Nanoparticles have high proportion of surface to volume in comparison with bulk samples and this unique characteristic has made them proper for many medical and industrial applications. There are considerable studies on vast application of Fe3O4 nanoparticles such as cancer therapy, drug delivery (1,2) MR imaging (3) and biotechnology (4). There are many methods can be used for synthesizing nanoparticles such as non-alkoxide sol-gel method, low temperature solid state reaction and microwave method (5-8).
Nanoparticles are usually smaller than biological structures such as cell, virus, protein or a gene. This tiny size provided them adequate for fast distribution in many tissues and considerable cellular uptake phenomenon (9). Recently, nanoparticles were coated with biocompatible materials in order to increase their stability in blood circulation and biological organs. They are many materials which are used for coating such as BSA (Bovine serum albumin), DMSA (Dimercaptosuccinic Acid C4S2O4H6), dextran, polyethylene glycol, chitosan and aspartic acid (10-13). Presence of these coatings at the surface of nanomaterials increases the cell entrance and reduces their toxicity effects (14).
There are small numbers of studies on Fe3O4 toxicity especially under in vivo conditions and also, some studies reported controversial results. As an illustration, some researchers have reported non-toxicity under in vivo conditions and some others have shown minimal toxicity at same concentrations (14-17). Moreover, it is interesting to assess the Fe3O4 toxicity for any specific coating compounds separately by considering the variety of biocompatible materials (14). Therefore, it seems it is essential to investigate the toxicity effects of Fe3O4 nanoparticles and coated Fe3O4 nanoparticles in both in vitro and in vivo conditions.
BSA is a serum albumin protein derived from cows. It is often used as a protein concentration standard. It is a globular protein that is used in numerous biochemical applications due to its stability and lack of interference within biological reactions. DMSA is a nontoxic substance that recently used in some patients in order to absorb additional elements of the body (chelating agent) (15). Using these substances creates an anionic coating around the nanoparticle surface and prevents opsonization (accumulation of blood proteins around the nanoparticle). Therefore, these coating materials can potentially help nanoparticles for more stability in blood circulation and less deletion by reticuloendothelial system of liver and spleen. In fact, presence of these substances on the surface of nanomaterials decreases direct contact with cells and cellular components which led to reduction of toxicity effects. Moreover, they increase the tissue distribution and cell absorption (17).
In this study an attempts is to evaluate the effect of BSA and DMSA (as a coating on the surface of Fe3O4 nanoparticles) on the toxicity effects of Fe3O4 nanoparticles.
3.1. Synthesis of Fe3O4 nanoparticles
Fe3O4 nanoparticles were synthesized by wetted chemical method. In this method, three solutions of FeCl2 (0.01 M equals to 1.98 g), FeCl3 (0.02 M equals to 5.41 g) and NaOH (0.08 M equals to 3.2 g) (all from Merck company) were prepared in the distilled deionized water. For this purpose, FeCl2 solution was poured into a triple neck round balloon and meanwhile, FeCl3 solution was added to the same balloon, under vigorous magnetic stirring. Then, every three or four seconds, one droplet of ME (Mercaptoethanol) solution was added via magnetic stirrer. Finally, NaOH solution was added to the balloon by the same way. The resulting solution was washed by deionized water and then was centrifuged in order to remove any aggregate as impurity. All processes were done at room temperature (18).
3.2. Coating of Fe3O4 nanoparticles with BSA and DMSA
0.5 gr of BSA was diluted to 50 ml normal saline and the solution was added to 100 ml ofFe3O4 solution which prepared before and allowed the interaction to be completed for 3 hour under rapid stirring with ultrasonic. The coated nanoparticles were separated from the uncoated nanoparticles and extra BSA by centrifugation for 30 min. Then, for more accuracy, 100 ml of distilled water was added and the centrifugation was repeated. The same processes were done in order to prepare the DMSA coated Fe3O4 nanoparticles except to ultrasonic process which was done just for 1 hour. All processes were done at room temperature (18, 19).
3.3. Quality assurance and measurement of properties of the samples
XRD (X-Ray Diffraction, Bruker D8 ADVANCE λ=0.154nm Cu Kα radiation) was used in order to evaluate the size and crystalline structure. The accelerating voltage and the applied current were 40 kV and 40 mA, respectively. Data were recorded at a scan rate for two seconds in steps of 0.04° for 2 . The crystalline size was calculated from X-ray line broadening analysis by the Debye-Scherrer equation for the full-width at half-maximum of the strongest reflection Where, D is the crystalline size in nm, λ is the Cu-Kα wavelength (0.154 nm), β is the half-maximum breadth, and θ is the Bragg angle of the (311) plane (20):
The room temperature magnetization measurements up to a maximum field of 10 kOe were carried out using AGFM (Alternating Gradient-Force Magnetometer, Meghnatis Daghigh Kavir Co, Iran). TEM (Transmission Electron Microscope) was used for size and size distribution (12). The coating chemical interactions were assessed by FTIR (Fourier Transform Infrared Spectroscopy, JASCO FT/IR-680 PLUS) (21).
3.4. Nanoparticles injection to mice
For this purpose, 240 mice of Balb/c strain were prepared from RAZI Vaccine and Serum Research Institute. They were 3-months old and were kept in natural light and humid at 22-24 ºC temperature. They were divided into 16 equal groups (each group contains 15 mice). One group was injected with normal saline as a control group and the 15 remain groups were received Fe3O4, DMSA, BSA, Fe3O4@ DMSA and Fe3O4@BSA. Different concentrations of 50, 100 and 200 mg per kg of mice weight were intra peritoneally injected. The animal studies were performed in accordance with regulatory guidance on the care and use of experimental animals. Mice’s weight were measured and recorded at the time of injection and every week up to one month.
3.5. Measurement of Liver factors
Blood samples were taken directly from heart under mild anesthesia with ketamine at the time intervals of 7, 15 and 30 days post injection. Blood samples were poured into the special pipes which contain EDTA (Edetic Acid) anticoagulant agent. Then, liver factors such as SGPT (Serum Glutamic Pyruvate Transaminase), SGOT (Serum Glutamic Oxaloacetic Transaminase), LDH (Lactate Dehydrogenase) and ALP (Alkaline phosphatase) were measured by Automatic Analyzer (RA1000 Technicon, America).
3.6. Statistical analysis
The mean values of liver factors (with treatment dose segregation) were compared by ANOVA test (analysis of variance) and t-test by SPSS (version 15) computer program in all groups. Results are the mean value of 5 separate experiments for each group.
4.1. Physical properties and quality assurance of coated Fe3O4 nanoparticles
The structure of all samples was assessed by XRD. Figure 1 indicates the XRD pattern of the uncoated, DMSA coatedand BSA coated Fe3O4 nanoparticles. As can be seen, all samples are single phase and also have the ferrite spinel structure. The intensity of XRD background toward peak is higher in the sample coated with BSA in comparison with that of DMSA. It is probably due to the DMSA and BSA structures. The mean size of the particles was determined by Debye-Scherer formula. It was calculated 11nm for uncoated Fe3O4 nanoparticles, 17 nm for DMSA coatedand 25 nm for BSA coated Fe3O4 nanoparticles.
TEM photograph of the uncoated Fe3O4 nanoparticles is shown in figure 2. This photograph indicates that the sizes of the particles are around 10 nm with approximately uniform size distribution. This is compatible with the results of the XRD patterns (Figure 1) because the particle size increases by coating process.
Magnetic properties of the nanoparticles were investigated by AGFM and it was proved that all samples are superparamagnetic. Figure 3 shows the uncoated, DMSA and BSA coated Fe3O4 nanoparticles results. The saturation magnetization was determined by extrapolation of magnetization curve on the basis of when . It was measured 62, 27 and 23 emu/gr for uncoated, DMSA and BSA coated nanoparticles respectively (21).
FTIR curves of the Fe3O4, DMSA coated and BSA coated Fe3O4 nanoparticles are demonstrated in figure 4. It can be seen that, 1628 cm-1 and 3419 cm-1 peaks in the Fe3O4 curve, are related to OH junctions and it can be concluded that there is water molecule in the material structure. The 581 cm-1 peak indicates that the spinel structure was formed and we will see it is compatible with the XRD curve results. Moreover, 1619 cm-1 and 1376 cm-1 peaks in DMSA coated Fe3O4 nanoparticles curve, are related to the asymmetry and symmetry stresses of COO group, respectively. By considering that these peaks are in a close relation with the 1699 cm-1 and 1421 cm-1 peaks in the DMSA curve, it is proved that the DMSA has coated the surface of the Fe3O4 nanoparticles. Furthermore, the 581 cm-1 peak decrease is another reason for this conjunction (20).
By the same way, it can be concluded that BSA has also coated the surface of the Fe3O4 nanoparticles. For instance, 2919 cm-1 and 3520 cm-1 peaks in the BSA coated Fe3O4 nanoparticles curve are the deformed peaks of 2923 cm-1 and 3419 cm-1 in Fe3O4 curve. The 588 cm-1 peak is related to the spinel structure which is the 581 cm-1 deformed peak in the Fe3O4 FTIR. The peak of 1495 cm-1 in the BSA coated Fe3O4 is related to the albumin conjunctions which is absent in the Fe3O4 and DMSA coated Fe3O4 FTIR. It is another reason for the accuracy of the coating process between the BSA and Fe3O4 nanoparticles.
4.2. Liver enzymes measurement
SGOT, SGPT, ALP and LDH liver enzymes were measured 7, 15, 30 days post injection in all 16 groups. The results of the day of 7 and 15 are so much similar and therefore the results of the day 7 are not shown here. Figure 5, 6, 7 and 8 show the SGOT, SGPT, ALP and LDH measurement results 15 days post injection.
As can be seen, there is not any meaningful change in the groups received less than 100 mg/kg BSA, DMSA and uncoated Fe3O4 nanoparticles in comparison with control. There is significant change for DMSA coated nanoparticles treated group even in 50 mg/kg concentration but BSA coated nanoparticles treated group shows meaningful change just in 200 mg/kg concentration (By considering the p-value which is more than 0.05). Therefore, it can be resulted that BSA has high compatibility with biological systems.
Figures 9, 10, 11 and 12 demonstrates the results of SGOT, SGPT, ALP and LDH liver enzymes measurements 30 days post injection. It can be seen that there are less differences between the treated and control groups for all enzymes in comparison with that of 7 and 15 days post injection. In the groups who received albumin there is not any meaningful difference even in high doses (200 mg/kg). Moreover, in the concentration of 50 mg/kg there is not any meaningful difference in all treated groups. These findings resulted that the liver enzymes values intended to come back to the normal value after a month and most of the differences are reversible.
The findings seem to prove that coated nanoparticles are more affected on liver function in comparison with uncoated iron oxidenanoparticles. This probably occurred, due to their more stability in blood circulation and consequently more penetration in different organs and cells. Therefore, it seems that the use of such coating materials (DMSA and BSA) on the surface of the Fe3O4 nanoparticles increases their stability and side effects on liver enzymes.
Also, by considering the p-value which is more than 0.05 for all variables there is not any significant change in mice’s weight in all groups during a month (Results are not shown here).
By surveying the results of 30 days post injection, it can be concluded that most values are returning to the normal value and it is expected that all measured enzymes return to normal value in near future. This point shows that coated and uncoated iron oxidenanoparticles do not create any irreversible effect or disorder in liver function even in high doses (200 mg/kg).
These results are so much similar to that of Kim et al., which they injected less than 100 mg/kg concentrations of silica (SiO2) coated nanoparticles (CoFe2O4) intra peritoneally into mice and realized the presence and distribution of nanoparticles in mice organs. No specific disorder was found in liver enzymes 30 days post injection and also no weight changes were observed (17). Hafeli and Pauer intra spinally injected poly lactic acid coated Fe3O4 into rats. They didn’t observed any mortality, toxicity or abnormality in animals’ behavior during one year post injection. Animal’s growth and weight were reported normal (16).
On the other hand, Sadeghiani et al., reported inflammatory responses due to the intravenously injection of poly aspartic acid coated Fe3O4 nanoparticles into mice. Moreover, they observed lymphocytes, monocytes and neutrophils increase and also some disorders in the maturation process of red blood cells were occurred. These effects occurred during the first to 15th day post injection and remained until one month (14). In another study, intravenously injection of DMSA coated Fe3O4 nanoparticles into mice entering lungs (respiratory bronchioles and alveolar sac) while crossing the blood air dam and led to inflammatory responses, but the severity of these changes reduced after three months (15). The most important and remarkable point which is mentioned in most studies is small amount usage of Fe3O4 nanoparticles in medicine do not create any severe effect.
In this study, the effects of intra peritoneally injection of BSA and DMSA coated Fe3O4 nanoparticles (up to 200 mg/kg) on mice’s liver enzymes were assessed during a month. The results indicate that, most of liver enzymes were changed meaningful and significantly. However, this situation was temporary and most of them returned to their normal range after a month. The findings seem to prove the fact that coated iron oxidenanoparticles were more affected in comparison with uncoated nanoparticles. This phenomenon happened probably due to their more stability in blood circulation and consequently more penetration in different organs and cells. Moreover, DMSA coated nanoparticles were more toxic in comparison with BSA coated nanoparticles.
From findings here, low concentrations in vivo application of Fe3O4 nanoparticles (less than 200 mg/kg) do not create any serious or sever toxic effect. Furthermore, there was not any meaningful change in mice’s weight during a month post injection. According to the controversial reports in different studies, it seems that more investigations with different biocompatible coatings are necessary.
The authors are grateful from Falavarjan Azad University for their cooperation and supplying the experimental equipments (Most experimental results are from research code No. 305/2231).
All authors have participated equally.
There is no conflict of interest.
Funding / Support
The study is self-funded.