Biodistribution and Acute Toxicity of Intravenous Multifunctional 125 I-Radiolabeled Fe 3 O 4-Ag Heterodimer Nanoparticles in Mice

Department of Nuclear Medicine, The First Affiliated Hospital of Soochow University, Suzhou, 215006 Jiangsu, China Department of Applied Chemistry, Anhui Agricultural University, Hefei, 230036 Anhui, China Key Laboratory of Organic Synthesis of Jiangsu Province, College of Chemistry, Chemical Engineering and Materials Science & Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Suzhou 215123, China


Introduction
In recent years, a great deal of attention has been paid to silver nanoparticles (AgNPs) since they are used as popular antibacterial and antifungal agents in the light of an enormously increasing bacterial resistance against repeatedly and excessively used classical antibiotics.AgNPs can effectively eliminate bacteria at a relatively low concentration [1][2][3].Besides antimicrobial ability, AgNPs are effective in the field of photothermal cancer therapy and/or surfaceenhanced Raman spectroscopy [4].
Magnetic iron oxide (Fe 3 O 4 ) NPs have been widely used in many important fields due to their unique characteristics, such as biochemical properties, superparamagnetism and low price [5][6][7][8].Fe 3 O 4 -Ag heterodimer NPs possess magnetic functionality and antimicrobial ability at the same time [2].
Our group has successfully developed Fe 3 O 4 -Ag 125 I heterostructured radionuclide NPs as novel dual-modality imaging agents for magnetic resonance imaging (MRI) and single-photon emission computerized tomography (SPECT) [9].The Fe 3 O 4 -Ag 125 I heterostructured radionuclide NPs demonstrate high radiolabeling efficiency and clearly reduced T 2 -MRI signal intensity.
We aimed to apply this material to medical imaging.However, no study has investigated the distribution and toxicity of Fe 3 O 4 -AgNPs in animals.Moreover, previous studies show inconsistent results, indicating that the distribution and toxicity of Fe 3 O 4 or Ag NPs are highly dependent on the various factors, such as shape, size, coating agent of the NPs, duration after drug administration, and animal gender [10][11][12][13][14][15].Therefore, we investigated the biodistribution and toxicity of Fe 3 O 4 -Ag 125 I NPs in mice after intravenous injection.The bioaccumulation of Fe 3 O 4 -Ag 125 I NPs was studied via in vivo experiments.The serum biochemistry and hematology were analyzed to reveal potential functional changes.The histopathological changes were observed by using an electron microscope.[9].Briefly, the Fe 3 O 4 NPs were synthesized by thermal decomposition of ironoleate complex, and then the AgNPs were grown onto the cubic Fe 3 O 4 NPs by adding the silver acetate into the reaction system.Subsequently, the Fe 3 O 4 -AgNPs were functionalized by hydrophilic mPEG-LA polymers and phase transferred from hexane to water.Finally, Fe 3 O 4 -Ag 125 I NPs were produced by reacting the Ag component of the heterostructured NPs with 125 I.

Materials and Methods
The labeling efficiency and radiochemical purity were analyzed using paper chromatography.The fractions containing 125 I-labeled Fe 3 O 4 -Ag were determined using a gamma counter to calculate the radiolabeling yield (%).The solution was filtered through a 0.22 μm pore-size membrane in order to avoid potential bacterial and dust particles for in vivo studies.
2.4.Biodistribution of Fe 3 O 4 -Ag 125 I NPs.Kunming mice (n = 5 per time point) were intravenously injected with Fe 3 O 4 -Ag 125 I NPs (100 μL/4.92-6.99MBq) via the tail vein once daily and sacrificed by exsanguination under ether anesthesia at 1, 2, 8, 24, and 48 h after injection.Blood samples (approximately 100 μL each) were collected via retroorbital bleeding, and main organs, such as the blood, lung, brain, kidney, liver, pancreas, spleen, stomach, thyroid, intestine, bone, and muscle, were dissected from anesthetized mice and weighed at 1, 2, 8, 24, and 48 h postinjection.The radioactivity of the tissue was measured in a γ-counter (Shanghai Nucleus Research Institute Rihuan Photoelectric Instrument Co. Ltd.).The uptake in organs was calculated as the proportion of injected dose per gram of tissue (%ID/g).

2.5.
In Vivo SPECT Imaging.SPECT scans were performed using the IRIX (Philips, Netherlands) equipped with highresolution low-energy parallel-hole collimator.Briefly, after injection of Fe 3 O 4 -Ag 125 I NPs, mice were anesthetized using isoflurane.The SPECT scans were performed at various time points.Images were acquired with 1 × 10 5 counts on a 128 × 128 matrix.The energy peak for the camera was set to 37 keV, and the energy window was set to peak energy ±30%, which was 26-48 keV.
2.6.Serum Biochemistry and Hematology.The mice were sacrificed after injection of Fe 3 O 4 -Ag 125 I NPs (40 mg/mL) for seven consecutive days.The blood was collected from the retroorbital sinus.For hematological analysis, the blood samples were combined with EDTA-3K for anticoagulation.The hematological measurements were performed using an automated hematology analyzer (BC-5800, Mindray Co., Shenzhen, China) following the standard protocols.
For serum biochemistry analysis, the blood samples were centrifuged at 3000 rpm for 15 min within 1 h, and the supernatant was collected.All the biochemical parameters were determined on a clinical automatic chemistry analyzer (Chemray360, Rayto Co., Shenzhen, China) following the standard protocols.

Transmission Electron Microscopy (TEM).
For TEM analysis of the spleen, heart, liver, and kidney, small pieces of tissue samples (∼1 mm 3 ) were fixed in 2.5% glutaraldehyde solution overnight and washed with phosphate-buffered saline (PBS).Postfixation was performed with 1% osmium tetroxide for 2 h.Then, the samples were washed with PBS and dehydrated with a graded series of alcohols (50%, 70%, 80%, 95%, and 100%), followed by rinsing with acetone.Ultrathin sections from each tumor sample were prepared and examined under JEOL-JEM-2100F TEM operating at 200 kV.

Statistical Analysis.
The results were expressed as the mean ± standard deviation (SD).Data were analyzed by oneway ANOVA and Student's t-test.p < 0 05 was considered as statistically significant.All statistical tests were two sided.
A TEM image (Figure 1) confirmed that the average size of Fe 3 O 4 -AgNPs was 24.53 ± 2.99 nm.The addition of a radionuclide into the Fe 3 O 4 -AgNPs did not change the morphology of the samples.Journal of Nanomaterials 48 h after injection) and spleen (41.87 ± 6.73%ID/g at 1 h after injection, 41.41 ± 13.32%ID/g at 2 h after injection, 39.49 ± 11.37%ID/g at 8 h after injection, 19.07 ± 13.22%ID/ g at 24 h after injection, and 15.34 ± 6.82%ID/g at 48 h after injection).These findings indicated that the injected 125 Ilabled conjugates were mainly taken up by the reticuloendothelial system (RES).
The activity level in the abdominal region (particularly the spleen and liver) was high in the first five static images, which was generally consistent with the results of in vivo biodistribution studies, indicating that the injected Fe 3 O 4 -Ag 125 I NPs were mainly sequestered by the RES.   3 Journal of Nanomaterials Little radioactivity was observed in the thyroid region during the early imaging procedure.However, there were slight increases in thyroid at the end of the imaging procedure, suggesting that this compound was deiodinated in vivo just as the results of biodistribution.

Discussion
During the past few decades, there are increasing applications of AgNPs in various fields.However, AgNPs have several shortcomings, including agglomeration, easy oxidation, low penetration into tissue, and cytotoxicity [16,17].Iron oxide NPs can add a magnetic functionality and prevent agglomeration to AgNPs.It has been reported that the bactericidal efficiency of Fe 3 O 4 -AgNPs is stronger than Fe 2 O 3 -Ag heterodimers or plain Ag [18].Despite the advantages of Fe 3 O 4 -AgNPs, the biodistribution and of Fe 3 O 4 -AgNPs remain largely unexplored.
In the present study, we systematically investigated the biodistribution of Fe 3 O 4 -AgNPs in mice after intravenous injection by noninvasive nuclear imaging techniques.Our study confirmed that the majority of Fe 3 O 4 -Ag 125 I NPs were accumulated in the spleen and liver, and such pattern could be attributed to uptake by the B cells and macrophages in the spleen and the Kupffer cells in the liver, which are part of the mononuclear phagocyte system.These results were consistent with some previous studies on biodistribution of nontargeted AgNPs and Fe 3 O 4 NPs [19][20][21][22].Chrastina and Schnitzer have radiolabeled AgNPs with 125 I to track the in vivo tissue uptake of AgNPs after systemic administration by biodistribution analysis and SPECT imaging.Their results have also revealed the uptake of AgNPs in the liver and spleen [23].
Recently, toxicity of Fe 3 O 4 NPs or AgNPs has been widely studied.Fe 3 O 4 NPs are generally considered as biocompatible, safe, and nontoxic materials.Median lethal dose (LD-50) of the uncoated Fe 3 O 4 NPs is 300-600 mg Fe/kg body weight [24].However, the toxicity of AgNPs based on in vivo studies is controversial.Maneewattanapinyo et al. have investigated the acute oral toxicity of AgNPs by in vivo experiments and found that the LD-50 of colloidal AgNPs is greater than 5000 mg/kg body weight [25].Another study has also revealed that no obvious changes in serum chemistry, hematology, and histopathology are found after SD rats are administered with up to 36 mg/kg AgNPs by oral gavage for 13 weeks [14].However, other studies have demonstrated that short-term administration of AgNPs can significantly increase ALT or/and AST [15,26,27].Tiwari et al. have investigated the toxic effect of various doses of AgNPs on Wistar rats and indicated that AgNPs at lower dose (<10 mg/kg) are safe, while its higher dose (>20 mg/ kg) is toxic [28].Recently, Ghaseminezhad et al. have compared the cytotoxicities of AgNPs and Ag/Fe 3 O 4 nanocomposites to human fibroblasts and found that Ag/Fe 3 O 4 nanocomposites are less cytotoxic than AgNPs [29].The Ag/Fe 3 O 4 nanocomposites show lower release of Ag ions and less ROS production compared with AgNPs.In the present study, the activities of liver enzymes (ALT and AST) were increased in the Fe 3 O 4 -Ag 125 I NP-challenged groups compared with the control groups, indicating that liver tissues were damaged following administration of Fe 3 O 4 -Ag 125 I NPs.
Some studies have suggested that the toxicity of AgNPs depends on surface capping.It has been demonstrated that polysaccharide-coated AgNPs induce more severe damages compared with uncoated AgNPs [30], whereas carboncoated AgNPs are less cytotoxic towards macrophages [31].Therefore, in order to develop the Fe 3 O 4 -Ag 125 I heterostructured radionuclide NPs as dual-modality imaging agents, NPs need to be coated with special compounds in the future.Additional studies are required in order to reshape the surface of Fe 3 O 4 -Ag to modify their characteristics.
Collectively, our present study investigated the biodistribution and acute toxicity of 125 I-radiolabeled Fe 3 O 4 -Ag heterodimer NPs in mice.We found that the liver and spleen were the major target organs for the accumulation of Fe 3 O 4 -Ag 125 I NPs.Damage of liver tissue was observed in the Fe 3 O 4 -Ag 125 I NP-challenged groups compared with the control groups.Further studies on surface coating of Fe 3 O 4 -Ag with targeted materials are highly necessary for safe medical applications of Fe 3 O 4 -AgNPs as dual-modality imaging agents.
Fe 3 O 4 -Ag 125 I NPs in Mice.
Figure 2   presents the biodistribution data of Fe 3 O 4 -Ag 125 I NPs in different organs at various time points postinjection.The uptake of Fe 3 O 4 -Ag 125 I was high in the liver (31.98 ± 3.74%ID/g at 1 h after injection, 31.00 ± 9.42%ID/g at 2 h after injection, 22.51 ± 4.57%ID/g at 8 h after injection, 5.79 ± 4.24%ID/g at 24 h after injection, and 4.48 ± 2.20%ID/g at 2

3. 4 .
Toxicity Evaluations.Haematological and serum biochemistry parameters were analyzed after exposure to Fe 3 O 4 -Ag 125 I NPs.Table 1 lists the data.Most parameters remained within the normal ranges at 7 days after the intravenous injection of Fe 3 O 4 -Ag 125 I NPs.Significant changes were only observed for alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

3. 5 .
TEM Analysis.TEM analysis was performed on the spleen, heart, liver, and kidney from the Fe 3 O 4 -Ag 125 I NPsadministered mice and negative control mice (Figure4).The results demonstrated that the Fe 3 O 4 -Ag 125 I NPs agglomerated in the spleen.In the liver, Fe 3 O 4 -Ag 125 I NPs were scattered throughout the parenchyma.In line with the result of biodistribution, less Fe 3 O 4 -Ag 125 I NPs were detected in the heart and kidney.

Table 1 :
Haematological and serum biochemistry parameters of the mice exposed to Fe 3 O 4 -Ag 125 I. Data represent means ± SD (n = 5).

Figure 4 :
Figure 4: TEM images of the spleen, heart, liver, and kidney of Fe 3 O 4 -Ag 125 I NPs-treated mice: (a) spleen, (b) liver, (c) kidney, and (d) heart.Arrows in black color show NPs.
2.1.Ethics Statement.Male Kunming mice (6 weeks of age) were purchased from the Center for Experimental Animal of Soochow University.Animal experiments were preapproved by the institutional review board and the Experimental Animal Center of the First Affiliated Hospital of Soochow University.All SPECT scans were performed under general anesthesia, and all efforts were made to minimize animal suffering.2.2.Materials.All the reagents for the synthesis of Fe 3 O 4 -AgNPs were purchased from Sigma-Aldrich.125I was obtained from Chengdu Gaotong Isotope Corporation (Chengdu, China).All other chemicals were prepared with analytical-grade reagents dissolved in deionized water prepared by LabWater (Shanghai Hejie Technology Co. Ltd.).2.3.Synthesis ofFe 3 O 4 -Ag 125 I NPs.Fe 3 O 4 -Ag 125 I NPs were synthesized as previously reported 9/L)