Introduction

High proportion of surface to volume of the nanoparticles in comparison of bulk samples, have made them adequate to be used in many medical and industrial applications. There are extensive researches on wide application of Fe3O4 nanostructures such as MR imaging tumor therapy [1], drug delivery and gene transfer to tissues and cells [2], labeling cells, and macromolecules [3]. There are many methods which can be used for synthesizing Fe3O4 and other nanaoparticles such as gamma-irradiation [4], electron beam radiation [5], co-precipitation, low temperature solid state reaction, and microwave method [6–10].

Nanoparticles are often smaller than cellular structures, virus, protein, or a gene. This unique characteristic provided them proper for quick distribution in most organs and tissues, and therefore very strong cellular uptake phenomenon [11]. In recent years, in order to increase the stability of nanoparticles in biological systems and blood circulation, they are coated with biocompatible materials such as albumin, dextran [12], polyethylene glycol, polyethylene oxide [13], aspartic acid, and dimercaptosuccinic acid (DMSA) (C4S2O4H6) [14]. Presence of such coatings increases the entrance to cells and decreases the toxicity effects [15].

There are few reports on Fe3O4 toxicity especially under in vivo conditions and moreover some researchers showed controversial results. For instance, some studies have reported minimal toxicity in some concentrations and some others have shown non-toxicity effects under in vivo conditions [16–18]. Therefore, it is essential to assess the toxicity effects of Fe3O4 nanoparticles in both in vitro and in vivo conditions. Moreover, considering the variety of biocompatible compounds can be used, it is interesting to study the tissue distribution and Fe3O4 toxicity for any particular coating compounds separately [15].

DMSA is a nontoxic substance that nowadays is used in some patients to absorb additional elements of the body (chelating agent) [16]. Using this substance creates an anionic coating around the nanoparticle surface and prevents opsonization (accumulation of blood proteins around the nanoparticle). Therefore, DMSA helps nanoparticles for less deletion by reticuloendothelial system of liver and spleen. Presence of this substance on nanoparticles surface increases the cell absorption and tissue distribution and also decreases direct contact with cells and cellular components which led to reduction of toxicity effects [18].

On the other hand, Technetium-99m (99mTc) is one of the most important radioisotopes in nuclear medicine which has wide application [19]. Radiolabeling of coated Fe3O4 with 99mTc can potentially help to achieve more desirable tissue distribution by applying magnetic fields due to super paramagnetic properties of Fe3O4 nanoparticles [20]. Radiolabeling of nanoparticles may reduce their affects on biodistribution and toxicity. In this study an attempt is to produce the coated Fe3O4 nanoparticles which labeled with 99mTc and also assess the biodistribution and toxicity effect of the nanoparticles in mice. At the end, in vitro and in vivo stability of final prepared nanoparticles also were investigated.

Experimental

Synthesis of DMSA coated Fe3O4 nanoparticles

DMSA-coated Fe3O4 nanoparticles (Fe3O4@DMSA) were synthesized by wet-chemical method. For this purpose, three solutions of FeCl2 (0.01 M), FeCl3 (0.02 M), and NaOH (0.08 M) (all from Merck Company) were prepared in the distilled deionized water, under vigorous stirring. At first, FeCl2 solution was poured into a three spout balloon container. Meanwhile, FeCl3 solution was added to the same balloon. After that, 0.01 M DMSA solution was prepared in deoxygenated deionized water. In the construction process, every 3 or 4 s, one droplet of DMSA solution was added via nitrogen bubbling and magnetic stirrer. Finally, FeCl3 solution was added to the balloon by the same way and under atmosphere control condition (N2). The resulting solution was washed by deionized water and then was centrifuged in order to remove any impurity aggregate. Then, the precipitated sample was dried at room temperature. All processes were done at room temperature. The details are described in references [21, 22].

Radiolabeling of Fe3O4@DMSA with 99mTc radioisotope

In the present study, in order to radiolabeling of Fe3O4@DMSA nanoparticles, the 99mTc, with activity of 1.3 GBq was prepared from 99Mo–99mTc generator. It was injected into a small sterile and vacuuming vial under the observance of radiation protection rules. Subsequently, 0.5 ml of stannous chloride (2.0 mg/ml) solution in 0.1 N HCl, was injected into the same vial, and stirred for several seconds. The stennous chloride, which mainly contains SnCl2, is a reducing agent that reduces surface charge of 99mTc to a suitable condition for labeling with other molecules. Then, 1 ml diluted Fe3O4@DMSA solution was added into the vial containing 99mTc and stannous chloride. The mixture was kept at room temperature and radio labeled and coated Fe3O4 nanoparticles (Fe3O4@DMSA–99mTc) were prepared after several minutes. The details are described in references [23, 24].

Quality assurance of Fe3O4@DMSA–99mTc

The chemical interaction between Fe3O4 and DMSA were investigated by Fourier transform infrared spectroscopy (FTIR, JASCO FT/IR-680 PLUS). The labeling efficiency was assessed by instant thin layer chromatography (ITLC) which silica gel and acetone solvent were used as instant and mobile phase, respectively. The radioactivity of each part of ITLC strips was determined by High Purity Germanium spectroscopy (HPGe spectroscopy, Canberra). Magnetic and structure properties of Fe3O4@DMSA–99mTc nanoparticles were investigated by alternating gradient-force magnetometer (AGFM, Meghnatis Daghigh Kavir Co, Iran) and X-ray diffraction (XRD, Bruker D8 ADVANCE λ = 0.154 nm Cu Kα radiation) [22].

Breeding animals and treatments

The animal studies were performed in accordance with regulatory guidance’s on the care and use of experimental animals. For this purpose, 55 male and female mice of Balb/C strain were provided from RAZI Vaccine and Serum Research Institute. Studying their life cycle was performed in natural light and humid and at 22–24 °C temperature. In order to investigate the toxicity effects 25 mice were randomly selected. They were divided into five equal groups. Four groups were intraperitoneally injected by 50, 100, 200, and 300 mg/kg of Fe3O4@DMSA–99mTc, respectively, and 5th group just was injected with normal saline (control group). Rest mice were used for biodistribution and stability assessments.

In vitro and in vivo stability

In order to assessment of in vivo stability, 100 MBq of Fe3O4@DMSA–99mTc intravenously injected to five mice (each mouse received 100 MBq). After that blood samples were collected at different time intervals which were then subjected to ITLC. For In vitro stability investigation, 50 MBq of Fe3O4@DMSA–99mTc were mixed separately to saline and mice serum and then incubated at 37 °C. Samples were subjected to ITLC at different time intervals.

Measurement of blood, kidney, and liver factors

Four days after treatment, blood samples were taken directly from heart under mild anesthesia with ether. Samples were poured into the special pipes which contain edetic acid (EDTA) anti coagulation agent. After that, cell blood counting (CBC) test was done (Sysmex K-100) for all blood samples in order to assessment the number and percentage of red blood cells, white blood cells, platelets, neutrophils, and lymphocytes. Kidney and liver factors such as lactate dehydrogenase (LDH), serum glutamic pyruvate transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), uric acid, creatinine, and urea were measured by Hitachi Automatic Analyzer (Roche 902).

Tissue section preparation for Perl’s method staining

For this purpose, several mice were dissected immediately after bleeding (4 days after treatment) and tissue sections were prepared from their kidney and liver. These organs were selected because the liver is the center of drug metabolism and kidneys are responsible for drug exclusion from the blood circulation. Then, the prepared tissue sections were stained using specific iron Prussian blue method (Perl’s Method) to observe nanoparticles accumulation. Accumulation of iron oxide nanoparticles were showed in tissue sections as dark blue grains under microscope light.

Biodistribution of Fe3O4@DMSA–99mTc

For evaluating localization of Fe3O4@DMSA–99mTc in different tissues and organs, 25 mice were divided into five equal groups that each group contains five mice. Each mouse was intravenously injected through the tail vein with 200 kBq of Fe3O4@DMSA–99mTc. The tissue distribution studies were evaluated after 20 min, 1, 2, 4, and 10 h post injection. At these time intervals, animals were sacrificed and subsequently, tissues (brain, lung, spleen, kidney, liver, and blood) were removed and washed with normal saline. The radioactivity of each organ was counted with Gamma counter (Berthold-LB2111, Germany) and showed as percent injected dose per gram of tissue [25].

Statistical analysis

The mean values of blood, kidney, and 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 of five separate experiments for each group.

Results and discussion

Physical properties assessment and quality assurance of Fe3O4@DMSA–99mTc

Figure 1 shows the transmission electron microscope (TEM) photograph of the Fe3O4@DMSA nanoparticles. This picture demonstrates that the size of the particles is around 10–15 nm with approximately uniform size distribution. The mean size of the nanoparticles was also determined from XRD pattern by Debye–Scherer formula. It was calculated around 15 nm which has good agreement with that TEM result (XRD curve is not shown here) [22].

Fig. 1
figure 1

TEM photograph of the Fe3O4@DMSA nanoparticles

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Magnetic properties of the nanoparticles were assessed by AGFM and it was found that they have the super paramagnetic property. The saturation magnetization was determined by extrapolation of magnetization curve on the basis of \( 1/H \) when \( 1/H \to 0 \). It was measured 24 emu/g (AGFM curve is not shown here) [22].

Figure 2 indicates the FTIR curve of the Fe3O4, DMSA, and Fe3O4@DMSA. As can be seen, in the Fe3O4 curve, 1,628 and 3,419 peaks are related to OH junctions and it means that there is water in the material structure. The 581 peak shows that the spinel structure was formed and we will see it has a good agreement with XRD results. On the other hand, in the Fe3O4@DMSA curve, 1,619 and 1,376 peaks are related to the asymmetry and symmetry stresses of COO group, respectively. If we compare them with the 1,699 and 1,421 peaks in the DMSA curve, it can be concluded that the DMSA has coated the surface of the Fe3O4 nanoparticles. Also, decrease in the 581 peak is the other reason for this conjunction [22].

Fig. 2
figure 2

a FTIR curve of Fe3O4. b FTIR curve of Fe3O4@DMSA. c FTIR curve of DMSA

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The labeling efficiency of nanoparticles was verified by using ITLC method, since it is an easy way and well accepted process to assess the radiolabeling quality in nuclear medicine. The labeling efficiency was obtained more than 98 %. The same ITLC tests were done after 2, 4, and 8 h and the labeling efficiency of 90 % was determined.

In vitro and in vivo stability studies

In vitro stability study indicated that the labeled conjugate was approximately stable up to 10 h both in serum (around 88 %) and normal saline (around 90 %) which had good agreement with the in vivo stability studies (around 92 %).

Results of blood, kidney, and liver factors measurement

Table 1 shows the results of CBC tests 4 days after treatment. As can be seen, the WBC and lymphocyte mean numbers are decreased for samples treated over 100 mg/kg concentration. The similar event has happened for RBC mean number over 200 mg/kg and for platelets just in 300 mg/kg concentration. On the other hand, neutrophil percentage is slowly increased for all treatment doses.

Table 1 Cell Blood Counting (CBC) test 4 days after treatment with coated and radio labeled Fe3O4 nanoparticles (intraperitoneally injection)
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The results of kidney and liver factors 4 days after treatment are shown in Table 2. By considering the P value which is more than 0.05 for all variables, it can be seen that, there is not any meaningful difference for kidney and liver factors (such as LDH, SGPT, SGOT, Uric Acid, Creatinine, and Urea) between the treated and control groups in all treatment doses. This point means that Fe3O4 nanoparticles do not create any disorder in the kidney and liver function even in high doses (300 mg/kg).

Table 2 Results of kidney and liver factors measurement 4 days after treatment with coated and radio labeled Fe3O4 nanoparticles (intraperitoneally injection)
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Stained kidney and liver tissue sections observation

Figure 3, displays aggregated Fe3O4@DMSA–99mTc nanoparticles in the stained liver tissue sections 4 days after treatment. As can be seen, these nanoparticles were detected in the sinusoids and hepatocytes of the liver tissue of treatment mice.

Fig. 3
figure 3

The stained liver tissue sections (Perl’s method) 4 days after treatment (Magnification ×40). a Control group. b, c 50 mg/kg treated group. Dark blue dots show the accumulation of iron oxide nanoparticles in the sinusoids (S), hepatocytes (H) and liver blood vessels (V). (Color figure online)

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Figure 4 shows accumulated Fe3O4@DMSA–99mTc nanoparticles in the stained kidney tissue sections 4 days after treatment. It can be seen that they were found in the glomerules, nephron wall cells, internal nephron canal, and kidney blood vessels. These results indicate that, the Fe3O4@DMSA–99mTc nanoparticles were passed through the membrane of different cells.

Fig. 4
figure 4

The stained kidney tissue sections (Perl’s method) 4 days after treatment (Magnification ×40). a Control group. b, c 50 mg/kg treated group. Dark blue dots show the accumulation of iron oxide nanoparticles in the glomerules (G), nephron wall cells (P & D), internal nephron canal (L) and kidney blood vessels (V). (Color figure online)

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Biodistribution of Fe3O4@DMSA–99mTc in normal mice

Tissue distribution of Fe3O4@DMSA–99mTc between 20 min and 10 h in healthy mice of Balb/c is represented in Table 3. As can be seen, the major accumulation is observed in liver. The second organs which have high accumulation are kidneys, lungs, and spleen. There is considerable difference between their accumulations with that of liver. Negligible uptake is found in brain. Liver and lung indicated a reduction accumulation after 20 min whereas spleen and kidney showed an increased uptake after 2 h post injection and then decreased. High accumulation of liver is related to the phagocytic uptake in the Kupffer cell of the liver. Many studies reported that most of the intravenously injected particles are removed from blood circulation by the mononuclear phagocyte system (MPS) [16, 17]. The result is in accordance with the earlier reports which shown Fe3O4 localization coating with other biocompatible materials or labeling with other radioisotopes [24, 26].

Table 3 Biodistribution of Fe3O4@DMSA–99mTc in mice following intravenous injection
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Conclusions

From the Figs. 3, 4 it is concluded that, the Fe3O4@DMSA–99mTc nanoparticles were passed through the membrane of different cells and dramatically penetrate to liver and kidney after 4 days. But they did not create any abnormality or functional disorder in the kidney and liver tissues.

From Table 2 it is found that, there is not any meaningful variation in the liver and kidney enzymes or factors even in high treatment doses such as 300 mg/kg (P ≥ 0.05). This is probably for the reason that nanoparticles were eliminated slowly from the body by excretion or may be lack of absorption.

On the other hand, the number and percentage of different blood cells were decreased specially in high doses. It is noteworthy that these high doses never used in medicine especially for diagnostic applications and as we know 99mTc only used for diagnostic applications.

From findings here, there is not any serious harmful side effect for Fe3O4@DMSA–99mTc nanoparticles in order to in vivo applications.