1.1 Background of nanoparticles and iron oxide nanoparticles
Nanoparticles can be engineered or found naturally in the environment. In recent years, the rapid development of nanotechnology has attracted global attention. The structure of nanoparticles is on the nanometer scale, with size of 1-100 nm. Due to the nanoscale dimensions, nanoparticles possess extremely high surface to volume ratio compared with bulky forms of the same materials. Therefore, nanoparticles exhibit unique properties, such as greater strength, stability and biological activity (Boverhof et al., 2010). Nanotechnology has been widely used in biological, environmental, material and medical fields.Engineered nanoparticles, due to their specific physicochemical properties, are increasingly used for biological and medical purpose, including diagnostic and therapeutic applications (Liu et al., 2012; Schladt et al., 2011). In particular, iron oxide nanoparticles has been used as contrast agents for magnetic resonance imaging (MRI), intracellular labeling, drug delivery and cancer therapy (Chouly et al., 1996; Liong et al., 2008; Xie et al., 2009; Yu et al., 2008). The commercial iron oxide nanoparticles, Resovist® contains a crystalline core composed of Fe3O4 (magnetite) and γ-Fe2O3 (maghemite) coated with carboxydextran. The superparamagnetic property of iron oxide nanoparticles can generate heat in an alternating magnetic field and guide specific targeting by an external magnetic field.
The property is very crucial for the development of contrast enhancement, targeted delivery of drugs or genes, tissue engineering, cancer thermal therapy, magnetic transfections, iron detection, chelation therapy and tissue engineering (Bulte et al., 2001;
Gupta et al., 2005; Hamm et al., 1994; Hautot et al., 2007; Huber, 2005; Ito et al., 2005;
Liu et al., 2006). Iron oxide nanoparticles were rapidly taken up by phagocytes, and
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mainly accumulated in the liver with approximately 80% of the injected dose, the spleen with 8-10%, bone marrow, and lymph nodes with 10 % within few minutes post systemic administration (Grazioli et al., 2009; Hamm et al., 1994). Hence, iron oxide nanoparticles are an organ-specific MRI contrast agent, using for the diagnosis of organs of reticuloendothelial system associated organs. Malignant tumors typically lack of a substantial number of phagocytic cells, they appear hyperintense or bright images compared with the hypointense or black images of normal tissues. Because of this property, iron oxide nanoparticles are employed as a diagnostic tool for distinguishing between tumor and normal tissues.
1.2 Toxicity of nanoparticles
Due to several characteristics of nanoparticles such as extremely small size and high surface to volume ratio, nanomaterials can enter the human body easily and have the potential to alter cellular responses. The toxicity of nanoparticles is dependent on many factors, including the bulk forms, shape, surface structure, surface charge, size, chemical composition, aggregation and solubility (Oberdörster et al., 2007). Previous studies have shown that engineered carbon nanoparticles can activate platelets and cause vascular thrombosis (Radomski et al., 2005). Respiratory exposure of titanium dioxide (TiO2), carbon, diesel exhaust particles and iron oxide nanoparticles causes lung inflammation, including the stimulation of proinflammatory cytokines production, cellular proliferation, fibro-proliferative effects and development of lung tumors (Cho et al., 2009; Gustafsson et al., 2011; Li et al., 2010; Warheit et al., 2006). Mechanistic studies reported that nanoparticles impaired mitochondrial functions, and induced formation of apoptosis bodies, leakage of lactate dehydrogenase, chromosome condensation, generation of reactive oxygen species (ROS) and DNA damage (Singh et al., 2010).
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1.3 Effects of nanoparticles on immune cells
Because nanoparticles are rapidly taken up by phagocytes of the immune system, understanding the interaction between nanoparticles and the immune system is important. Accumulating evidence suggests that nanoparticles possess a wide spectrum of immunomodulatory effects. For example, poly-hydroxylated metallofullerenol ([Gd@C82(OH)22]n) nanoparticles induced the maturation and affected the phenotype of dendritic cells (DC) to activate T help 1 (Th 1) immune responses (Yang et al., 2010a).
As to T cell-immunity, [Gd@C82(OH)22]n nanoparticlespossess anti-tumor ability by promoting T cells to differentiate into Th1 cells, shifting the Th1/Th2 balance to the Th1 direction and activating tumor necrosis factor α (TNF-α) mediated cellular immunity (Liu et al., 2009). Gold nanoparticles have been reported accumulated in DC and inhibited the production of TNF-α after lipopolysaccharide (LPS) stimulation, but did not alter the cell phenotype (Villiers et al., 2010). Gold nanoparticles induced proinflammatory responses and oxidative stress, and inhibited toll-like receptor 9 (TLR-9) signaling in macrophages (Abdelhalim et al., 2011; Ma et al., 2010; Nishanth et al., 2011; Tsai et al., 2012; Yen et al., 2009). Notably, iron oxide nanoparticles have been shown to modulate the functionality of various immune cells, including macrophages, DC and T lymphocytes. Exposure to iron oxide nanoparticles inhibited the phagocytic activity of Raw 264.7 cells, a murine macrophage line and increased the production of TNF-α and nitric oxide (NO) (Hsiao et al., 2008). Iron oxide nanoparticles induced apoptosis in primary macrophages and murine macrophage cell line J774 via oxidative stress (Lunov et al., 2010a; Naqvi et al., 2010). A single intratracheal exposure to iron oxide nanoparticles elicited inflammatory and pro-oxidative responses in mice (Park et al., 2010). In addition, iron oxide nanoparticles suppressed the functions of DC, such as antigen presentation and stimulation of cluster
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of differentiation 4+ (CD 4+) T cells (Blank et al., 2011). As to the effect on T cells, mice intravenously administered with iron oxide nanoparticles showed an increased cellularity of CD4+ and CD8+ T cells, and an enhanced production of interleukin 2 (IL-2) and interferon γ (IFN-γ) by T cells (Blank et al., 2011). Recently, our laboratory reported that iron oxide nanoparticles compromised antigen-specific immune responses in ovalbumin (OVA)-sensitized mice. A single intravenous administration of iron oxide nanoparticles markedly inhibited antigen-specific antibodies, including OVA-specific IgG1 and IgG2a. The cell viability and IFN-γ production by splenocytes re-stimulated with the sensitized antigen OVA were attenuated by iron oxide nanoparticles (Shen et al., 2011b). In contrast, IL-4 was unaffected, indicating that iron oxide nanoparticles switched the Th1/Th2 balance toward Th2-dominant immune responses. In addition, iron oxide nanoparticle-mediated inhibition of IFN-γ was associated with diminished intracellular levels of glutathione (GSH) (Shen et al., 2011a). Collectively, these results clearly demonstrated that iron oxide nanoparticles exhibited a broad spectrum of immunomodulatory effects.
1.4 The potential toxicity of nanoparticles to the central nervous system (CNS) Nanoparticles may enter the CNS via several pathways, one of which is the nose-to-brain transport through olfactory epithelium, trigeminal nerve (Mistry et al., 2009; Oberdorster et al., 2004). Previous studies have shown that nanoparticles can transfer from olfactory epithelium and trigeminal nerve into CNS, including manganese oxide, gold, carbon, iron oxide nanoparticle and TiO2. Hence, intranasal exposure is a potential health concern. The other concern on the nose-to-brain transport is due to the potential of nanoparticles as delivering vehicles for mucosal immunization via the intranasal route (Hutter et al., 2010; Mistry et al., 2009; Sayin et al., 2009). The other pathway is penetrating the blood-brain barrier (BBB). The capability of nanoparticles
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across BBB dependent on its physicochemical properties. For example, the lipophilic nanoparticles can pass directly from blood capillaries into BBB and charge-bearing and hydrophilic nanoparticles require gated channels and receptors to across BBB. The application of nanoparticles in CNS, such as imaging, or therapeutics in nano-oncology and neurodegenerative diseases, is achieved via systemic administration, and implantation of nano-enabled drug delivery system (Nunes et al., 2012; Yang et al., 2010d). Several lines of evidence has indicated that exposure to nanoparticles caused neurotoxicity in the CNS (Hu et al., 2010; Nunes et al., 2012; Yang et al., 2010d). For example, quantum dots were internalized by macrophages and microglia within glioma cells and altered in the responsiveness of glia and neurons after intravenous administration (Jackson et al., 2007). Quantum dots induced apoptosis in neuroblastoma cell lines, which was mediated by mitochondrial-dependent pathways, oxidative stress and inhibited survival signals, such as Ras, Raf-1 and ERK (Chan et al., 2006; Jan et al., 2008). The intracerebral injection of quantum dots caused the activation of astrocytes (Maysinger et al., 2007). Collectively, these studies clearly demonstrated that quantum dot nanoparticles affected the CNS.
Iron oxide nanoparticles have various diagnostic and potential therapeutic applications in the CNS, including tumor imaging and molecular imaging to evaluate the efficacy of therapy and hyperthermal therapy in glioma treatment (Engberink et al., 2010; Weinstein et al., 2010). Several lines of evidence has indicated that exposure to iron oxide nanoparticles may induce neurotoxicity. For example, intranasal administration of iron oxide nanoparticles accumulated in the olfactory bulb and hippocampus, and induced cell morphological changes, including endoplasmic reticulum (ER) dilation and an increased amount of lysosomes (Wang et al., 2011c).
Furthermore, iron oxide nanoparticles induced neuron damage via oxidative stress, as evidenced by an increased activity of antioxidants and a decreased level of the total
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GSH (Wang et al., 2007; Wang et al., 2009). As to in vitro studies, iron oxide nanoparticles inhibited cell viability and the neurite generation induced by nerve growth factor stimulation in rat pheochromocytoma cell line, PC12 cells (Pisanic et al., 2007).
In addition to neurons, iron oxide nanoparticles caused microglial proliferation, activation and recruitment in the olfactory bulb after intranasal instillation. Iron oxide nanoparticles induce cell proliferation, phagocytosis and oxidative stress in BV-2 cells, a murine microglial cell line (Wang et al., 2011c). However, the study pertaining to the effect of iron oxide nanoparticles on the immune in the CNS is limited.
1.5 Microglia
Microglia are the resident immune cells in the CNS and considered to serve as a guardian of the brain. In the adult brain and under physiological conditions, resting microglia have low expression of surface antigens and ramified morphology, including a small cellular body and ramified processes. Resting microglia possess a variety of ability to support neurogenesis, including the secretion of neurotrophic factor and phagocytosis (Saijo et al., 2011). The morphological changes from resting ramified form to activated amoeboid form, including rod-cell shaped and hypertrophy cellular body. The earliest responses of activated microglia are proliferation, recruitment peripheral macrophages and migration to the injury and inflammatory sites. Microglia are also the predominant phagocyte in the CNS. They engulfed pathogens and removed the apoptotic cells and cellular debris to maintain homeostasis in the CNS. In response to pathogen infection, injury and foreign particle invasion, microglia are activated and enhance the expression of surface receptors, including major histocompatibility complex (MHC) and complement receptors. Activated microglia are capable of generating several cytotoxic factors, such as NO, ROS, proteases, and other inflammatory mediators. The cytokines produced by activated microglia play an
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essential role in regulating the inflammatory responses in the CNS. The proinflammatory cytokines, such as IL-1β, IL-6, IL-8 and TNF-α, are produced by activated microglia to promote inflammatory responses (Kim et al., 2005). The antigen presentation of microglia is to serve as a link between the innate and adaptive immune systems. After engulfing antigen-containing pathogens and infected cells, microglia have abilities to process antigen and present peptides on the cell surface to stimulate T cells. MHC class-II and co-stimulatory molecules such as B7 are expressed together with the processed antigens (Garden et al., 2006). Though microglia serve as the main immune effector cells in the CNS, over-activation of microglia is participated in a variety of neurologic diseases including multiple sclerosis, Parkinson's disease, Alzheimer's disease, human immunodeficiency virus dementia and stroke. For example, the amyloid-β protein that is considered as a possible etiologic factor in Alzheimer's disease recruits and activates microglia. The activation of microglia plays a role in the initiation stage of disease progression and neuronal death (Garden et al., 2006; Kim et al., 2005; Minagar et al., 2002).
1.6 Immunological impacts of nanoparticles on microglia
Nanoparticles can enter into the CNS and alter the function of microglia, including gold, silica, TiO2 and iron oxide nanoparticles. For example, gold nanoparticles were internalized into the lysosomes of murine microglial N9 cells and enhanced IL-1α and granulocyte/macrophage colony stimulating factor (GM-CSF) secretion. In addition, intranasal administration of gold nanoparticles elicited the activation of microglia and up-regulation of TLR-2 in the olfactory bulb (Hutter et al., 2010). Silica nanoparticles (SiNP) can be internalized and accumulated in the cytoplasm and phagocytic vacuoles of microglia. Microglia exposed to SiNP did not affect cell viability and the phagocytic activity, but elicited oxidative stress and the production of IL-1β. SiNP increased the
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gene expression of cyclooxygenase-2 and inhibited TNF-α expression (Choi et al., 2010). Exposure of BV-2 microglial cells to TiO2 nanoparticles elicited ROS production within few minutes followed by an increase in the mitochondrial membrane potential and apoptotic cell death. TiO2 nanoparticles also enhanced the expression of the inflammation-associated genes in microglia (Long et al., 2006; Long et al., 2007).
Likewise, iron oxide nanoparticles have been demonstrated to alter the functionality of microglia. Iron oxide nanoparticles can be engulfed by BV-2 microglial cells and caused the disappearance of mitochondrial cristae and swelling of ER. The internalized iron oxide nanoparticles elicited proliferation and the production of ROS and NO. In addition, intranasal administration of iron oxide nanoparticles to mice resulted in neuron loss and microglial activation in the olfactory bulb, hippocampus and striatum (Wang et al., 2011c).
1.7 Objective of the study
Iron oxide nanoparticles have been used for biomedical research and clinical diagnosis. Numerous reports have demonstrated that iron oxide nanoparticles affect various immune functions. (Blank et al., 2011; Hsiao et al., 2008; Wang et al., 2011a).
Recently, our laboratory reported that iron oxide nanoparticles inhibited the antigen-specific antibody production and T lymphocytes responses in OVA-sensitized mice (Shen et al., 2011b). In addition, iron oxide nanoparticles inhibited the IFN-γ production in antigen-primed splenocytes associated with a diminished intracellular level of GSH (Shen et al., 2011a). These results suggest that iron oxide nanoparticles alter antigen-specific immunity and T cell functionality.
Iron oxide nanoparticles are employed for various diagnostic and potential therapeutic applications in the CNS, including imaging, targeted cancer therapy and hyperthermal therapy in glioma treatment (Engberink et al., 2010; Nunes et al., 2012;
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Silva et al., 2011; Silva, 2008; Spuch et al., 2012). Iron oxide nanoparticles have been shown to induce oxidative stress and neuron damage in mouse brain (Wang et al., 2009).
Iron oxide nanoparticles also induced cell proliferation and caused oxidative stress in microglia (Wang et al., 2011c). These results suggest that iron oxide nanoparticles could activate microglia and induce neurotoxicity. To date, most of studies investigate the effect of nanoparticles on resting microglia. Studies pertaining to the effect of nanoparticles on the defense capacity of microglia against pathogens are scarce.
Therefore, the objective of the study was to investigate the effect of iron oxide nanoparticles on the functionality of LPS-stimulated microglia. LPS is the major outer membrane component of gram-negative bacteria cell wall, which can activate microglia, thus serve as standard agents in mimicking bacterial infections (Draheim et al., 1999;
Prinz et al., 1999).
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