9, 14-15]. Zinc nanoparticles are used in cosmetics ointment, sunscreens, food additive, antimicrobial, fungicide, UV protection coatings, and as a catalyst [15].SiO2 nanoparticles have found extensive applications in chemical mechanical polishing and as the additives to drugs, cosmetics, printer toners, varnishes, and food. In recent years, the use of SiO2 nanoparticles has been extended to biomedical and biotechnological fields [16]. Fe3O4 nanoparticles have been used as the contrast agents in Magnetic resonance imaging (MRI) angiography and perfusion imaging [17].

In this study, the induced oxidative hematotoxic effects of different doses of nano ZnO, nano Fe3O4, and nano SiO2 have been studied on human RBC. .

MATERIAL AND METHODS

Nano particles characterization

Silicon Dioxide (SiO2) Nanopowder (99+%, 20-30 nm, amorphous) was purchased from Nanosany Corporation, Iranian Nanomaterials Pioneers Company, Mashhad, Iran.   Figure 1 and Figure 2 represent transmission electron microscope (TEM) and scanning electron microscope (SEM) images of nano SiO2.

Iron Oxide (Fe3O4) Nanopowder (99+%, 20- 40 nm, spherical) was purchased from Nanosany Corporation, Iranian Nanomaterials Pioneers Company, Mashhad, Iran. Figure 3 represent the TEM image of nano Fe3O4.

The nano-zinc metal powder (99.99+%, 20-40 nm) was purchased from the Intelligent Materials Pvt. Ltd., Nanoshel LLC, Wilmington, DE, USA.  Figure 4 represent transmission electron the TEM image of nano ZnO.

All the other chemicals used in this experiment were analytical grade with the highest purity .

Blood samples preparation

Blood samples were collected from healthy male volunteers in heparinized tubes and centrifuged at 3000 rpm for 25 min. Plasma and the buffy coats of blood were discarded. RBCs were washed three times with phosphate-buffered normal saline and then exposed to different concentrations (50, 100, 250 mg/ml) of nano ZnO, nano Fe3O4, and nano SiO2 before incubation at 4C° for 24h. Each experiment was performed in triplicate.

Lipid peroxidation assay

 Lipid peroxidation in human erythrocytes was quantified by measuring the formation of TBARS. Erythrocytes were mixed with trichloroacetic acid 20% (1:1). Samples were centrifuged (600g×10min) and then  thiobarbituric acid 15% was added to the supernatants. Finally,  the samples were heated at temperature 100°c for15 min and the absorbance of the supernatant was measured at 532 nm. The quantities of TBARS were presented as the percentage of TBARS production over the control.

Quantification of intracellular GSH levels

Cellular level of reduced GSH was determined using the GSH colorimetric assay kit. This method is based on a chemical reaction between GSH and 5, 50-dithiobis (2-nitrobenzoic acid) (DTNB) that results in  generating glutathione disulfide (GSSG) and-nitro-5-thiobenzoicacid, a yellow colored product. Thus, GSH concentration in a sample solution can be determined by the absorbance measurement at 412 nm [18].

 Statistical analysis

One-way analysis of variance (ANOVA) was used to compare the results. P value greater than 0.05 was considered insignificant.

Figure 5 indicates schematic of the quantification of oxidative stress induction assay.