# Treatment of Anemia Progression via Magnetite and Folate NanoparticlesIn Vivo

Authors: Hanaa Hussein Elsayed; Al Sayed A. M. Al-Sherbini; Eman Elsayed Abd-Elhady; Kawkab Abd El Aziz. Ahmed
Journal: ISRN Nanotechnology
 (2014)

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## Abstract

Iron deficiency anemia is a major global public health problem. Food fortification with iron (Fe) can be an effective strategy to control iron deficiency. An iron oxide nanoparticle (NP) is a new physical and chemical property form. These properties (small particle size, unique physical properties) make nanoiron a great scientific interest especially in the treatment of anemia. The study aimed to reduce anemia by nanoparticles (NPs). Forty-eight adult female Sprague-Dewily rats were divided into four groups (12 rats each). Group A represented a negative control. Other groups were fed standard diet iron free and three time of require zinc to reach anemic. Group B fed standard diet with ferrous sulfate until the improvement of the situation of anemia or for 8 weeks. Groups C and D were divided into three subgroups; each subgroup was fed a dose from magnetite or folate coated magnetite NPs. Results showed that symptoms of loss of appetite and severe lethargy demonstrate that magnetite and folate-coated magnetite nanoparticles have serious toxicological effectsin vivo. Some doses from NPs improve blood picture during 2 weeks but change in histopathology examinations were occur in some groups within 2 weeks. Nanoparticles were considered the toxicological hazards especially the size of less than 54 nm.


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## Body

## 1. Introduction

Nanotechnologies enable scientists to manipulate matter at the nanoscale (one thousand millionth of a meter). Within this size-range, materials can exhibit new and unusual properties, such as altered chemical reactivity, or changed electronic, optical, or magnetic behavior [1].Nanotechnology has also the potential to affect many aspects of food and agricultural systems. Food security, packaging materials, disease treatment, delivery systems, bioavailability, new tools for molecular and cellular biology, and new materials for pathogen detection are examples of the important items that are linked with nanotechnology within the food production chain [2, 3].The term “nanofood” describes food which has been cultivated, produced, processed, or packaged using nanotechnology techniques or tools, or to which manufactured nanomaterials have been added [4]. Examples of nanoingredients and manufactured nanomaterial additives include nanoparticles of iron or zinc and nanocapsules containing ingredients like coenzyme Q10 or Omega 3.Directive 2002/46/EC European Commission defined food supplements as concentrated sources of nutrients and other substances developed to supplement the normal diet and have nutritional or physiological effect whether used alone or combined. It is marketed in dose forms, namely, forms such as capsules, pastilles, tablets, pills, and other similar forms, sachets of powder, ampoules of liquid, drop suspending bottles, and other similar forms of liquids and powders designed to be taken in measured small unit quantities, such as nutrients, vitamins and minerals are meant.Nanoparticles (NPs) can be more chemically reactive and more bioactive than larger particles. Because of their very small size, NPs also have much greater access to our bodies, so they are more likely than larger particles to enter cells, tissues, and organs. These novel properties offer many new opportunities for food industry applications, for example, as potent nutritional additives, stronger flavorings, and colorings, or antibacterial ingredients for food packaging. However, these same properties may also result in greater toxicity risks for human health and the environment [5].Nanotechnologies may also present new risks because of their novel properties, as well as potential benefits to consumers. There is a wide variety of nanomaterials, and while many types of nanomaterials may well prove to be harmless, others may present a higher risk.Iron-deficiency anemia is a common anemia (low red blood cell level) caused by insufficient dietary intake and absorption of iron and/or iron loss from intestinal bleeding, parasitic infection, menstruation, and so forth. Red blood cells contain iron, and they are not formed when iron is deficient [6].The main criterion for diagnosing iron deficiency or iron deficiency anemia is that other causes of anemia have been ruled out, such as B12/folate deficiency (http://www.medicines.org.uk). Iron supplementation is indicated when diet alone cannot restore deficient iron levels to normal within an acceptable timeframe. Supplements are especially important when an individual is experiencing clinical symptoms of iron deficiency anemia. The goals of providing oral iron supplements are to supply sufficient iron to restore normal storage levels of iron and to replenish hemoglobin deficits [7].Supplemental iron is available in two forms: ferrous and ferric. Ferrous iron salts (ferrous fumarate, ferrous sulfate, and ferrous gluconate) are the best-absorbed forms of iron supplements [8].Nanoparticles (NPs) have emerged as a promising strategy for the efficient delivery of drugs used in the treatment of different types of diseases because of their many advantages over classical delivery matrices. The important technological advantages of nanoparticles used as drug carriers are high stability, high carrier capacity, feasibility of incorporation of both hydrophilic and hydrophobic substances, and feasibility of variable routes of administration, including oral application and inhalation. Nanoparticles (NPs), because of their small size, have distinct properties compared to the bulk form of the same material, thus offering many new developments in the fields of biosensors, biomedicine, and bionanotechnology. The adsorption of protein on NPs and its consequences on the structure and function are strongly dependent on the size and shape of the NPs [9].Recent research on the gastrointestinal absorption of nanoparticles Hoffart et al. [10] has focused on enhancing the absorption of drugs, vaccines, and nutrients that either are degraded by the digestive process or poorly absorbed. Nel et al. [11] reported that there are toxicity concerns with nanoparticles. As most studies in this area have focused on airborne nanoparticles, there are only limited data on orally ingested trace element nanoparticles.The focus in this semiexperimental research is to test the best absorbed forms of iron supplements (ferrous sulfate, magnetite, and folate coated magnetite) and to assess other effects from various doses and the best period for treatment.


## 2. Materials and Methods

### 2.1. Materials

All chemicals were of analytical reagent grade and were used without further purification. Nanostructured materials have been a subject of intensive research during the past 10 years [12, 13]. The nanocomposite phases with the dimensions smaller than 100 nm exhibit extraordinary material characteristics, including mechanical, chemical, structural, optical, and electric/magnetic properties.


### 2.2. Nanomaterial

Preparation of magnetite and folate-coated magnetite: the photochemical method was carried out by dissolving the mixture of ferric oxalate with FeCl3·6H2O in predetermined concentration ratio in 100 mL of aqueous hydrogen peroxide solution or 0.1 mol dm-3 folate aqueous hydrogen peroxide solution. The mixture was stirred in sunlight until the color changed. The pH of the solution was adjusted to 13 by addition of 1 mol dm-3 of KOH portionwise to the product. The black precipitates were collected by magnet and the product was washed several times with distilled water for several times and finally dried in the air.


### 2.3. Spectroscopic Characterization

Transmission electron microscopy of the produced samples was carried out by using Joel-100S transmission electron microscope, of resolution of 0.3 nm. X-ray Diffract meter (Rigaku, Japan) performed crystallographic study on iron oxide powder. UV-Visble absorption spectra.Figures2(a) and 2(b) show that the absorption spectra of folic acid show two bands, the first one is at λ
max
⁡ = 366 nm and the other one pointed at λ
max
⁡ = 279 nm (Figure 2(a)). The absorption spectrum of folate attached magnetite nanoparticles shows the decreasing of the absorption bands for folic acid with another absorption band observed at λ
max
⁡ = 339 nm, (Figure 2(b)).


#### 2.3.1. FTIR

The FTIR spectra of magnetite and folate coated magnetite were obtained as shown in Figures2(a) and 2(b). For magnetite, the two strong absorption bands at around 633 and 562 cm−1 result from the split of the ν
1 band at 570 cm−1, which corresponds to the Fe–O bond of bulk magnetite. The band at 447 cm−1 comes from the shifting of the ν
2 band of the Fe–O bond of bulk magnetite at around 375 cm−1, (Figure 2(a)). The high frequency bands ν
1 (Tetrahedral bands) and ν
2 (Octahedral bands) are attributed to the vibrations of iron ions in both tetrahedral and octahedral positions, respectively. The surface groups of folic acid-modified magnetite are characterized by FTIR (Figure 2(b)). The absorption peaks at 596 and 445 cm−1 are attributed to Fe–O bond absorption. The absorption peak at 1643 cm−1 can be attributed to N–H bending vibration, which is also favorable evidence for the existence of primary amine in molecules. Absorption at 1604 cm−1 corresponded to the aromatic ring stretching vibration in the folic acid molecule, absorption at 1396 cm−1 corresponded to the benzoic vibrations in folic acid molecule, and absorption at 1533 cm−1 can be attributed to the characteristic absorption of amide. Infrared data proved that folic acid molecules have successfully modified the surface of Fe3O4 nanoparticles.


### 2.4. Animals

Forty-eight adult female Sprague-Dewily rats (weighing 80 ± 10 g) were obtained from Vaccine and Immunity Organization, Hellwan farm, Cairo, Egypt. Rats were housed individually in stainless steel cages with grated stainless steel floors under healthy environmental conditions. Rats consumed deionized water and diet ad-libitum. Nanoiron was applied at three doses (87,174, and 348 mg/kg diet). Nanoiron plus folate was at 87.3, 174.5, and 349 mg/kg diet according to Reeves et al., [14]. These doses represented 1/2 the requirement and two-time from the requirement daily intake of iron in standard diet.


### 2.5. Chemicals and Other Materials

Casein “>85% protein,” corn starch, DL–methionine, choline chloride, vitamins, minerals, and other required chemicals obtained from El-Gomhorya Company for Chemicals and Drugs, Cairo, Egypt.


### 2.6. Standard Diet

Standard diet was prepared from fine ingredients per 100 g. The diet had the following composition.Sunflower oil 10%, salt mixture 4% [15], vitamin mixture 1% [16], choline chloride 0.2%, protein 12%, DL-methionine 0.3%, and corn starch up to 100 g according to Reeves et al. [14].


### 2.7. Experimental Design in Figure1

Figure 1
Experimental design.Absorption spectra of (a) aqueous solution of folic acid and (b) colloidal solution of foliate coated SPIO nanoparticles.
(a)
(b)Rats were divided into four groups (12 rats each). Group A (n
=
12) that served as negative control was anesthetized and sacrificed. Blood samples were taken from hepatic portal vein in heparinized tube to analysis blood picture. Liver, spleen, and lungs were isolated immediately, plotted free from adhering blood, washed with cooled saline (0.9% sodium chloride/deionized water), and dried between filter paper. All organs were weighed and then put in 10% formalin until further histopathology examination. The other 36 rats were fed ironfree diet where three times the recommended zinc requirement was added to rats’ diet to get anemic.Anemic rats were divided into three groups. Group B (n
=
12) was fed standard diet with iron as ferrous sulfate until treated from anemia. Group C was divided into three subgroups; (four rats each) fed basal diet with three doses of magnetite. In addition, group D (n
=
12) was fed standard diet with three doses of folate coated magnetite for the treatment of anemia. Every week, blood was collected from eye vein of each rat to assess blood picture. Blood treated with heparin was used for analyzing hematologic parameters such as erythrocyte count (red blood cell [RBC]), hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (PLT) count, and total leucocyte count (white blood cell [WBC]) were determined on day 14 and every week for 8 weeks or for treatment of anemia.After reaching the normal level of hemoglobin, rats were anesthetized and sacrificed; all organs were weighed and put in 10% formalin until further histopathology examination. Body weights of rats were recorded twice a week to monitor percentage body weight change as follows.(i)
Blood picture analysis was done by using blood cell counter “GRMA-210.”
(ii)
% Body weight change was calculated as Final body weight − Initial body weight/Initial body weight × 100.
(iii)
Relative weight of organs was calculated as organ weight/final body weight.


### 2.8. Histopathology Examination

Tissue samples (liver, kidneys, spleen, and lung) were fixed in neural buffered formalin 10%, dehydrated in ascending concentrations of ethanol, cleaned in xylene, and embedded in paraffin. Sections4-5
μ thick were prepared and stained with Hematoxylin and Eosin [17].


### 2.9. Statistical Analysis

All data are presented as mean ± SE. Differences between groups means were assessed by an unpaired Student’st-test for single comparisons or by ANOVA for multiple comparisons using SPSS 16.0. Values of P
<
0.05 were considered significant.


## 2.1. Materials

All chemicals were of analytical reagent grade and were used without further purification. Nanostructured materials have been a subject of intensive research during the past 10 years [12, 13]. The nanocomposite phases with the dimensions smaller than 100 nm exhibit extraordinary material characteristics, including mechanical, chemical, structural, optical, and electric/magnetic properties.


## 2.2. Nanomaterial

Preparation of magnetite and folate-coated magnetite: the photochemical method was carried out by dissolving the mixture of ferric oxalate with FeCl3·6H2O in predetermined concentration ratio in 100 mL of aqueous hydrogen peroxide solution or 0.1 mol dm-3 folate aqueous hydrogen peroxide solution. The mixture was stirred in sunlight until the color changed. The pH of the solution was adjusted to 13 by addition of 1 mol dm-3 of KOH portionwise to the product. The black precipitates were collected by magnet and the product was washed several times with distilled water for several times and finally dried in the air.


## 2.3. Spectroscopic Characterization

Transmission electron microscopy of the produced samples was carried out by using Joel-100S transmission electron microscope, of resolution of 0.3 nm. X-ray Diffract meter (Rigaku, Japan) performed crystallographic study on iron oxide powder. UV-Visble absorption spectra.Figures2(a) and 2(b) show that the absorption spectra of folic acid show two bands, the first one is at λ
max
⁡ = 366 nm and the other one pointed at λ
max
⁡ = 279 nm (Figure 2(a)). The absorption spectrum of folate attached magnetite nanoparticles shows the decreasing of the absorption bands for folic acid with another absorption band observed at λ
max
⁡ = 339 nm, (Figure 2(b)).


### 2.3.1. FTIR

The FTIR spectra of magnetite and folate coated magnetite were obtained as shown in Figures2(a) and 2(b). For magnetite, the two strong absorption bands at around 633 and 562 cm−1 result from the split of the ν
1 band at 570 cm−1, which corresponds to the Fe–O bond of bulk magnetite. The band at 447 cm−1 comes from the shifting of the ν
2 band of the Fe–O bond of bulk magnetite at around 375 cm−1, (Figure 2(a)). The high frequency bands ν
1 (Tetrahedral bands) and ν
2 (Octahedral bands) are attributed to the vibrations of iron ions in both tetrahedral and octahedral positions, respectively. The surface groups of folic acid-modified magnetite are characterized by FTIR (Figure 2(b)). The absorption peaks at 596 and 445 cm−1 are attributed to Fe–O bond absorption. The absorption peak at 1643 cm−1 can be attributed to N–H bending vibration, which is also favorable evidence for the existence of primary amine in molecules. Absorption at 1604 cm−1 corresponded to the aromatic ring stretching vibration in the folic acid molecule, absorption at 1396 cm−1 corresponded to the benzoic vibrations in folic acid molecule, and absorption at 1533 cm−1 can be attributed to the characteristic absorption of amide. Infrared data proved that folic acid molecules have successfully modified the surface of Fe3O4 nanoparticles.


## 2.3.1. FTIR

The FTIR spectra of magnetite and folate coated magnetite were obtained as shown in Figures2(a) and 2(b). For magnetite, the two strong absorption bands at around 633 and 562 cm−1 result from the split of the ν
1 band at 570 cm−1, which corresponds to the Fe–O bond of bulk magnetite. The band at 447 cm−1 comes from the shifting of the ν
2 band of the Fe–O bond of bulk magnetite at around 375 cm−1, (Figure 2(a)). The high frequency bands ν
1 (Tetrahedral bands) and ν
2 (Octahedral bands) are attributed to the vibrations of iron ions in both tetrahedral and octahedral positions, respectively. The surface groups of folic acid-modified magnetite are characterized by FTIR (Figure 2(b)). The absorption peaks at 596 and 445 cm−1 are attributed to Fe–O bond absorption. The absorption peak at 1643 cm−1 can be attributed to N–H bending vibration, which is also favorable evidence for the existence of primary amine in molecules. Absorption at 1604 cm−1 corresponded to the aromatic ring stretching vibration in the folic acid molecule, absorption at 1396 cm−1 corresponded to the benzoic vibrations in folic acid molecule, and absorption at 1533 cm−1 can be attributed to the characteristic absorption of amide. Infrared data proved that folic acid molecules have successfully modified the surface of Fe3O4 nanoparticles.


## 2.4. Animals

Forty-eight adult female Sprague-Dewily rats (weighing 80 ± 10 g) were obtained from Vaccine and Immunity Organization, Hellwan farm, Cairo, Egypt. Rats were housed individually in stainless steel cages with grated stainless steel floors under healthy environmental conditions. Rats consumed deionized water and diet ad-libitum. Nanoiron was applied at three doses (87,174, and 348 mg/kg diet). Nanoiron plus folate was at 87.3, 174.5, and 349 mg/kg diet according to Reeves et al., [14]. These doses represented 1/2 the requirement and two-time from the requirement daily intake of iron in standard diet.


## 2.5. Chemicals and Other Materials

Casein “>85% protein,” corn starch, DL–methionine, choline chloride, vitamins, minerals, and other required chemicals obtained from El-Gomhorya Company for Chemicals and Drugs, Cairo, Egypt.


## 2.6. Standard Diet

Standard diet was prepared from fine ingredients per 100 g. The diet had the following composition.Sunflower oil 10%, salt mixture 4% [15], vitamin mixture 1% [16], choline chloride 0.2%, protein 12%, DL-methionine 0.3%, and corn starch up to 100 g according to Reeves et al. [14].


## 2.7. Experimental Design in Figure1

Figure 1
Experimental design.Absorption spectra of (a) aqueous solution of folic acid and (b) colloidal solution of foliate coated SPIO nanoparticles.
(a)
(b)Rats were divided into four groups (12 rats each). Group A (n
=
12) that served as negative control was anesthetized and sacrificed. Blood samples were taken from hepatic portal vein in heparinized tube to analysis blood picture. Liver, spleen, and lungs were isolated immediately, plotted free from adhering blood, washed with cooled saline (0.9% sodium chloride/deionized water), and dried between filter paper. All organs were weighed and then put in 10% formalin until further histopathology examination. The other 36 rats were fed ironfree diet where three times the recommended zinc requirement was added to rats’ diet to get anemic.Anemic rats were divided into three groups. Group B (n
=
12) was fed standard diet with iron as ferrous sulfate until treated from anemia. Group C was divided into three subgroups; (four rats each) fed basal diet with three doses of magnetite. In addition, group D (n
=
12) was fed standard diet with three doses of folate coated magnetite for the treatment of anemia. Every week, blood was collected from eye vein of each rat to assess blood picture. Blood treated with heparin was used for analyzing hematologic parameters such as erythrocyte count (red blood cell [RBC]), hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (PLT) count, and total leucocyte count (white blood cell [WBC]) were determined on day 14 and every week for 8 weeks or for treatment of anemia.After reaching the normal level of hemoglobin, rats were anesthetized and sacrificed; all organs were weighed and put in 10% formalin until further histopathology examination. Body weights of rats were recorded twice a week to monitor percentage body weight change as follows.(i)
Blood picture analysis was done by using blood cell counter “GRMA-210.”
(ii)
% Body weight change was calculated as Final body weight − Initial body weight/Initial body weight × 100.
(iii)
Relative weight of organs was calculated as organ weight/final body weight.


## 2.8. Histopathology Examination

Tissue samples (liver, kidneys, spleen, and lung) were fixed in neural buffered formalin 10%, dehydrated in ascending concentrations of ethanol, cleaned in xylene, and embedded in paraffin. Sections4-5
μ thick were prepared and stained with Hematoxylin and Eosin [17].


## 2.9. Statistical Analysis

All data are presented as mean ± SE. Differences between groups means were assessed by an unpaired Student’st-test for single comparisons or by ANOVA for multiple comparisons using SPSS 16.0. Values of P
<
0.05 were considered significant.


## 3. Results

### 3.1. Characterization of Material Nanoparticles (MNPs)

#### 3.1.1. Transmission Electron Micrograph (TEM)

Transmission electron microscopy for the prepared SPIO NPs showed that particles have approximately (~) spherical shapes and their sizes were in the range of ~54 ± 3 nm, using UV/VIS spectroscopic characterization (Figure2(a)).


#### 3.1.2. X-Ray Diffraction Analysis

The XRD spectrum of the prepared SPIO nanoparticles shows that six characteristic peaks for Fe3O4 (2θ = 30.10, 35.50, 43.10, 53.40, 57.00, and 62.60), marked by their indices 220, 311, 400, 422, 511, and 440, were observed for all samples using FTIR spectroscopic characterization (Figure 2(b)).


### 3.2. Biology Results

No animal mortality was found in rats treated with various iron-supplemented dietsbut there were some symptoms such as loss of appetite and severe lethargy which were observed during the experimental period of the rat, after administration of nanoparticle suspension. Results presented in Table1 indicated that the body weights change and different relative weight rats received iron-supplemented diets. Results showed that the relative weight liver of group (D2) was the nearest to normal group. The highest relative weights of lung and spleen were in group (C3) but they were similar to normal group in relative weight renal. The highest percent of body weight change was in the group fed first dose of magnetite while the lowest percent showed in group (D2).Table 1
Relative weight and body weight change.
Relative wt. liver
Relative wt. spleen
Relative wt. lung
Relative wt. renal
Body weight change%
Normal G (A)
3.5
±
0.07
0.27
±
0.09
0.54
±
0.08
0.81
±
0.08
17.3
±
3.5
FeSO4 G (B)
4.01
±
0.2
0.41
±
0.04
0.76
±
0.09
0.77
±
0.09
11.0
±
4.5
Fe G (C1)
4.1
±
0.5
0.47
±
0.08
0.68
±
0.06
0.87
±
0.06
24.0
±
5.7
Fe G (C2)
4.3
±
0.5
0.5
±
0.07
0.85
±
0.09
0.79
±
0.02
20.3
±
6.6
Fe G (C3)
4.63
±
0.5
0.63
±
0.1
0.93
±
0.08
0.81
±
0.09
9.3
±
5.4
Fe + Fo G (D1)
3.98
±
0.2
0.44
±
0.03
0.86
±
0.1
0.84
±
0.1
19.5
±
7.5
Fe + Fo G (D2)
3.93
±
0.2
0.47
±
0.05
0.79
±
0.2
0.91
±
0.3
7.7
±
1.1
Fe + Fo G (D3)
4.48
±
0.1
0.54
±
0.02
0.59
±
0.02
0.80
±
0.04
18.5
±
8.8


### 3.3. Biochemical Results

#### 3.3.1. Hematology

Dietary iron deficiency with adding two-times zinc requirement for 2 to 3 weeks led to nutritional iron deficiency anemia in rats.Results presented in Figures3 and 4 indicated that the Hb and Hct were corrected in 75% of rats fed ferrous sulfate (Group B) during three weeks, while 25% fluctuated between up and down in the following weeks failing to reach normal value. Group C1 rats treated with 1/2 iron requirement dose from magnetite showed gradual decrease in Hb level throughout most of the follow-up weeks and were sacrificed at 7 weeks without showing improvement in iron status. Rats in Groups C2 and C3 reached normal Hb and Hct values by the 4th week followed by marked decreased Hb and Hct values afterwards.Figure 3
Hemoglobin (Hb) level in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure 4
Hematocrit (HCT) level in rat blood fed different diet with or without nanoparticles.N = Normal level.On the other hand, Group D1 (recipient of 1/2 dose of iron requirements and folat) reached normal Hb and Hct levels within four weeks. The other two Groups D2 and D3 showed improvement in Hb and Hct level within three weeks followed by decline in Hb and Hct values.Figures5, 6, 7, and 8 demonstrated normal values of RBCs, MCV, MCH, and MCHC within 3 weeks in Group B reaching plateau afterwards. Group D2 did not show any improvement in RBC level for the duration of the study. All groups exceeded normal values at different speeds or durations.Figure 5
Red blood cells (RBCs) level in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure 6
Mean corpuscular volume (MCV) in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure 7
Mean corpuscular hemoglobin (MCH) in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure 8
Mean corpuscular hemoglobin concentration (MCHC) in rat blood fed different diet with or without nanoparticles.N = Normal level.The rats fed 1st dose of magnetite and 3rd dose of folate-coated magnetite the results of MCV were less than normal volume to 7 wk. while the 2nd dose of magnetite showed that the normal dose fruition at 4 wk. after that increased about normal level. The rats received the 3rd dose of iron nanoparticles treated at two weeks and then the results fluctuated between up and down. The 1st dose of folate-coated magnetite indicated that normal level was at 5 weeks.MCH was less than normal level among all treatment groups, showed in Figure6.A bell shaped curve of MCHC parameter was noticed in Groups B, C2, D1, and D3 around the third week.Platelets level explained that increased in-group fed ferrous sulfate at third wk. after that decreased level in same group. Steady raise of PLT level until 5th week in group fed 1st dose of magnetite. Level of PLT increased from 3rd to 4th week, and then decreased from 5th to 7th week, but at eighth week, the increase was more than normal level of group fed 2nd dose from magnetite. Steady increase in group that received 3rd dose of magnetite. The 1st dose added to diet of folate-coated magnetite clarified that increased in PLT level at 2nd wk. and decreased after that but it steal higher than normal value. Rats fed 2nd and 3rd doses of folate-coated magnetite showed that the level of PLT fluctuated between up and down.MPV % demonstrated that the normal level was at the 3rd week with group fed 1st dose of magnetite and ferrous sulfate but the 2nd and 3rd doses of magnetite and 1st dose of folate-coated magnetite showed the normality at 2 week, while the 2nd and 3rd doses of folate-coated magnetite increased more than normal and after that decreased less than normal.Figures11 and 12 displayed that treated groups with nanomaterial the result fluctuated between increase and decrease than normal level of WBC. The largest increase shows in the group fed the 2nd dose of folate-coated magnetite.Lymph finding showed that all group fluctuated between increase and decrease than normal level (Figure12).


### 3.4. Histopathology Results

#### 3.4.1. Liver

Microscopically, examined sections of liver from rat treated with magnetite (1st dose) revealed cytoplasmic vacuolizations of hepatocytes, infiltration of centrolobular hepatocytes with leucocytic inflammatory cells (Figure13), sinusoidal leukocytosis (Figure 14) and portal infiltration with leucocytes. Moreover, at the 2nd dose and 3rd dose, examined liver revealed Kupffer cells activation, focal hepatic necrosis associated with leucocytic cells infiltration (Figure 15) cytoplasmic vacuolizations of hepatocytes, apoptosis of hepatocytes (Figure 16), as well as portal infiltration with leucocytes. Liver sections of rat treated with both magnetite and folate coated magnetite (1st and 2nd doses) showed Kupffer cells activation, cytoplasmic vacuolization of hepatocytes and focal hepatic necrosis associated with leucocytic cells infiltration (Figure 17). At the 3rd dose, in addition to the previously mentioned changes, examined sections revealed apoptosis of hepatocytes. Examined sections of liver from rats either treated with sulphate or control, untreated rat revealed no histopathological changes (Figure 18).


#### 3.4.2. Kidneys

Microscopically, examined sections of kidneys of rat treated with magnetite (1st, 2nd, and 3rd doses) revealed the same histopathological alterations which summarized as vacuolations of epithelial lining renal tubules as well as endothelial lining glomerular tufts (Figure 19), focal tubular necrosis associated with leucocytic inflammatory cells infiltration (Figure 20), and tubular necrosis associated with calcification (Figure 21). Improvement in the histopathological picture was noticed in examined sections of rat treated with both folate coated magnetite. At the 1st dose, examined sections showed vacuolations of epithelial lining renal tubules and endothelial lining glomerular tufts together with focal tubular necrosis associated with leucocytic cells infiltration (Figure 22). Meanwhile, at 2nd and 3rd doses, kidneys sections revealed vacuolations of epithelial lining renal tubules as well as endothelial lining glomerular tufts (Figure 23). However, examined sections either treated with sulphate or control, untreated rat revealed no histopathological changes (Figure 24).


#### 3.4.3. Lung

Histopathology, examined sections of lung of rat treated with magnetite (1st, 2nd, and 3rd doses) revealed hyperplasia of bronchial epithelium with intraluminal accumulation of mucous exudate (Figure25), interstitial pneumonia (Figure 26), perivasculitis (Figure 27), and hyperplasia of peribronchial lymphocytes. Improvement in the histopathological picture was observed in examined sections from rat treated with both nanoiron and folic acid. Examined lung (1st, 2nd, and 3rd doses) revealed a slight thickening of interstitial tissue (Figure 28) and perivasculitis (Figure 29). However, examined sections either treated with sulphate or control, untreated rat revealed no histopathological changes (Figure 30).


#### 3.4.4. Spleen

Microscopically, examined sections of spleen of rat treated with magnetite (1st, 2nd, and 3rd doses) revealed lymphocytic necrosis and depletion (Figure31). Meanwhile, examined spleen of rats treated with folate coated magnetite or treated with sulphate or those from control, untreated group revealed no histopathology changes (Figure 32).


## 3.1. Characterization of Material Nanoparticles (MNPs)

### 3.1.1. Transmission Electron Micrograph (TEM)

Transmission electron microscopy for the prepared SPIO NPs showed that particles have approximately (~) spherical shapes and their sizes were in the range of ~54 ± 3 nm, using UV/VIS spectroscopic characterization (Figure2(a)).


### 3.1.2. X-Ray Diffraction Analysis

The XRD spectrum of the prepared SPIO nanoparticles shows that six characteristic peaks for Fe3O4 (2θ = 30.10, 35.50, 43.10, 53.40, 57.00, and 62.60), marked by their indices 220, 311, 400, 422, 511, and 440, were observed for all samples using FTIR spectroscopic characterization (Figure 2(b)).


## 3.1.1. Transmission Electron Micrograph (TEM)

Transmission electron microscopy for the prepared SPIO NPs showed that particles have approximately (~) spherical shapes and their sizes were in the range of ~54 ± 3 nm, using UV/VIS spectroscopic characterization (Figure2(a)).


## 3.1.2. X-Ray Diffraction Analysis

The XRD spectrum of the prepared SPIO nanoparticles shows that six characteristic peaks for Fe3O4 (2θ = 30.10, 35.50, 43.10, 53.40, 57.00, and 62.60), marked by their indices 220, 311, 400, 422, 511, and 440, were observed for all samples using FTIR spectroscopic characterization (Figure 2(b)).


## 3.2. Biology Results

No animal mortality was found in rats treated with various iron-supplemented dietsbut there were some symptoms such as loss of appetite and severe lethargy which were observed during the experimental period of the rat, after administration of nanoparticle suspension. Results presented in Table1 indicated that the body weights change and different relative weight rats received iron-supplemented diets. Results showed that the relative weight liver of group (D2) was the nearest to normal group. The highest relative weights of lung and spleen were in group (C3) but they were similar to normal group in relative weight renal. The highest percent of body weight change was in the group fed first dose of magnetite while the lowest percent showed in group (D2).Table 1
Relative weight and body weight change.
Relative wt. liver
Relative wt. spleen
Relative wt. lung
Relative wt. renal
Body weight change%
Normal G (A)
3.5
±
0.07
0.27
±
0.09
0.54
±
0.08
0.81
±
0.08
17.3
±
3.5
FeSO4 G (B)
4.01
±
0.2
0.41
±
0.04
0.76
±
0.09
0.77
±
0.09
11.0
±
4.5
Fe G (C1)
4.1
±
0.5
0.47
±
0.08
0.68
±
0.06
0.87
±
0.06
24.0
±
5.7
Fe G (C2)
4.3
±
0.5
0.5
±
0.07
0.85
±
0.09
0.79
±
0.02
20.3
±
6.6
Fe G (C3)
4.63
±
0.5
0.63
±
0.1
0.93
±
0.08
0.81
±
0.09
9.3
±
5.4
Fe + Fo G (D1)
3.98
±
0.2
0.44
±
0.03
0.86
±
0.1
0.84
±
0.1
19.5
±
7.5
Fe + Fo G (D2)
3.93
±
0.2
0.47
±
0.05
0.79
±
0.2
0.91
±
0.3
7.7
±
1.1
Fe + Fo G (D3)
4.48
±
0.1
0.54
±
0.02
0.59
±
0.02
0.80
±
0.04
18.5
±
8.8


## 3.3. Biochemical Results

### 3.3.1. Hematology

Dietary iron deficiency with adding two-times zinc requirement for 2 to 3 weeks led to nutritional iron deficiency anemia in rats.Results presented in Figures3 and 4 indicated that the Hb and Hct were corrected in 75% of rats fed ferrous sulfate (Group B) during three weeks, while 25% fluctuated between up and down in the following weeks failing to reach normal value. Group C1 rats treated with 1/2 iron requirement dose from magnetite showed gradual decrease in Hb level throughout most of the follow-up weeks and were sacrificed at 7 weeks without showing improvement in iron status. Rats in Groups C2 and C3 reached normal Hb and Hct values by the 4th week followed by marked decreased Hb and Hct values afterwards.Figure 3
Hemoglobin (Hb) level in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure 4
Hematocrit (HCT) level in rat blood fed different diet with or without nanoparticles.N = Normal level.On the other hand, Group D1 (recipient of 1/2 dose of iron requirements and folat) reached normal Hb and Hct levels within four weeks. The other two Groups D2 and D3 showed improvement in Hb and Hct level within three weeks followed by decline in Hb and Hct values.Figures5, 6, 7, and 8 demonstrated normal values of RBCs, MCV, MCH, and MCHC within 3 weeks in Group B reaching plateau afterwards. Group D2 did not show any improvement in RBC level for the duration of the study. All groups exceeded normal values at different speeds or durations.Figure 5
Red blood cells (RBCs) level in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure 6
Mean corpuscular volume (MCV) in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure 7
Mean corpuscular hemoglobin (MCH) in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure 8
Mean corpuscular hemoglobin concentration (MCHC) in rat blood fed different diet with or without nanoparticles.N = Normal level.The rats fed 1st dose of magnetite and 3rd dose of folate-coated magnetite the results of MCV were less than normal volume to 7 wk. while the 2nd dose of magnetite showed that the normal dose fruition at 4 wk. after that increased about normal level. The rats received the 3rd dose of iron nanoparticles treated at two weeks and then the results fluctuated between up and down. The 1st dose of folate-coated magnetite indicated that normal level was at 5 weeks.MCH was less than normal level among all treatment groups, showed in Figure6.A bell shaped curve of MCHC parameter was noticed in Groups B, C2, D1, and D3 around the third week.Platelets level explained that increased in-group fed ferrous sulfate at third wk. after that decreased level in same group. Steady raise of PLT level until 5th week in group fed 1st dose of magnetite. Level of PLT increased from 3rd to 4th week, and then decreased from 5th to 7th week, but at eighth week, the increase was more than normal level of group fed 2nd dose from magnetite. Steady increase in group that received 3rd dose of magnetite. The 1st dose added to diet of folate-coated magnetite clarified that increased in PLT level at 2nd wk. and decreased after that but it steal higher than normal value. Rats fed 2nd and 3rd doses of folate-coated magnetite showed that the level of PLT fluctuated between up and down.MPV % demonstrated that the normal level was at the 3rd week with group fed 1st dose of magnetite and ferrous sulfate but the 2nd and 3rd doses of magnetite and 1st dose of folate-coated magnetite showed the normality at 2 week, while the 2nd and 3rd doses of folate-coated magnetite increased more than normal and after that decreased less than normal.Figures11 and 12 displayed that treated groups with nanomaterial the result fluctuated between increase and decrease than normal level of WBC. The largest increase shows in the group fed the 2nd dose of folate-coated magnetite.Lymph finding showed that all group fluctuated between increase and decrease than normal level (Figure12).


## 3.3.1. Hematology

Dietary iron deficiency with adding two-times zinc requirement for 2 to 3 weeks led to nutritional iron deficiency anemia in rats.Results presented in Figures3 and 4 indicated that the Hb and Hct were corrected in 75% of rats fed ferrous sulfate (Group B) during three weeks, while 25% fluctuated between up and down in the following weeks failing to reach normal value. Group C1 rats treated with 1/2 iron requirement dose from magnetite showed gradual decrease in Hb level throughout most of the follow-up weeks and were sacrificed at 7 weeks without showing improvement in iron status. Rats in Groups C2 and C3 reached normal Hb and Hct values by the 4th week followed by marked decreased Hb and Hct values afterwards.Figure 3
Hemoglobin (Hb) level in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure 4
Hematocrit (HCT) level in rat blood fed different diet with or without nanoparticles.N = Normal level.On the other hand, Group D1 (recipient of 1/2 dose of iron requirements and folat) reached normal Hb and Hct levels within four weeks. The other two Groups D2 and D3 showed improvement in Hb and Hct level within three weeks followed by decline in Hb and Hct values.Figures5, 6, 7, and 8 demonstrated normal values of RBCs, MCV, MCH, and MCHC within 3 weeks in Group B reaching plateau afterwards. Group D2 did not show any improvement in RBC level for the duration of the study. All groups exceeded normal values at different speeds or durations.Figure 5
Red blood cells (RBCs) level in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure 6
Mean corpuscular volume (MCV) in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure 7
Mean corpuscular hemoglobin (MCH) in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure 8
Mean corpuscular hemoglobin concentration (MCHC) in rat blood fed different diet with or without nanoparticles.N = Normal level.The rats fed 1st dose of magnetite and 3rd dose of folate-coated magnetite the results of MCV were less than normal volume to 7 wk. while the 2nd dose of magnetite showed that the normal dose fruition at 4 wk. after that increased about normal level. The rats received the 3rd dose of iron nanoparticles treated at two weeks and then the results fluctuated between up and down. The 1st dose of folate-coated magnetite indicated that normal level was at 5 weeks.MCH was less than normal level among all treatment groups, showed in Figure6.A bell shaped curve of MCHC parameter was noticed in Groups B, C2, D1, and D3 around the third week.Platelets level explained that increased in-group fed ferrous sulfate at third wk. after that decreased level in same group. Steady raise of PLT level until 5th week in group fed 1st dose of magnetite. Level of PLT increased from 3rd to 4th week, and then decreased from 5th to 7th week, but at eighth week, the increase was more than normal level of group fed 2nd dose from magnetite. Steady increase in group that received 3rd dose of magnetite. The 1st dose added to diet of folate-coated magnetite clarified that increased in PLT level at 2nd wk. and decreased after that but it steal higher than normal value. Rats fed 2nd and 3rd doses of folate-coated magnetite showed that the level of PLT fluctuated between up and down.MPV % demonstrated that the normal level was at the 3rd week with group fed 1st dose of magnetite and ferrous sulfate but the 2nd and 3rd doses of magnetite and 1st dose of folate-coated magnetite showed the normality at 2 week, while the 2nd and 3rd doses of folate-coated magnetite increased more than normal and after that decreased less than normal.Figures11 and 12 displayed that treated groups with nanomaterial the result fluctuated between increase and decrease than normal level of WBC. The largest increase shows in the group fed the 2nd dose of folate-coated magnetite.Lymph finding showed that all group fluctuated between increase and decrease than normal level (Figure12).


## 3.4. Histopathology Results

### 3.4.1. Liver

Microscopically, examined sections of liver from rat treated with magnetite (1st dose) revealed cytoplasmic vacuolizations of hepatocytes, infiltration of centrolobular hepatocytes with leucocytic inflammatory cells (Figure13), sinusoidal leukocytosis (Figure 14) and portal infiltration with leucocytes. Moreover, at the 2nd dose and 3rd dose, examined liver revealed Kupffer cells activation, focal hepatic necrosis associated with leucocytic cells infiltration (Figure 15) cytoplasmic vacuolizations of hepatocytes, apoptosis of hepatocytes (Figure 16), as well as portal infiltration with leucocytes. Liver sections of rat treated with both magnetite and folate coated magnetite (1st and 2nd doses) showed Kupffer cells activation, cytoplasmic vacuolization of hepatocytes and focal hepatic necrosis associated with leucocytic cells infiltration (Figure 17). At the 3rd dose, in addition to the previously mentioned changes, examined sections revealed apoptosis of hepatocytes. Examined sections of liver from rats either treated with sulphate or control, untreated rat revealed no histopathological changes (Figure 18).


### 3.4.2. Kidneys

Microscopically, examined sections of kidneys of rat treated with magnetite (1st, 2nd, and 3rd doses) revealed the same histopathological alterations which summarized as vacuolations of epithelial lining renal tubules as well as endothelial lining glomerular tufts (Figure 19), focal tubular necrosis associated with leucocytic inflammatory cells infiltration (Figure 20), and tubular necrosis associated with calcification (Figure 21). Improvement in the histopathological picture was noticed in examined sections of rat treated with both folate coated magnetite. At the 1st dose, examined sections showed vacuolations of epithelial lining renal tubules and endothelial lining glomerular tufts together with focal tubular necrosis associated with leucocytic cells infiltration (Figure 22). Meanwhile, at 2nd and 3rd doses, kidneys sections revealed vacuolations of epithelial lining renal tubules as well as endothelial lining glomerular tufts (Figure 23). However, examined sections either treated with sulphate or control, untreated rat revealed no histopathological changes (Figure 24).


### 3.4.3. Lung

Histopathology, examined sections of lung of rat treated with magnetite (1st, 2nd, and 3rd doses) revealed hyperplasia of bronchial epithelium with intraluminal accumulation of mucous exudate (Figure25), interstitial pneumonia (Figure 26), perivasculitis (Figure 27), and hyperplasia of peribronchial lymphocytes. Improvement in the histopathological picture was observed in examined sections from rat treated with both nanoiron and folic acid. Examined lung (1st, 2nd, and 3rd doses) revealed a slight thickening of interstitial tissue (Figure 28) and perivasculitis (Figure 29). However, examined sections either treated with sulphate or control, untreated rat revealed no histopathological changes (Figure 30).


### 3.4.4. Spleen

Microscopically, examined sections of spleen of rat treated with magnetite (1st, 2nd, and 3rd doses) revealed lymphocytic necrosis and depletion (Figure31). Meanwhile, examined spleen of rats treated with folate coated magnetite or treated with sulphate or those from control, untreated group revealed no histopathology changes (Figure 32).


## 3.4.1. Liver

Microscopically, examined sections of liver from rat treated with magnetite (1st dose) revealed cytoplasmic vacuolizations of hepatocytes, infiltration of centrolobular hepatocytes with leucocytic inflammatory cells (Figure13), sinusoidal leukocytosis (Figure 14) and portal infiltration with leucocytes. Moreover, at the 2nd dose and 3rd dose, examined liver revealed Kupffer cells activation, focal hepatic necrosis associated with leucocytic cells infiltration (Figure 15) cytoplasmic vacuolizations of hepatocytes, apoptosis of hepatocytes (Figure 16), as well as portal infiltration with leucocytes. Liver sections of rat treated with both magnetite and folate coated magnetite (1st and 2nd doses) showed Kupffer cells activation, cytoplasmic vacuolization of hepatocytes and focal hepatic necrosis associated with leucocytic cells infiltration (Figure 17). At the 3rd dose, in addition to the previously mentioned changes, examined sections revealed apoptosis of hepatocytes. Examined sections of liver from rats either treated with sulphate or control, untreated rat revealed no histopathological changes (Figure 18).


## 3.4.2. Kidneys

Microscopically, examined sections of kidneys of rat treated with magnetite (1st, 2nd, and 3rd doses) revealed the same histopathological alterations which summarized as vacuolations of epithelial lining renal tubules as well as endothelial lining glomerular tufts (Figure 19), focal tubular necrosis associated with leucocytic inflammatory cells infiltration (Figure 20), and tubular necrosis associated with calcification (Figure 21). Improvement in the histopathological picture was noticed in examined sections of rat treated with both folate coated magnetite. At the 1st dose, examined sections showed vacuolations of epithelial lining renal tubules and endothelial lining glomerular tufts together with focal tubular necrosis associated with leucocytic cells infiltration (Figure 22). Meanwhile, at 2nd and 3rd doses, kidneys sections revealed vacuolations of epithelial lining renal tubules as well as endothelial lining glomerular tufts (Figure 23). However, examined sections either treated with sulphate or control, untreated rat revealed no histopathological changes (Figure 24).


## 3.4.3. Lung

Histopathology, examined sections of lung of rat treated with magnetite (1st, 2nd, and 3rd doses) revealed hyperplasia of bronchial epithelium with intraluminal accumulation of mucous exudate (Figure25), interstitial pneumonia (Figure 26), perivasculitis (Figure 27), and hyperplasia of peribronchial lymphocytes. Improvement in the histopathological picture was observed in examined sections from rat treated with both nanoiron and folic acid. Examined lung (1st, 2nd, and 3rd doses) revealed a slight thickening of interstitial tissue (Figure 28) and perivasculitis (Figure 29). However, examined sections either treated with sulphate or control, untreated rat revealed no histopathological changes (Figure 30).


## 3.4.4. Spleen

Microscopically, examined sections of spleen of rat treated with magnetite (1st, 2nd, and 3rd doses) revealed lymphocytic necrosis and depletion (Figure31). Meanwhile, examined spleen of rats treated with folate coated magnetite or treated with sulphate or those from control, untreated group revealed no histopathology changes (Figure 32).


## 4. Discussion

### 4.1. Hematology

Findings of current study in Figures3 and 4 are consistent with previous reports. Singh [18] who illustrated that generally the internalization of NPs within cells is likely to occur in a time-dependent manner followed by plateau when cells reach maximum saturation. Similarly, Fischer et al. [19] reported that cell viability subsequent to nanoparticle uptake is expected to be either unaffected or decreased as a function of time. The transient cell growth and proliferation observed in the study may be due to additional nutrients present within the cells in the form of iron and phospholipids for the viable cells stimulating cell growth. Soenen et al. [20] explained the bell-shaped curve observed as an initial lag phase in cellular activity where the cells have to deal with sudden exposure and internalization of NPs, middle log phase marked by a period (6–10 h) of cellular growth as the cells resume their cellular activities, and finally the cell number either stabilizes or there is reduced cell viability as a result of cell toxicity.Regarding Figure5, among the causes of low RBC are the nutritional deficiency, bone marrow disorders or damage, chronic inflammatory disease, and kidney failure. On the other hand, high RBC level also known as polycythemia could be caused by dehydration, lung (pulmonary) disease, kidney, or other tumor that produces excess erythropoietin [21].With regard to MCV value in Figure6, low results of MCV indicate that RBCs are smaller than normal (microcytic), caused by iron deficiency anemia or thalassemia, but the high findings indicated that RBCs are larger than normal (macrocytic), for example, in anemia caused by vitamin B12 or folate deficiency [21].The low results of MCH illustrate mirrors MCV results; small red cells would have a lower normal value [21].The low concentration may be low when MCV is low; decreased MCHC values (hypochromia) are seen in conditions such as iron deficiency anemia but high concentration, increased MCHC values (hyperchromia) are seen in conditions where the hemoglobin is more concentrated inside the red cells [21]. However, the criteria to define toxicity of NPs needs to be clearly defined [22], particularly as emerging studies have begun to highlight aberrant cellular responses including DNA damage, oxidative stress, and mitochondrial membrane Dysfunction.Results showed that the level of PLT fluctuated between up and down for all treated groups that were demonstrated in Figure9. Low result is known as thrombocytopenia (viral infection, platelet autoantibody, cirrhosis). Causes of high result are known as thrombocytosis (cancer (lung, gastrointestinal, breast, ovarian, and lymphoma), iron deficiency anemia, and hemolytic anemia). Sadeghiani et al. [23] showed that uncoated iron oxide NPs have very low solubility that can lead to precipitation (if not sufficiently small) due to gravitation forces and also a high rate of agglomeration under physiological conditions that can impede blood vessels particularly in a clinical exposure setting. Vieira et al. [24] study the Fe3O4 NPs that could potentially be used as novel therapeutic agents in the treatment of protein myeloid aggregation-associated human pathologies.Figure 9
Platelets level (PLT) in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure10 illustrated MPV % in all rats groups. Causes of low value of MPV indicate that average size of platelets is small and a low MPV may mean that a condition is affecting the production of platelets by the bone marrow. Causes of high result indicate a high number of larger, younger platelets in the blood; this may be due to the bone marrow producing and releasing platelets rapidly into circulation [21].Figure 10
Mean Platelets volume (MPV) in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure 11
White blood cells level (WBCs) in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure 12
Lymph level in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure 13
Liver of rat treated with nanoiron (1st dose) showing cytoplasmic vacuolizations of hepatocytes, infiltration of centrilobular hepatocytes with leucocytic inflammatory cells (H & E × 400).Figure 14
Liver of rat treated with nanoiron (1st dose) showing sinusoidal leukocytosis (H & E × 400).Figure 15
Liver of rat treated with nanoiron (2nd dose) showing focal hepatic necrosis associated with leucocytic cells infiltration (H & E × 400).Figure 16
Liver of rat treated with nanoiron (3rd dose) showing Kupffer cells activation, cytoplasmic vacuolizations of hepatocytes, and apoptosis of hepatocytes (H & E × 400).Figure 17
Liver of rat treated with nanoiron and folic acid (1st dose) showing cytoplasmic vacuolizations of hepatocytes and focal hepatic necrosis associated with leucocytes infiltration (H & E × 400).Figure 18
Liver of rat treated with sulphate showing no histopathological changes (H & E × 400).Figure 19
Kidneys of rat treated with nanoiron (1st dose) showing vacuolations of epithelial lining renal tubules as well as endothelial lining glomerular tufts (H & E × 400).Figure 20
Kidneys of rat treated with nanoiron (1st dose) showing focal tubular necrosis associated with leucocytic inflammatory cells infiltration (H & E × 400).Figure 21
Kidneys of rat treated with nanoiron (3rd dose) showing tubular necrosis associated with calcification (H & E × 400).Figure 22
Kidneys of rat treated with nanoiron and folic acid (1st dose) showing focal necrosis of renal tubules associated with leucocytic cells infiltration (H & E × 400).Figure 23
Kidneys of rat treated with nanoiron and folic acid (2nd dose) showing vacuolations of epithelial lining renal tubules as well as endothelial lining glomerular tufts (H & E × 400).Figure 24
Kidneys of rat treated with sulphate showing no histopathological changes (H & E × 400).Figure 25
Lung of rat treated with nanoiron (1st dose) showing hyperplasia of bronchial epithelium with intraluminal accumulation of mucous exudate (H & E × 200).Figure 26
Lung of rat treated with nanoiron (1st dose) showing interstitial pneumonia (H & E X 400).Figure 27
Lung of rat treated with nanoiron (2nd dose) showing perivasculitis (H & E × 400).Figure 28
Lung of rat treated with nanoiron and folic acid (1st dose) showing slight thickening of interstitial tissue (H & E × 400).Figure 29
Lung of rat treated with nanoiron and folic acid (3rd dose) showing perivasculitis (H & E × 400).Figure 30
Lung of rat treated with sulphate showing no histopathological changes (H & E × 400).Figure 31
Spleen of rat treated with nanoiron (2nd dose) showing lymphocytic necrosis and depletion (H & E × 400).Figure 32
Spleen of rat treated with nanoiron and folic acid (2nd dose) showing no histopathological changes (H & E × 400).Figure11 showed WBC results of normal and treatment group. Low result of WBC level is known as neutropenia (reaction to drugs, chemotherapy, bone marrow damage, and cancer that spreads to the bone marrow). Causes of high result are known as neutrophilia (acute bacterial infections, inflammation, and tissue death [21]).Result of lymph showed in Figure12. Low result of lymph is known as lymphocytopenia (autoimmune disorders, infections, and bone marrow damage). Causes of high result are Known as lymphocytosis (acute viral infections, certain bacterial infections, toxoplasmosis, and chronic inflammatory disorder) [21]. Häfeli et al. [25] reported that exposure to nanoparticles (NPs) has been associated with significant toxic effects such as inflammation, impaired mitochondrial function (MTT), membrane leakage of lactate dehydrogenase (LDH assay), and generation of reactive oxygen species (ROS). Singh [19] showed that the exposure to these NPs and the effect that the range of surface coatings utilized for functionality are crucial. Therefore, there is a considerable need to address biocompatibility and biosafety concerns associated with their usage in a variety of applications. Buscombe et al. [26] explanation that the sentinel lymph node (SLN) is defined as the first regional lymph node receiving lymphatic drainage from a malignant tumor and the first node to which metastatic cells are likely to anchor. Therefore, accurate detection and characterization of the SLN is of major importance for cancer staging and for the choice of therapy in patients.


### 4.2. Histopathology

#### 4.2.1. Liver

Findings showed in Figures from13 to 18 agree with some studies on nanoparticles. Increasing numbers of studies demonstrated that many types of nanoparticles have toxic effects mainly on liver, kidneys, and spleen tissues [27]. In liver tissue, the hydropic degeneration around the central vein was prominent and the spotty necrosis of hepatocyte was also found in the female mice postexposure 2 weeks to the 80 nm and fine TiO2 particles. Pasupuleti et al. [28] showed that the Liver with nanozinc oxide at dose of 2000 mg/kg B.W. appears focal necrosis of liver. Esmaeillou et al. [29] showed the histopathology changes of liver with nanozinc oxide, such as cellular necrosis, congestion, and glycogen accumulation in liver.


#### 4.2.2. Kidneys

Wang et al. [27] reported that the histopathology changes of kidneys in the female mice are shown in the 80 nm TiO2 group; the renal tubule was filled with the proteinic liquids. In addition, the serious swelling in the renal glomerulus was observed in the group treated with fine particles. Esmaeillou et al. [29] reported that the rats’ group exposure to nanozinc oxide explained swelling in epithelial cells of proximal tubules, glomeruli segmentation, hydropic degeneration in kidneys’ epithelial cells, and necrosis of epithelial cells in kidney tubules. The previous study had agreement with the results in this research.


#### 4.2.3. Lung

Histopathology, examined sections of lung of rat treated with both magnetite and folate- coated magnetite had different changes (Figures25, 26, 27, 28, 29, and 30). Esmaeillou et al. [29] indicated that rats group after exposure to nanozinc oxide showed the histopathology changes of lung like this serous inflammation, severe hyperemia in alveoli, and edema in lung. Warheit et al. [30] administrated that the size range <100 nm of a-quartz particles produce more potent inflammatory responses when compared to larger particles of similar chemistry at the same mass concentrations or doses particles in rats: toxicity is not dependent upon particle size but on surface characteristics.


#### 4.2.4. Spleen

Interestingly, trace damage of reproductive organs was detected in further histopathology examinations. The atretic/healthy follicle ratio was significantly higher in examined sections of treated mice spleen compared with control group. The possible mechanism through which ZnO nanoparticles exert their toxic effects on human liver cells was investigated by Sharma et al. [31]. The results demonstrate that ZnO nanoparticles accumulate in the tissues and induce intracellular reactive oxygen species (ROS) generation. ROS trigger a decrease in mitochondrial membrane potential (MMP) with a simultaneous increase in the ratio of Bax/Bcl2 leading to mitochondrial mediated apoptosis. Moreover, ROS can also induce DNA damage. It seems that different nanoparticles exert their toxic effects on different tissues through similar oxidative mechanisms, this result is consistent with the group fed standard diet and three doses of magnetite while disagree with other groups.


## 4.1. Hematology

Findings of current study in Figures3 and 4 are consistent with previous reports. Singh [18] who illustrated that generally the internalization of NPs within cells is likely to occur in a time-dependent manner followed by plateau when cells reach maximum saturation. Similarly, Fischer et al. [19] reported that cell viability subsequent to nanoparticle uptake is expected to be either unaffected or decreased as a function of time. The transient cell growth and proliferation observed in the study may be due to additional nutrients present within the cells in the form of iron and phospholipids for the viable cells stimulating cell growth. Soenen et al. [20] explained the bell-shaped curve observed as an initial lag phase in cellular activity where the cells have to deal with sudden exposure and internalization of NPs, middle log phase marked by a period (6–10 h) of cellular growth as the cells resume their cellular activities, and finally the cell number either stabilizes or there is reduced cell viability as a result of cell toxicity.Regarding Figure5, among the causes of low RBC are the nutritional deficiency, bone marrow disorders or damage, chronic inflammatory disease, and kidney failure. On the other hand, high RBC level also known as polycythemia could be caused by dehydration, lung (pulmonary) disease, kidney, or other tumor that produces excess erythropoietin [21].With regard to MCV value in Figure6, low results of MCV indicate that RBCs are smaller than normal (microcytic), caused by iron deficiency anemia or thalassemia, but the high findings indicated that RBCs are larger than normal (macrocytic), for example, in anemia caused by vitamin B12 or folate deficiency [21].The low results of MCH illustrate mirrors MCV results; small red cells would have a lower normal value [21].The low concentration may be low when MCV is low; decreased MCHC values (hypochromia) are seen in conditions such as iron deficiency anemia but high concentration, increased MCHC values (hyperchromia) are seen in conditions where the hemoglobin is more concentrated inside the red cells [21]. However, the criteria to define toxicity of NPs needs to be clearly defined [22], particularly as emerging studies have begun to highlight aberrant cellular responses including DNA damage, oxidative stress, and mitochondrial membrane Dysfunction.Results showed that the level of PLT fluctuated between up and down for all treated groups that were demonstrated in Figure9. Low result is known as thrombocytopenia (viral infection, platelet autoantibody, cirrhosis). Causes of high result are known as thrombocytosis (cancer (lung, gastrointestinal, breast, ovarian, and lymphoma), iron deficiency anemia, and hemolytic anemia). Sadeghiani et al. [23] showed that uncoated iron oxide NPs have very low solubility that can lead to precipitation (if not sufficiently small) due to gravitation forces and also a high rate of agglomeration under physiological conditions that can impede blood vessels particularly in a clinical exposure setting. Vieira et al. [24] study the Fe3O4 NPs that could potentially be used as novel therapeutic agents in the treatment of protein myeloid aggregation-associated human pathologies.Figure 9
Platelets level (PLT) in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure10 illustrated MPV % in all rats groups. Causes of low value of MPV indicate that average size of platelets is small and a low MPV may mean that a condition is affecting the production of platelets by the bone marrow. Causes of high result indicate a high number of larger, younger platelets in the blood; this may be due to the bone marrow producing and releasing platelets rapidly into circulation [21].Figure 10
Mean Platelets volume (MPV) in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure 11
White blood cells level (WBCs) in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure 12
Lymph level in rat blood fed different diet with or without nanoparticles.N = Normal level.Figure 13
Liver of rat treated with nanoiron (1st dose) showing cytoplasmic vacuolizations of hepatocytes, infiltration of centrilobular hepatocytes with leucocytic inflammatory cells (H & E × 400).Figure 14
Liver of rat treated with nanoiron (1st dose) showing sinusoidal leukocytosis (H & E × 400).Figure 15
Liver of rat treated with nanoiron (2nd dose) showing focal hepatic necrosis associated with leucocytic cells infiltration (H & E × 400).Figure 16
Liver of rat treated with nanoiron (3rd dose) showing Kupffer cells activation, cytoplasmic vacuolizations of hepatocytes, and apoptosis of hepatocytes (H & E × 400).Figure 17
Liver of rat treated with nanoiron and folic acid (1st dose) showing cytoplasmic vacuolizations of hepatocytes and focal hepatic necrosis associated with leucocytes infiltration (H & E × 400).Figure 18
Liver of rat treated with sulphate showing no histopathological changes (H & E × 400).Figure 19
Kidneys of rat treated with nanoiron (1st dose) showing vacuolations of epithelial lining renal tubules as well as endothelial lining glomerular tufts (H & E × 400).Figure 20
Kidneys of rat treated with nanoiron (1st dose) showing focal tubular necrosis associated with leucocytic inflammatory cells infiltration (H & E × 400).Figure 21
Kidneys of rat treated with nanoiron (3rd dose) showing tubular necrosis associated with calcification (H & E × 400).Figure 22
Kidneys of rat treated with nanoiron and folic acid (1st dose) showing focal necrosis of renal tubules associated with leucocytic cells infiltration (H & E × 400).Figure 23
Kidneys of rat treated with nanoiron and folic acid (2nd dose) showing vacuolations of epithelial lining renal tubules as well as endothelial lining glomerular tufts (H & E × 400).Figure 24
Kidneys of rat treated with sulphate showing no histopathological changes (H & E × 400).Figure 25
Lung of rat treated with nanoiron (1st dose) showing hyperplasia of bronchial epithelium with intraluminal accumulation of mucous exudate (H & E × 200).Figure 26
Lung of rat treated with nanoiron (1st dose) showing interstitial pneumonia (H & E X 400).Figure 27
Lung of rat treated with nanoiron (2nd dose) showing perivasculitis (H & E × 400).Figure 28
Lung of rat treated with nanoiron and folic acid (1st dose) showing slight thickening of interstitial tissue (H & E × 400).Figure 29
Lung of rat treated with nanoiron and folic acid (3rd dose) showing perivasculitis (H & E × 400).Figure 30
Lung of rat treated with sulphate showing no histopathological changes (H & E × 400).Figure 31
Spleen of rat treated with nanoiron (2nd dose) showing lymphocytic necrosis and depletion (H & E × 400).Figure 32
Spleen of rat treated with nanoiron and folic acid (2nd dose) showing no histopathological changes (H & E × 400).Figure11 showed WBC results of normal and treatment group. Low result of WBC level is known as neutropenia (reaction to drugs, chemotherapy, bone marrow damage, and cancer that spreads to the bone marrow). Causes of high result are known as neutrophilia (acute bacterial infections, inflammation, and tissue death [21]).Result of lymph showed in Figure12. Low result of lymph is known as lymphocytopenia (autoimmune disorders, infections, and bone marrow damage). Causes of high result are Known as lymphocytosis (acute viral infections, certain bacterial infections, toxoplasmosis, and chronic inflammatory disorder) [21]. Häfeli et al. [25] reported that exposure to nanoparticles (NPs) has been associated with significant toxic effects such as inflammation, impaired mitochondrial function (MTT), membrane leakage of lactate dehydrogenase (LDH assay), and generation of reactive oxygen species (ROS). Singh [19] showed that the exposure to these NPs and the effect that the range of surface coatings utilized for functionality are crucial. Therefore, there is a considerable need to address biocompatibility and biosafety concerns associated with their usage in a variety of applications. Buscombe et al. [26] explanation that the sentinel lymph node (SLN) is defined as the first regional lymph node receiving lymphatic drainage from a malignant tumor and the first node to which metastatic cells are likely to anchor. Therefore, accurate detection and characterization of the SLN is of major importance for cancer staging and for the choice of therapy in patients.


## 4.2. Histopathology

### 4.2.1. Liver

Findings showed in Figures from13 to 18 agree with some studies on nanoparticles. Increasing numbers of studies demonstrated that many types of nanoparticles have toxic effects mainly on liver, kidneys, and spleen tissues [27]. In liver tissue, the hydropic degeneration around the central vein was prominent and the spotty necrosis of hepatocyte was also found in the female mice postexposure 2 weeks to the 80 nm and fine TiO2 particles. Pasupuleti et al. [28] showed that the Liver with nanozinc oxide at dose of 2000 mg/kg B.W. appears focal necrosis of liver. Esmaeillou et al. [29] showed the histopathology changes of liver with nanozinc oxide, such as cellular necrosis, congestion, and glycogen accumulation in liver.


### 4.2.2. Kidneys

Wang et al. [27] reported that the histopathology changes of kidneys in the female mice are shown in the 80 nm TiO2 group; the renal tubule was filled with the proteinic liquids. In addition, the serious swelling in the renal glomerulus was observed in the group treated with fine particles. Esmaeillou et al. [29] reported that the rats’ group exposure to nanozinc oxide explained swelling in epithelial cells of proximal tubules, glomeruli segmentation, hydropic degeneration in kidneys’ epithelial cells, and necrosis of epithelial cells in kidney tubules. The previous study had agreement with the results in this research.


### 4.2.3. Lung

Histopathology, examined sections of lung of rat treated with both magnetite and folate- coated magnetite had different changes (Figures25, 26, 27, 28, 29, and 30). Esmaeillou et al. [29] indicated that rats group after exposure to nanozinc oxide showed the histopathology changes of lung like this serous inflammation, severe hyperemia in alveoli, and edema in lung. Warheit et al. [30] administrated that the size range <100 nm of a-quartz particles produce more potent inflammatory responses when compared to larger particles of similar chemistry at the same mass concentrations or doses particles in rats: toxicity is not dependent upon particle size but on surface characteristics.


### 4.2.4. Spleen

Interestingly, trace damage of reproductive organs was detected in further histopathology examinations. The atretic/healthy follicle ratio was significantly higher in examined sections of treated mice spleen compared with control group. The possible mechanism through which ZnO nanoparticles exert their toxic effects on human liver cells was investigated by Sharma et al. [31]. The results demonstrate that ZnO nanoparticles accumulate in the tissues and induce intracellular reactive oxygen species (ROS) generation. ROS trigger a decrease in mitochondrial membrane potential (MMP) with a simultaneous increase in the ratio of Bax/Bcl2 leading to mitochondrial mediated apoptosis. Moreover, ROS can also induce DNA damage. It seems that different nanoparticles exert their toxic effects on different tissues through similar oxidative mechanisms, this result is consistent with the group fed standard diet and three doses of magnetite while disagree with other groups.


## 4.2.1. Liver

Findings showed in Figures from13 to 18 agree with some studies on nanoparticles. Increasing numbers of studies demonstrated that many types of nanoparticles have toxic effects mainly on liver, kidneys, and spleen tissues [27]. In liver tissue, the hydropic degeneration around the central vein was prominent and the spotty necrosis of hepatocyte was also found in the female mice postexposure 2 weeks to the 80 nm and fine TiO2 particles. Pasupuleti et al. [28] showed that the Liver with nanozinc oxide at dose of 2000 mg/kg B.W. appears focal necrosis of liver. Esmaeillou et al. [29] showed the histopathology changes of liver with nanozinc oxide, such as cellular necrosis, congestion, and glycogen accumulation in liver.


## 4.2.2. Kidneys

Wang et al. [27] reported that the histopathology changes of kidneys in the female mice are shown in the 80 nm TiO2 group; the renal tubule was filled with the proteinic liquids. In addition, the serious swelling in the renal glomerulus was observed in the group treated with fine particles. Esmaeillou et al. [29] reported that the rats’ group exposure to nanozinc oxide explained swelling in epithelial cells of proximal tubules, glomeruli segmentation, hydropic degeneration in kidneys’ epithelial cells, and necrosis of epithelial cells in kidney tubules. The previous study had agreement with the results in this research.


## 4.2.3. Lung

Histopathology, examined sections of lung of rat treated with both magnetite and folate- coated magnetite had different changes (Figures25, 26, 27, 28, 29, and 30). Esmaeillou et al. [29] indicated that rats group after exposure to nanozinc oxide showed the histopathology changes of lung like this serous inflammation, severe hyperemia in alveoli, and edema in lung. Warheit et al. [30] administrated that the size range <100 nm of a-quartz particles produce more potent inflammatory responses when compared to larger particles of similar chemistry at the same mass concentrations or doses particles in rats: toxicity is not dependent upon particle size but on surface characteristics.


## 4.2.4. Spleen

Interestingly, trace damage of reproductive organs was detected in further histopathology examinations. The atretic/healthy follicle ratio was significantly higher in examined sections of treated mice spleen compared with control group. The possible mechanism through which ZnO nanoparticles exert their toxic effects on human liver cells was investigated by Sharma et al. [31]. The results demonstrate that ZnO nanoparticles accumulate in the tissues and induce intracellular reactive oxygen species (ROS) generation. ROS trigger a decrease in mitochondrial membrane potential (MMP) with a simultaneous increase in the ratio of Bax/Bcl2 leading to mitochondrial mediated apoptosis. Moreover, ROS can also induce DNA damage. It seems that different nanoparticles exert their toxic effects on different tissues through similar oxidative mechanisms, this result is consistent with the group fed standard diet and three doses of magnetite while disagree with other groups.


## 5. Conclusion

Different studies on the toxicological effects of nanoparticles have indicated that these particles act with the similar oxidative toxicological mechanisms and these effects are dependent on their physicochemical properties such as specific surface area, the properties of the metal concerned, as well as nanoparticle size. Furthermore, different exposure routes, such as oral administration, dermal contact, and inhalation, have various specific toxicological impacts. Symptoms of vomiting, loss of appetite, and severe lethargy demonstrate that magnetite and folate-coated magnetite nanoparticles have serious toxicological effectsin vivo. The biochemical and pathological examinations revealed that lung, liver, and kidney are target organs for these particles. According to the present results, magnetite and folate-coated magnetite nanoparticles can induce their toxicological effects on different organs and some parameter of blood picture within a 3-week timeframe in the rats. Therefore, it is necessary for all researchers who are regularly exposed to nanoparticles such as these nanoparticles to consider the toxicological hazards especially the size of 54 nm.



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*Source: 287575-2014-03-04.xml*