Toxicity and Tissue Distribution of Magnetic Nanoparticles in Mice
http://www.100md.com
《毒物学科学杂志》
Laboratory of Toxicology, College of Veterinary Medicine and School of Agricultural Biotechnology, Materials Chemistry Laboratory, Chemical Biology Laboratory, School of Chemistry (NS60), Seoul National University, Seoul 151–742
Laboratory of Molecular Oncology, Korea Institute of Radiological & Medical Sciences, Seoul 139–240, Korea
ABSTRACT
The development of technology enables the reduction of material size in science. The use of particle reduction in size from micro to nanoscale not only provides benefits to diverse scientific fields but also poses potential risks to humans and the environment. For the successful application of nanomaterials in bioscience, it is essential to understand the biological fate and potential toxicity of nanoparticles. The aim of this study was to evaluate the biological distribution as well as the potential toxicity of magnetic nanoparticles to enable their diverse applications in life science, such as drug development, protein detection, and gene delivery. We recently synthesized biocompatible silica-overcoated magnetic nanoparticles containing rhodamine B isothiocyanate (RITC) within a silica shell of controllable thickness [MNPs@SiO2(RITC)]. In this study, the MNPs@SiO2(RITC) with 50-nm thickness were used as a model nanomaterial. After intraperitoneal administration of MNPs@SiO2(RITC) for 4 weeks into mice, the nanoparticles were detected in the brain, indicating that such nanosized materials can penetrate blood–brain barrier (BBB) without disturbing its function or producing apparent toxicity. After a 4-week observation, MNPs@SiO2(RITC) was still present in various organs without causing apparent toxicity. Taken together, our results demonstrated that magnetic nanoparticles of 50-nm size did not cause apparent toxicity under the experimental conditions of this study.
Key Words: magnetic nanoparticles; blood-brain barrier.
INTRODUCTION
The recent shift in the focus of developing nanoparticles from microscale to nanoscale is essential for future advances in both the digital revolution and modern biology and may change the very foundations of education, medicine, and industrial manufacture and have a potential harmful effect on the environment. Such a rapid development in nanotechnology will result in several changes in areas such as nanoscale visualization, insight into living systems, revolutionary biotechnology, synthesis of new drugs as their targeted delivery, and regenerative medicine and offer many other benefits (Roco, 2003).
Reducing the particle size of materials is an efficient and reliable tool for improving the bioavailability of a gene or drug delivery system. In fact, nanotechnology helps in overcoming the limitations of size and can change the outlook of the world regarding science (Chad and Andrew, 2000). Further, nanomaterials can be modified for better efficiency to facilitate their applications in different fields such as bioscience and medicine. For example, magnetic nanoparticles (MNPs) have been studied with an intent to apply them in bioscience, because they offer benefits such as separation and gathering of materials of interest by using a magnetic force (Hongwei et al., 2003). However, the lack of information regarding the toxicity of manufactured nanoparticles poses serious problems. Therefore, it is necessary that specialists and researchers in toxicology, chemistry, and other fields are aware of the importance of analyzing the positive aspects of nanomaterials while avoiding their potential toxic effects (Oberdorster et al., 2005).
Our team, consisting of experts in multidisciplinary areas, recently synthesized silica-overcoated MNPs containing rhodamine B isothiocyanate (RITC) within a silica shell of controllable thickness, MNPs@SiO2(RITC), for future applications such as drug or gene delivery (Yoon et al., 2005). However, for the application of MNPs for targeted delivery, sufficient data regarding the toxicity and biological fate of the MNPs should be accumulated. Both the toxicity and tissue distribution of MNPs@SiO2(RITC) should be evaluated because nanoparticles are expected to be applicable to biomedicine. Therefore, in this study, MNPs@SiO2(RITC) with a 50-nm thickness were used as a model nanomaterial for the evaluation of in vivo biological distribution and potential toxicity. Here, we report that nanosized MNPs@SiO2(RITC) can penetrate the blood–brain barrier without any apparent toxicity. These results support the hypothesis that the MNPs can be applied to the field of biomedicine and further suggest that extensive studies are required to clarify the potential chronic toxicity of MNPs.
MATERIALS AND METHODS
Preparation of magnetic nanoparticles.
Fifty-nm size MNPs labeled with RITC (Sigma-Aldrich, St. Louis, MO) were synthesized according to a previously described method (Yoon et al., 2005). In brief, cobalt ferrite (Sigma-Aldrich) was added to polyvinylpyrolidone (PVP, Sigma-Aldrich) solution. The PVP-stabilized cobalt ferrite nanoparticles were separated by the addition of acetone and centrifugation. The precipitated particles were redispersed in ethanol. Trimethoxysilane (Gelest, Morrisville, PA) modified by RITC was prepared from 3-aminopropyltriethoxysilane (APS; Gelest) and RITC under nitrogen. Such synthesized dye solution was mixed with tetraethoxysilane (TEOS, Gelest) and injected into the PVP-stabilized cobalt ferrite ethanol solution and, subsequently, polymerized on the surface of the PVP-stabilized cobalt ferrites by adding ammonia as a catalyst to form RITC-incorporated MNPs [MNPs@SiO2(RITC)]. The silica shell thickness was successfully adjusted such that it can be reproduced by controlling the concentration of TEOS. All MNPs@SiO2(RITC) were confirmed by a transmission electron microscope (TEM).
Animals and MNPs@SiO2(RITC) treatment.
Male ICR mice (6 weeks old) were purchased from Joongang Laboratory Animal Inc. (Seoul, Korea). Our laboratory animal facility is maintained under a 12-h light/dark cycle at a temperature of 20–22°C, and a relative humidity of 20–50%. All methods used in this study were approved by the Animal Care and Use Committee at Seoul National University (SNU) and conformed to the NIH guidelines (NIH publication No. 86–23, revised 1985). Based on systematic studies for identification of dose-range as well as practical consideration of the synthetic capacity of MNPs@SiO2(RITC), three different concentrations of MNPs@SiO2(RITC), i.e., 100, 50, and 25 mg/kg, were administered to the mice intraperitoneally for 4 weeks. For the blood–brain barrier (BBB) study, a concentration of 10 mg/kg was selected because this concentration was sufficient to test the BBB function. During the study period, any clinical signs were carefully observed. The mice were sacrificed after designated time periods (every week: thus, at four time points during the study) to analyze the tissue distribution and perform toxicity studies.
Serum biochemical analysis.
Blood samples were collected every week after MNP treatment (four time points of analysis in total). Using a biochemical autoanalyzer (VITALAB, Merck, The Netherlands), serum biochemical analysis was carried out to determine the serum level of total protein, albumin, total bilirubin, aspartate transaminase (AST), alanine transaminase (ALT), glucose, cholesterol, triglyceride, blood urea nitrogen, creatinine, calcium, phosphorus, and lactic acid dehydrogenase.
Hematological analysis.
As mentioned previously, blood samples were collected after MNP treatment at four points of time in total. Hematological parameters consisting of erythrocytes, leukocytes, neutrophils, lymphocytes, monocytes, eosinophils, basophils, platelets, hemoglobin, and hematocrit were determined using a hematological autoanalyzer (Coulter T540 hematology system; Fullerton, CA).
Histopathological analysis.
Mice were perfused with buffered (0.4 M phosphate buffer, pH 7.6) 4% paraformaldehyde. The brain, liver, lungs, kidneys, heart, spleen, testes (in males), and uterus (in females) were removed from each animal and immersion-fixed in the same fixative for 24 h at room temperature. Tissue sections (4 μm) were prepared after dehydration and were embedded in paraffin. The sections were stained with hematoxilin and eosin (H&E) and subsequently processed for histopathological examination under light microscope.
Fluorescent image analysis of MNP biodistribution.
Several representative organs such as the brain, liver, kidneys, lungs, and testes were removed and fixed in 4% paraformaldehyde solution. After fixation, the tissue samples were dehydrated with 30% sucrose for 24 h and then cryosectioned at 10-μm thickness (Mullen et al., 1992). Then, the slides were mounted in ultramount media (Dako, Carpinteria, CA) and incubated for 3 h at room temperature. After incubation, the slides were observed under a confocal laser scanning microscope (CLSM; Zeiss 510; Atlanta, GA). Serial cryosectioned slides of the brain were observed under CLSM at high power view (x1200; the section interval thickness was 10 μm). For image analysis, RITC excitation was performed using an argon laser at 543 nm. To confirm whether the presence of MNPs in the brain was a true indicator of their uptake by the neurons, an immunofluorescence study was performed with neuronal nuclei (NeuN), because this protein is highly expressed in the brain (Mullen et al., 1992). In brief, brain tissue sections were sequentially incubated with 10% normal goat serum in 0.1 M PBS for 30 min and with anti-NeuN antibody (1:20; Chemicon, Temecula, CA) overnight. After washing with phosphate-buffered saline (PBS), the sections were incubated with FITC-conjugated goat anti-mouse IgG (1:200; Serotec, Raleigh, NC). After a second washing with PBS, the sections were mounted with glycerol and observed under the CLSM. The dorsal cochlear nucleus (DCN) was observed as a representative of the whole brain.
Evaluation of blood–brain barrier (BBB) permeability.
To evaluate the potential effects of MNPs@SiO2(RITC) on BBB permeability, Evans blue (EB) dye was used as a marker of albumin extravasations because EB dye is known to bind to serum albumin. Mannitol was used as a positive control because hyperosmolar mannitol is known to disrupt the BBB permeability (Kaya et al., 2004). In brief, the EB dye (2% in saline, 4 ml/kg) was injected via the tail vein at 5 min prior to MNPs@SiO2(RITC) administration and was allowed to remain in circulation for 30 min. A solution of 25% mannitol (Sigma-Aldrich) dissolved in 0.9% saline was injected into the mice at 10 min after the intravenous injection of the EB dye. After 30 min, the thorax was opened under anesthesia. The mice were perfused transcardially with saline for approximately 10 min until the fluid from the right atrium became colorless. After decapitation, the brain was removed and dissected. After a careful examination for the presence of the EB dye, the dissected brain was homogenized in 2.5 ml PBS and mixed by vortexing for 2 min after the addition of 2.5 ml 60% trichloroacetic acid to precipitate the protein. The samples were then centrifuged for 30 min at 1000 x g. The absorbance of EB in the supernatants was measured at 610 nm for absorbance of EB using a spectrophotometer (Kaya et al., 2004).
Evaluation of genotoxicity.
Ames Assay Salmonella mutagenicity assay was performed according to the method of Maron and Ames (1983). Salmonella typhimurium strains TA97, TA98, TA100, and TA102 were purchased from Molecular Toxicology Inc. (Boone, NC). Each strain was tested for its genetic traits such as histidine requirement, deep rough (rfa) characteristic, UV sensitivity (uvrB mutation), and ampicillin- or tetracycline-resistance by R-factor prior to use. The strains were inoculated on oxoid nutrient broth and cultured for 15 h at 37°C with a continuous agitation at 150 rpm. The cell density was 1–2 x 105/ml. The concentrations of MNP@SiO2(RITC) tested were 0.25, 0.5, and 1.0 mg sample/plate. The S9 mixture and cofactor (Molecular Toxicology Inc.) were prepared, and the activity of the S9 mixture was confirmed by inducing mutagenesis using 2-aminoanthracene (2-AA). The positive controls were sodium azide (Sigma-Aldrich), ICR 191 acridine (Sigma-Aldrich), mitomycin C (Sigma-Aldrich), daunomycin (Perkin Elmer, Wellesley, MA), and 2-aminoanthracene (Sigma-Aldrich). They were dissolved in deionized distilled water (DDW) or dimethylsulfoxide (DMSO, Sigma-Aldrich). The direct plate incorporation method was used with three plates per concentration to be tested. Both 0.1 ml of the cell culture (1–2 x 105/ml) and 0.1 ml of sterilized DDW or DMSO suspension were mixed with/without 0.5 ml S9 mixture. Then, 2 ml of top agar containing histidine-biotin was mixed with minimal glucose agar and solidified. The plates were incubated at 37°C for 48 h, and the number of reverting colonies was counted. When the number of reverting colonies was more than double the number of the negative control and was dose dependent, the result was considered as positive.
Chromosome aberration assay.
Chinese hamster lung fibroblast cells (CHL) were plated in 60-mm dishes that contained 1.0 x 105 cells/plate for 3 days. After the 3-day incubation period, the cells were treated with saline (negative control), mitomycin C (positive control, 0.2 μg/ml), and various concentrations of MNPs (0.25, 0.5, and 1.0 mg/ml) for 6 h. After 2 h, the end of the treatment time, 0.2 μg/ml colcemid was added, and metaphase chromosomes were prepared as described previously (Tsutsui et al., 1984). For the determination of both chromosome aberrations, 500 metaphases per experimental group were scored. Structural chromosome aberrations observed in each experimental group were classified into six types: chromosome-type gap, chromosome-type break, chromosome-type exchange, chromatid-type gap, chromatid-type break, and chromatid-type exchange.
Statistical analysis.
Statistical significance of the differences in his+ reverting colonies among the various groups in the Ames assay was checked by the methods of Kim and Margolin (1999). Statistical analysis of all the remaining data was performed using Student's t-test (Graphpad Software, San Diego, CA). A value of p < 0.05 was considered significant and that of p < 0.01 was considered highly significant when compared to the corresponding control.
RESULTS
MNPs@SiO2(RITC) Did Not Induce Any Abnormal Clinical Signs in Laboratory Animals
During the study period, treatment with MNP@SiO2(RITC) for 4 weeks did not cause any adverse effects on growth because no statistically significant differences in the body weight gain were observed between the MNP-treated mice and control mice (Fig. 1). Further, no abnormal clinical signs and behaviors were detected in both the control and treated groups (data not shown). Considered together, MNP@SiO2(RITC) treatment did not induce any apparent toxicity in mice.
MNPs@SiO2(RITC) Were Distributed in Various Organs
MNPs were detected in diverse organs such as the brain, liver, lungs, kidneys, spleen, heart, testes, and uterus (Fig. 2). The particles were distributed in all organs, and the distribution pattern was time dependent. However, the distribution in the lungs was almost negligible. It is noteworthy that the distribution pattern in the female mice was almost identical to that in the male mice; therefore, the data have not been shown. Interestingly, MNPs@SiO2(RITC) could penetrate the BBB and gain access to the brain in a time-dependent manner, because the intensity of the fluorescence increased with time. To further confirm the uptake of MNPs@SiO2(RITC) into the brain, immunofluorescence study of the brain was performed with serial sections of the brain. This clearly demonstrated that MNPs@SiO2(RITC) could pass through the BBB and could be taken up by the neuron (Figs. 3A and 3B). To ensure whether the presence of MNPs represented true entrance into the brain cells, an additional immunofluorescence study was performed with anti-NeuN antibodies. As mentioned earlier, NeuN is an antibody that recognizes a neuron-specific antigen and is highly expressed in a brain cell; this was observed using selective staining of neuronal perikarya and nuclei. In Figure 3, FITC-stained NeuN images (left panels of Figs. 3C, 3D, 3E, and 3F) are clearly visible in the brain neuron. Merging the images of NeuN-FITC and MNP-RITC (right panels of Figs. 3C, 3D, 3E, and 3F) clearly demonstrated that the presence of the MNPs in the brain was due to true uptake of the MNPs by a brain neuron. Please note the MNP-RITC signals in the middle panels of Figures 3C, 3D, 3E, and 3F.
BBB Function Was Not Affected by MNPs@SiO2(RITC)
Penetration of the BBB by MNPs may give rise to concerns regarding the disruption of the BBB function. To evaluate the potential effects of MNPs@SiO2(RITC) on the BBB permeability, EB dye was injected intravenously as a BBB permeability tracer. After sacrificing the mice, the brain samples were sectioned, and the presence of the blue dye was examined. Our results strongly demonstrated that the EB dye was not present in the control (Fig. 4A) and MNPs@SiO2(RITC)-treated brains (Fig. 4B), since the fluorescence image was clear (Fig. 4D). Figure 4C shows the disruption of the BBB function due to administration of hyperosmolar mannitol. The spectrophotometrical analysis also demonstrated that the BBB permeability was intact (data not shown). Taken together, our results clearly demonstrated that MNPs@SiO2(RITC) could penetrate the BBB without affecting its function.
Treatment of MNPs@SiO2(RITC) Did Not Induce Any Apparent Toxicity
One of the main purposes of the current study was to evaluate the potential toxicity of MNPs@SiO2(RITC). In order to obtain detailed information of toxicity, we performed in vivo serum biochemical analysis, blood chemistry, histopathological examination, and in vitro mutagenecitiy assays. Every week, we performed hematological tests, clinical biochemistry tests, and histological analysis of various organs including the brain, liver, kidneys, heart, lungs, spleen, testes (in males), and uterus (in females) in all groups. In the hematological and clinical biochemistry tests, no significant changes were observed in the MNPs@SiO2(RITC)-treated groups (data not shown). Further, the treatment with MNPs did not induce any change in the relative and absolute organ weights (data not shown). The MNPs increased the number of revertants in the Salmonella mutation assay; however, the mutation pattern was neither reproducible nor concentration dependent (Table 1). In contrast, the MNPs did not induce any significant chromosome aberrations (Table 2). No abnormal histopathological gross lesions were observed in the treated groups (Fig. 5). The histopathological results shown in Figure 5 are of mice after 4 weeks of treatment. The remaining results of three different time points (1, 2, and 3 weeks) were identical; therefore, the data are not shown. Taken together, repeated administration of MNPs@SiO2(RITC) did not cause any apparent toxicity, and MNPs could penetrate the BBB without altering its function in our study.
DISCUSSION
The main reason for developing nanotechnology is to extend the limits of sustainable development at the nanoscale with less consumption of energy, water, and materials, and waste minimization. Nanotechnology will indeed develop in areas where potential advantages would exceed potential risks and where the potential risks are limited and can be addressed (Roco, 2003). Current approaches strongly suggest that consequences of nanotechnology are best addressed within the existing system applications such as biology, chemistry, or electronics. Nanoparticles can be produced from nearly any chemical. However, most nanoparticles that are currently being used are made from transition metals, silicon, carbon (single-walled carbon nanotubes), and metal oxides. Meanwhile, potential public and occupational exposures to manufactured nanoparticles should increase dramatically, because nanomaterials are supposed to improve the quality and performance of many consumer products as well as medical therapies. Therefore, it is time that information regarding the risk assessment of manufactured nanoparticles be collected. Critical questions regarding the potential human health and environmental impact of manufactured nanoparticles or nanomaterials have been raised only recently (Colvin, 2003).
Thus, the aim of current study is to determine the distribution pattern and potential toxicity of nanomaterials using MNPs@SiO2(RITC). As mentioned earlier, the MNPs will be applied to life science applications such as drug/gene delivery and bioimaging; therefore, MNPs have been used as a model nanomaterial in this study. Our results clearly indicate that MNPs are distributed in diverse organs. As shown in Figure 2, MNPs were found in almost all organs in a time-dependent manner. Interestingly, the distribution pattern of MNPs in the lungs is rather different. In our study, MNPs were administered to mice through the intraperitoneal route. Thus, the majority of the MNPs would be taken up by the liver via the first-pass effects and then redistributed from the liver to the other organs. Our results show that MNPs are rapidly and widely redistributed in the body except in the case of the lungs. We studied tissues that are enriched with the reticuloendothelial system (RES) such as the liver, spleen, and lungs and non-RES organs such as the heart and kidney. The highest concentrations of MNPs were observed in the liver and spleen. Among the RES organs, MNPs were distributed in small amounts only in the lungs, thereby suggesting that the localization of the MNPs in the liver, lungs, and spleen was not consistent with the RES system. Moreover, MNP concentration in several non-RES organs (i.e., testes, kidneys, and heart) was also high, suggesting that factors other than the RES system may also determine the tissue specificity of MNPs. More advanced and sophisticated kinetic models are required to take into account the differential tissue distribution of MNPs to delineate the detailed behavior of MNPs in different organs. Interestingly, the MNPs were found in the brain and testes, thereby indicating that they could penetrate the BBB as well as blood-testis barrier. These results suggest that MNPs used in this study have good biological characteristics to act as potential promising vectors for gene transfer and gene/drug delivery.
The principal basis of BBB is thought to be the specialized endothelial cells in the brain microvasculature, which are aided at least in part, by interactions with glia. Among the unique properties of these endothelial cells is the presence of tight junctions between cells, where the gaps between the junctions were approximately 4 nm (Kniesel and Wolburg, 2000). To gain entry to the brain, therefore, MNPs should probably pass through the cell membrane of the endothelial cells of the brain, rather than from between the endothelial cells. The penetration of molecules into the brain is largely related to their lipid solubility and to their ability to pass through the plasma membranes of the cells forming the barrier (Stewart, 2000). However, this is not the case for MNPs because they are highly water soluble. Moreover, the cells of the brain lack pinocytosis; therefore, MNPs should gain access to the brain by some other methods. In the mature central nervous system, the spinal and autonomic ganglia as well as a small number of other sites within the brain, called the circumventricular organs, are not protected by the BBB (Spencer, 2000). The discontinuity of the barrier allows entry of the anti-cancer drug doxorubicin into the brain. Probably, nanoparticles such as MNPs may adopt this method to pass the BBB without affecting its permeability. Such undisturbed BBB function by MNP treatment was further confirmed by the BBB function test using the EB dye (Fig. 4). To ensure that the penetration BBB by the MNPs represents true uptake by brain neurons, an additional immunofluorescence study has been performed using NeuN. DCN was selected as a representative of the whole brain. The DCN is an auditory nucleus that projects to the contralateral inferior colliculus and has a distinct laminar structure. The DCN and cerebellar cortex are developmentally derived from a common origin. In addition, the DCN has a cerebellar-like circuitry. The DCN is cytoarchitecturally and developmentally similar to the cerebellar cortex (Kaltenbach et al., 2004). Taken together, MNPs could penetrate the BBB without altering its function and were taken up by the brain cells.
Many in vitro tests have been developed to identify chemicals that can damage cellular DNA or cause mutations and, secondary to this, to identify potential carcinogens. By far, the test receiving maximum use and attention has been the Salmonella mutagenesis test because of its initial promise of high qualitative predictivity for cancer in rodents and, by extension, in humans. Therefore, in this study, Salmonella assay was adopted to predict the potential toxicity of MNPs. There were some significant changes in the revertants in the Salmonella assay (Table 1). However, such increased number of revertants may not reflex the toxicity of MNPs, because the increased pattern was neither concentration dependent nor reproducible. Our findings suggest that Salmonella assay data may not be relevant to predict the toxicity of MNPs. Our results can be further supported by the finding that the predictive relationship between the mutagenic potential determined by the Salmonella assay and genotoxicity was, at best, weak (Fetterman et al., 1997). In fact, the chromosome aberration assay also revealed that MNP had no mutagenic potential (Table 2). Taken together, our results clearly demonstrated that MNPs measuring approximately 50 nm did not cause clear toxicity under the current conditions.
In conclusion, we report here that MNPs@SiO2(RITC) can penetrate the BBB and persist in the body for a long time without causing toxicity. Thus far, the main activities at the interface between material and life sciences include functionalization and characterization of nanomaterials for their application to biosystems. Therefore, extensive studies are required in the future to provide the basis for a new class of nanomaterials for drugs, proteins, and gene delivery applications.
NOTES
1 Equal contributions to this work.
ACKNOWLEDGMENTS
This work was partly supported by the Nano Systems Institute-National Core Research Center (NSI-NCRC), Korea Science and Engineering Foundation (KOSEF). The author acknowledges that he/she has a research grant to do research in this area; the funding organization does not have control over the resulting publication.
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Laboratory of Molecular Oncology, Korea Institute of Radiological & Medical Sciences, Seoul 139–240, Korea
ABSTRACT
The development of technology enables the reduction of material size in science. The use of particle reduction in size from micro to nanoscale not only provides benefits to diverse scientific fields but also poses potential risks to humans and the environment. For the successful application of nanomaterials in bioscience, it is essential to understand the biological fate and potential toxicity of nanoparticles. The aim of this study was to evaluate the biological distribution as well as the potential toxicity of magnetic nanoparticles to enable their diverse applications in life science, such as drug development, protein detection, and gene delivery. We recently synthesized biocompatible silica-overcoated magnetic nanoparticles containing rhodamine B isothiocyanate (RITC) within a silica shell of controllable thickness [MNPs@SiO2(RITC)]. In this study, the MNPs@SiO2(RITC) with 50-nm thickness were used as a model nanomaterial. After intraperitoneal administration of MNPs@SiO2(RITC) for 4 weeks into mice, the nanoparticles were detected in the brain, indicating that such nanosized materials can penetrate blood–brain barrier (BBB) without disturbing its function or producing apparent toxicity. After a 4-week observation, MNPs@SiO2(RITC) was still present in various organs without causing apparent toxicity. Taken together, our results demonstrated that magnetic nanoparticles of 50-nm size did not cause apparent toxicity under the experimental conditions of this study.
Key Words: magnetic nanoparticles; blood-brain barrier.
INTRODUCTION
The recent shift in the focus of developing nanoparticles from microscale to nanoscale is essential for future advances in both the digital revolution and modern biology and may change the very foundations of education, medicine, and industrial manufacture and have a potential harmful effect on the environment. Such a rapid development in nanotechnology will result in several changes in areas such as nanoscale visualization, insight into living systems, revolutionary biotechnology, synthesis of new drugs as their targeted delivery, and regenerative medicine and offer many other benefits (Roco, 2003).
Reducing the particle size of materials is an efficient and reliable tool for improving the bioavailability of a gene or drug delivery system. In fact, nanotechnology helps in overcoming the limitations of size and can change the outlook of the world regarding science (Chad and Andrew, 2000). Further, nanomaterials can be modified for better efficiency to facilitate their applications in different fields such as bioscience and medicine. For example, magnetic nanoparticles (MNPs) have been studied with an intent to apply them in bioscience, because they offer benefits such as separation and gathering of materials of interest by using a magnetic force (Hongwei et al., 2003). However, the lack of information regarding the toxicity of manufactured nanoparticles poses serious problems. Therefore, it is necessary that specialists and researchers in toxicology, chemistry, and other fields are aware of the importance of analyzing the positive aspects of nanomaterials while avoiding their potential toxic effects (Oberdorster et al., 2005).
Our team, consisting of experts in multidisciplinary areas, recently synthesized silica-overcoated MNPs containing rhodamine B isothiocyanate (RITC) within a silica shell of controllable thickness, MNPs@SiO2(RITC), for future applications such as drug or gene delivery (Yoon et al., 2005). However, for the application of MNPs for targeted delivery, sufficient data regarding the toxicity and biological fate of the MNPs should be accumulated. Both the toxicity and tissue distribution of MNPs@SiO2(RITC) should be evaluated because nanoparticles are expected to be applicable to biomedicine. Therefore, in this study, MNPs@SiO2(RITC) with a 50-nm thickness were used as a model nanomaterial for the evaluation of in vivo biological distribution and potential toxicity. Here, we report that nanosized MNPs@SiO2(RITC) can penetrate the blood–brain barrier without any apparent toxicity. These results support the hypothesis that the MNPs can be applied to the field of biomedicine and further suggest that extensive studies are required to clarify the potential chronic toxicity of MNPs.
MATERIALS AND METHODS
Preparation of magnetic nanoparticles.
Fifty-nm size MNPs labeled with RITC (Sigma-Aldrich, St. Louis, MO) were synthesized according to a previously described method (Yoon et al., 2005). In brief, cobalt ferrite (Sigma-Aldrich) was added to polyvinylpyrolidone (PVP, Sigma-Aldrich) solution. The PVP-stabilized cobalt ferrite nanoparticles were separated by the addition of acetone and centrifugation. The precipitated particles were redispersed in ethanol. Trimethoxysilane (Gelest, Morrisville, PA) modified by RITC was prepared from 3-aminopropyltriethoxysilane (APS; Gelest) and RITC under nitrogen. Such synthesized dye solution was mixed with tetraethoxysilane (TEOS, Gelest) and injected into the PVP-stabilized cobalt ferrite ethanol solution and, subsequently, polymerized on the surface of the PVP-stabilized cobalt ferrites by adding ammonia as a catalyst to form RITC-incorporated MNPs [MNPs@SiO2(RITC)]. The silica shell thickness was successfully adjusted such that it can be reproduced by controlling the concentration of TEOS. All MNPs@SiO2(RITC) were confirmed by a transmission electron microscope (TEM).
Animals and MNPs@SiO2(RITC) treatment.
Male ICR mice (6 weeks old) were purchased from Joongang Laboratory Animal Inc. (Seoul, Korea). Our laboratory animal facility is maintained under a 12-h light/dark cycle at a temperature of 20–22°C, and a relative humidity of 20–50%. All methods used in this study were approved by the Animal Care and Use Committee at Seoul National University (SNU) and conformed to the NIH guidelines (NIH publication No. 86–23, revised 1985). Based on systematic studies for identification of dose-range as well as practical consideration of the synthetic capacity of MNPs@SiO2(RITC), three different concentrations of MNPs@SiO2(RITC), i.e., 100, 50, and 25 mg/kg, were administered to the mice intraperitoneally for 4 weeks. For the blood–brain barrier (BBB) study, a concentration of 10 mg/kg was selected because this concentration was sufficient to test the BBB function. During the study period, any clinical signs were carefully observed. The mice were sacrificed after designated time periods (every week: thus, at four time points during the study) to analyze the tissue distribution and perform toxicity studies.
Serum biochemical analysis.
Blood samples were collected every week after MNP treatment (four time points of analysis in total). Using a biochemical autoanalyzer (VITALAB, Merck, The Netherlands), serum biochemical analysis was carried out to determine the serum level of total protein, albumin, total bilirubin, aspartate transaminase (AST), alanine transaminase (ALT), glucose, cholesterol, triglyceride, blood urea nitrogen, creatinine, calcium, phosphorus, and lactic acid dehydrogenase.
Hematological analysis.
As mentioned previously, blood samples were collected after MNP treatment at four points of time in total. Hematological parameters consisting of erythrocytes, leukocytes, neutrophils, lymphocytes, monocytes, eosinophils, basophils, platelets, hemoglobin, and hematocrit were determined using a hematological autoanalyzer (Coulter T540 hematology system; Fullerton, CA).
Histopathological analysis.
Mice were perfused with buffered (0.4 M phosphate buffer, pH 7.6) 4% paraformaldehyde. The brain, liver, lungs, kidneys, heart, spleen, testes (in males), and uterus (in females) were removed from each animal and immersion-fixed in the same fixative for 24 h at room temperature. Tissue sections (4 μm) were prepared after dehydration and were embedded in paraffin. The sections were stained with hematoxilin and eosin (H&E) and subsequently processed for histopathological examination under light microscope.
Fluorescent image analysis of MNP biodistribution.
Several representative organs such as the brain, liver, kidneys, lungs, and testes were removed and fixed in 4% paraformaldehyde solution. After fixation, the tissue samples were dehydrated with 30% sucrose for 24 h and then cryosectioned at 10-μm thickness (Mullen et al., 1992). Then, the slides were mounted in ultramount media (Dako, Carpinteria, CA) and incubated for 3 h at room temperature. After incubation, the slides were observed under a confocal laser scanning microscope (CLSM; Zeiss 510; Atlanta, GA). Serial cryosectioned slides of the brain were observed under CLSM at high power view (x1200; the section interval thickness was 10 μm). For image analysis, RITC excitation was performed using an argon laser at 543 nm. To confirm whether the presence of MNPs in the brain was a true indicator of their uptake by the neurons, an immunofluorescence study was performed with neuronal nuclei (NeuN), because this protein is highly expressed in the brain (Mullen et al., 1992). In brief, brain tissue sections were sequentially incubated with 10% normal goat serum in 0.1 M PBS for 30 min and with anti-NeuN antibody (1:20; Chemicon, Temecula, CA) overnight. After washing with phosphate-buffered saline (PBS), the sections were incubated with FITC-conjugated goat anti-mouse IgG (1:200; Serotec, Raleigh, NC). After a second washing with PBS, the sections were mounted with glycerol and observed under the CLSM. The dorsal cochlear nucleus (DCN) was observed as a representative of the whole brain.
Evaluation of blood–brain barrier (BBB) permeability.
To evaluate the potential effects of MNPs@SiO2(RITC) on BBB permeability, Evans blue (EB) dye was used as a marker of albumin extravasations because EB dye is known to bind to serum albumin. Mannitol was used as a positive control because hyperosmolar mannitol is known to disrupt the BBB permeability (Kaya et al., 2004). In brief, the EB dye (2% in saline, 4 ml/kg) was injected via the tail vein at 5 min prior to MNPs@SiO2(RITC) administration and was allowed to remain in circulation for 30 min. A solution of 25% mannitol (Sigma-Aldrich) dissolved in 0.9% saline was injected into the mice at 10 min after the intravenous injection of the EB dye. After 30 min, the thorax was opened under anesthesia. The mice were perfused transcardially with saline for approximately 10 min until the fluid from the right atrium became colorless. After decapitation, the brain was removed and dissected. After a careful examination for the presence of the EB dye, the dissected brain was homogenized in 2.5 ml PBS and mixed by vortexing for 2 min after the addition of 2.5 ml 60% trichloroacetic acid to precipitate the protein. The samples were then centrifuged for 30 min at 1000 x g. The absorbance of EB in the supernatants was measured at 610 nm for absorbance of EB using a spectrophotometer (Kaya et al., 2004).
Evaluation of genotoxicity.
Ames Assay Salmonella mutagenicity assay was performed according to the method of Maron and Ames (1983). Salmonella typhimurium strains TA97, TA98, TA100, and TA102 were purchased from Molecular Toxicology Inc. (Boone, NC). Each strain was tested for its genetic traits such as histidine requirement, deep rough (rfa) characteristic, UV sensitivity (uvrB mutation), and ampicillin- or tetracycline-resistance by R-factor prior to use. The strains were inoculated on oxoid nutrient broth and cultured for 15 h at 37°C with a continuous agitation at 150 rpm. The cell density was 1–2 x 105/ml. The concentrations of MNP@SiO2(RITC) tested were 0.25, 0.5, and 1.0 mg sample/plate. The S9 mixture and cofactor (Molecular Toxicology Inc.) were prepared, and the activity of the S9 mixture was confirmed by inducing mutagenesis using 2-aminoanthracene (2-AA). The positive controls were sodium azide (Sigma-Aldrich), ICR 191 acridine (Sigma-Aldrich), mitomycin C (Sigma-Aldrich), daunomycin (Perkin Elmer, Wellesley, MA), and 2-aminoanthracene (Sigma-Aldrich). They were dissolved in deionized distilled water (DDW) or dimethylsulfoxide (DMSO, Sigma-Aldrich). The direct plate incorporation method was used with three plates per concentration to be tested. Both 0.1 ml of the cell culture (1–2 x 105/ml) and 0.1 ml of sterilized DDW or DMSO suspension were mixed with/without 0.5 ml S9 mixture. Then, 2 ml of top agar containing histidine-biotin was mixed with minimal glucose agar and solidified. The plates were incubated at 37°C for 48 h, and the number of reverting colonies was counted. When the number of reverting colonies was more than double the number of the negative control and was dose dependent, the result was considered as positive.
Chromosome aberration assay.
Chinese hamster lung fibroblast cells (CHL) were plated in 60-mm dishes that contained 1.0 x 105 cells/plate for 3 days. After the 3-day incubation period, the cells were treated with saline (negative control), mitomycin C (positive control, 0.2 μg/ml), and various concentrations of MNPs (0.25, 0.5, and 1.0 mg/ml) for 6 h. After 2 h, the end of the treatment time, 0.2 μg/ml colcemid was added, and metaphase chromosomes were prepared as described previously (Tsutsui et al., 1984). For the determination of both chromosome aberrations, 500 metaphases per experimental group were scored. Structural chromosome aberrations observed in each experimental group were classified into six types: chromosome-type gap, chromosome-type break, chromosome-type exchange, chromatid-type gap, chromatid-type break, and chromatid-type exchange.
Statistical analysis.
Statistical significance of the differences in his+ reverting colonies among the various groups in the Ames assay was checked by the methods of Kim and Margolin (1999). Statistical analysis of all the remaining data was performed using Student's t-test (Graphpad Software, San Diego, CA). A value of p < 0.05 was considered significant and that of p < 0.01 was considered highly significant when compared to the corresponding control.
RESULTS
MNPs@SiO2(RITC) Did Not Induce Any Abnormal Clinical Signs in Laboratory Animals
During the study period, treatment with MNP@SiO2(RITC) for 4 weeks did not cause any adverse effects on growth because no statistically significant differences in the body weight gain were observed between the MNP-treated mice and control mice (Fig. 1). Further, no abnormal clinical signs and behaviors were detected in both the control and treated groups (data not shown). Considered together, MNP@SiO2(RITC) treatment did not induce any apparent toxicity in mice.
MNPs@SiO2(RITC) Were Distributed in Various Organs
MNPs were detected in diverse organs such as the brain, liver, lungs, kidneys, spleen, heart, testes, and uterus (Fig. 2). The particles were distributed in all organs, and the distribution pattern was time dependent. However, the distribution in the lungs was almost negligible. It is noteworthy that the distribution pattern in the female mice was almost identical to that in the male mice; therefore, the data have not been shown. Interestingly, MNPs@SiO2(RITC) could penetrate the BBB and gain access to the brain in a time-dependent manner, because the intensity of the fluorescence increased with time. To further confirm the uptake of MNPs@SiO2(RITC) into the brain, immunofluorescence study of the brain was performed with serial sections of the brain. This clearly demonstrated that MNPs@SiO2(RITC) could pass through the BBB and could be taken up by the neuron (Figs. 3A and 3B). To ensure whether the presence of MNPs represented true entrance into the brain cells, an additional immunofluorescence study was performed with anti-NeuN antibodies. As mentioned earlier, NeuN is an antibody that recognizes a neuron-specific antigen and is highly expressed in a brain cell; this was observed using selective staining of neuronal perikarya and nuclei. In Figure 3, FITC-stained NeuN images (left panels of Figs. 3C, 3D, 3E, and 3F) are clearly visible in the brain neuron. Merging the images of NeuN-FITC and MNP-RITC (right panels of Figs. 3C, 3D, 3E, and 3F) clearly demonstrated that the presence of the MNPs in the brain was due to true uptake of the MNPs by a brain neuron. Please note the MNP-RITC signals in the middle panels of Figures 3C, 3D, 3E, and 3F.
BBB Function Was Not Affected by MNPs@SiO2(RITC)
Penetration of the BBB by MNPs may give rise to concerns regarding the disruption of the BBB function. To evaluate the potential effects of MNPs@SiO2(RITC) on the BBB permeability, EB dye was injected intravenously as a BBB permeability tracer. After sacrificing the mice, the brain samples were sectioned, and the presence of the blue dye was examined. Our results strongly demonstrated that the EB dye was not present in the control (Fig. 4A) and MNPs@SiO2(RITC)-treated brains (Fig. 4B), since the fluorescence image was clear (Fig. 4D). Figure 4C shows the disruption of the BBB function due to administration of hyperosmolar mannitol. The spectrophotometrical analysis also demonstrated that the BBB permeability was intact (data not shown). Taken together, our results clearly demonstrated that MNPs@SiO2(RITC) could penetrate the BBB without affecting its function.
Treatment of MNPs@SiO2(RITC) Did Not Induce Any Apparent Toxicity
One of the main purposes of the current study was to evaluate the potential toxicity of MNPs@SiO2(RITC). In order to obtain detailed information of toxicity, we performed in vivo serum biochemical analysis, blood chemistry, histopathological examination, and in vitro mutagenecitiy assays. Every week, we performed hematological tests, clinical biochemistry tests, and histological analysis of various organs including the brain, liver, kidneys, heart, lungs, spleen, testes (in males), and uterus (in females) in all groups. In the hematological and clinical biochemistry tests, no significant changes were observed in the MNPs@SiO2(RITC)-treated groups (data not shown). Further, the treatment with MNPs did not induce any change in the relative and absolute organ weights (data not shown). The MNPs increased the number of revertants in the Salmonella mutation assay; however, the mutation pattern was neither reproducible nor concentration dependent (Table 1). In contrast, the MNPs did not induce any significant chromosome aberrations (Table 2). No abnormal histopathological gross lesions were observed in the treated groups (Fig. 5). The histopathological results shown in Figure 5 are of mice after 4 weeks of treatment. The remaining results of three different time points (1, 2, and 3 weeks) were identical; therefore, the data are not shown. Taken together, repeated administration of MNPs@SiO2(RITC) did not cause any apparent toxicity, and MNPs could penetrate the BBB without altering its function in our study.
DISCUSSION
The main reason for developing nanotechnology is to extend the limits of sustainable development at the nanoscale with less consumption of energy, water, and materials, and waste minimization. Nanotechnology will indeed develop in areas where potential advantages would exceed potential risks and where the potential risks are limited and can be addressed (Roco, 2003). Current approaches strongly suggest that consequences of nanotechnology are best addressed within the existing system applications such as biology, chemistry, or electronics. Nanoparticles can be produced from nearly any chemical. However, most nanoparticles that are currently being used are made from transition metals, silicon, carbon (single-walled carbon nanotubes), and metal oxides. Meanwhile, potential public and occupational exposures to manufactured nanoparticles should increase dramatically, because nanomaterials are supposed to improve the quality and performance of many consumer products as well as medical therapies. Therefore, it is time that information regarding the risk assessment of manufactured nanoparticles be collected. Critical questions regarding the potential human health and environmental impact of manufactured nanoparticles or nanomaterials have been raised only recently (Colvin, 2003).
Thus, the aim of current study is to determine the distribution pattern and potential toxicity of nanomaterials using MNPs@SiO2(RITC). As mentioned earlier, the MNPs will be applied to life science applications such as drug/gene delivery and bioimaging; therefore, MNPs have been used as a model nanomaterial in this study. Our results clearly indicate that MNPs are distributed in diverse organs. As shown in Figure 2, MNPs were found in almost all organs in a time-dependent manner. Interestingly, the distribution pattern of MNPs in the lungs is rather different. In our study, MNPs were administered to mice through the intraperitoneal route. Thus, the majority of the MNPs would be taken up by the liver via the first-pass effects and then redistributed from the liver to the other organs. Our results show that MNPs are rapidly and widely redistributed in the body except in the case of the lungs. We studied tissues that are enriched with the reticuloendothelial system (RES) such as the liver, spleen, and lungs and non-RES organs such as the heart and kidney. The highest concentrations of MNPs were observed in the liver and spleen. Among the RES organs, MNPs were distributed in small amounts only in the lungs, thereby suggesting that the localization of the MNPs in the liver, lungs, and spleen was not consistent with the RES system. Moreover, MNP concentration in several non-RES organs (i.e., testes, kidneys, and heart) was also high, suggesting that factors other than the RES system may also determine the tissue specificity of MNPs. More advanced and sophisticated kinetic models are required to take into account the differential tissue distribution of MNPs to delineate the detailed behavior of MNPs in different organs. Interestingly, the MNPs were found in the brain and testes, thereby indicating that they could penetrate the BBB as well as blood-testis barrier. These results suggest that MNPs used in this study have good biological characteristics to act as potential promising vectors for gene transfer and gene/drug delivery.
The principal basis of BBB is thought to be the specialized endothelial cells in the brain microvasculature, which are aided at least in part, by interactions with glia. Among the unique properties of these endothelial cells is the presence of tight junctions between cells, where the gaps between the junctions were approximately 4 nm (Kniesel and Wolburg, 2000). To gain entry to the brain, therefore, MNPs should probably pass through the cell membrane of the endothelial cells of the brain, rather than from between the endothelial cells. The penetration of molecules into the brain is largely related to their lipid solubility and to their ability to pass through the plasma membranes of the cells forming the barrier (Stewart, 2000). However, this is not the case for MNPs because they are highly water soluble. Moreover, the cells of the brain lack pinocytosis; therefore, MNPs should gain access to the brain by some other methods. In the mature central nervous system, the spinal and autonomic ganglia as well as a small number of other sites within the brain, called the circumventricular organs, are not protected by the BBB (Spencer, 2000). The discontinuity of the barrier allows entry of the anti-cancer drug doxorubicin into the brain. Probably, nanoparticles such as MNPs may adopt this method to pass the BBB without affecting its permeability. Such undisturbed BBB function by MNP treatment was further confirmed by the BBB function test using the EB dye (Fig. 4). To ensure that the penetration BBB by the MNPs represents true uptake by brain neurons, an additional immunofluorescence study has been performed using NeuN. DCN was selected as a representative of the whole brain. The DCN is an auditory nucleus that projects to the contralateral inferior colliculus and has a distinct laminar structure. The DCN and cerebellar cortex are developmentally derived from a common origin. In addition, the DCN has a cerebellar-like circuitry. The DCN is cytoarchitecturally and developmentally similar to the cerebellar cortex (Kaltenbach et al., 2004). Taken together, MNPs could penetrate the BBB without altering its function and were taken up by the brain cells.
Many in vitro tests have been developed to identify chemicals that can damage cellular DNA or cause mutations and, secondary to this, to identify potential carcinogens. By far, the test receiving maximum use and attention has been the Salmonella mutagenesis test because of its initial promise of high qualitative predictivity for cancer in rodents and, by extension, in humans. Therefore, in this study, Salmonella assay was adopted to predict the potential toxicity of MNPs. There were some significant changes in the revertants in the Salmonella assay (Table 1). However, such increased number of revertants may not reflex the toxicity of MNPs, because the increased pattern was neither concentration dependent nor reproducible. Our findings suggest that Salmonella assay data may not be relevant to predict the toxicity of MNPs. Our results can be further supported by the finding that the predictive relationship between the mutagenic potential determined by the Salmonella assay and genotoxicity was, at best, weak (Fetterman et al., 1997). In fact, the chromosome aberration assay also revealed that MNP had no mutagenic potential (Table 2). Taken together, our results clearly demonstrated that MNPs measuring approximately 50 nm did not cause clear toxicity under the current conditions.
In conclusion, we report here that MNPs@SiO2(RITC) can penetrate the BBB and persist in the body for a long time without causing toxicity. Thus far, the main activities at the interface between material and life sciences include functionalization and characterization of nanomaterials for their application to biosystems. Therefore, extensive studies are required in the future to provide the basis for a new class of nanomaterials for drugs, proteins, and gene delivery applications.
NOTES
1 Equal contributions to this work.
ACKNOWLEDGMENTS
This work was partly supported by the Nano Systems Institute-National Core Research Center (NSI-NCRC), Korea Science and Engineering Foundation (KOSEF). The author acknowledges that he/she has a research grant to do research in this area; the funding organization does not have control over the resulting publication.
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