HHS Public Access Author manuscript Author Manuscript
Toxicol Res (Camb). Author manuscript; available in PMC 2017 May 01. Published in final edited form as: Toxicol Res (Camb). 2016 May 1; 5(3): 836–847. doi:10.1039/C5TX00421G.
Absence of Cytotoxicity towards Microglia of Iron Oxide (αFe2O3) Nanorhombohedra Crystal S. Lewis1,#, Luisa Torres2,#, Jeremy T. Miyauchi2,#, Cyrus Rastegar2, Jonathan M. Patete1, Jacqueline M. Smith1, Stanislaus S. Wong1,3,*, and Stella E. Tsirka2,* 1Department
of Chemistry, State University of New York at Stony Brook, Stony Brook,New York 11794-3400, USA
Author Manuscript
2Department
of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651, USA
3Condensed
Matter Physics and Materials Science Department, Building 480, Brookhaven National Laboratory, Upton, New York 11973, USA
Abstract
Author Manuscript
Understanding the nature of interactions between nanomaterials, such as commercially ubiquitous hematite (α-Fe2O3) Nanorhombohedra (N-Rhomb) and biological systems is of critical importance for gaining insight into the practical applicability of nanomaterials. Microglia represent the first line of defense in the central nervous system (CNS) during severe injury or disease such as Parkinson’s and Alzheimer’s disease as illustrative examples. Hence, to analyze the potential cytotoxic effect of nanorhombohedra exposure in the presence of microglia, we have synthesized Rhodamine B (RhB) labeled-α-Fe2O3 N-Rhomb, with lengths of 47 ± 10 nm and widths of 35 ± 8 nm. Internalization of RhB labeled-α-Fe2O3 N-Rhomb by microglia in the mouse brain was observed, and a dose-dependent increase in the cellular iron content as probed by cellular fluorescence was detected in cultured microglia after nanoparticle exposure. The cells maintained clear functional viability, exhibiting little to no cytotoxic effects after 24 and 48 hours at acceptable, physiological concentrations. Importantly, the nanoparticle exposure did not induce microglial cells to produce either tumor necrosis factor alpha (TNFα) or interleukin 1-beta (IL1β), two pro-inflammatory cytokines, nor did exposure induce the production of nitrites and reactive oxygen species (ROS), which are common indicators for the onset of inflammation. Finally, we propose that under the conditions of our experiments, i.e. in the presence of RhB labeled-α-Fe2O3 N-Rhomb maintaining concentrations of up to 100 µg/mL after 48 hours of incubation, the in vitro and in vivo internalization of RhB labeled-α-Fe2O3 N-Rhomb are likely to be clathrin-dependent, which represents a conventional mechanistic uptake route for most cells. Given the crucial role that microglia play in many neurological disorders, understanding the potential cytotoxic effects of these nanostructures is of fundamental importance if they are to be used in a therapeutic setting.
Author Manuscript *
; Email: [email protected]; ; Email: [email protected] #These authors contributed equally to the work.
Lewis et al.
Page 2
Author Manuscript
Keywords α-Fe2O3 nanostructures; Microglia; Cytotoxicity; Neurotoxicity
1. Introduction Nanomaterials, comprising nanoscale structures measuring between 1 and 100 nm in size, have attracted significant research interest due to their unique structure-dependent physical properties. Recently, concerns have been raised over the potentially deleterious effects of 1 3 these nanomaterials on human health and the environment. – From a toxicological perspective, nanoscale materials can induce different types of cellular responses, characterized by a variety of distinctive uptake mechanisms, such as endocytosis, mediated 4 6 for example by receptor-specific target sites. –
Author Manuscript Author Manuscript
For a given nanomaterial, morphology (e.g. in terms of its size and shape) is thought to be one of the key factors that can decisively determine the observed degree of its cytotoxicity and cellular uptake. Indeed, significant effort, including from one of our groups in particular, has been involved with systematically synthesizing novel motifs of diverse classes of nanomaterials, such as but not limited to derivatized carbon nanotubes (CNTs), rare earth ion-doped cerium phosphate (CePO4) nanowires, silicon dioxide (SiO2) nanotubes, titanium dioxide (TiO2) nanostructures, and zinc oxide (ZnO) nanowires and nanoparticles, to analyze their potential for biomedical applications. The objective of that prior body of work had been to correlate size, shape, morphology, and chemical composition of nanomaterials with their corresponding uptake mechanisms in an effort to probe and understand their 7 14 individual and collective impact upon cellular toxicity, in general. – In effect, we had been interested in determining the specific factors that control nanoscale toxicity.
Author Manuscript
The model system we study herein is related to a family of magnetic iron oxide (Fe3O4) nanostructures that has already been well studied. Indeed, nanoparticulate magnetite have previously been extensively investigated for incorporation into diverse applications, including for biological fluids, tissue-specific release of therapeutic agents, anti-cancer drug delivery systems, hyperthermia, and contrast enhancement for magnetic resonance imaging 15 18 (MRI). – In this context, the study of their potential toxicology to cells has served as a valuable means of gauging the viability, biocompatibility, and overall practicality of this 19 magnetic iron oxide platform for ubiquitous use in these assorted contexts. Nevertheless, the use of Fe3O4 for biomedical applications has been limited by issues associated not only with particle inhomogeneity and cost concerns but also with its inability to effectively differentiate between tumors and artifacts arising from bleeding, metal deposits, and/or 20 calcification in T2-weighted MRI images. A common, companion material to Fe3O4, i.e. hematite (Fe2O3), possesses a rhombohedral 21 crystal structure with a R3c space group. However, unlike Fe3O4, hematite can exist in different crystallographic forms such as alpha-hematite (α-Fe2O3), beta-hematite (β-Fe2O3), gamma-hematite (γ-Fe2O3), and epsilon-hematite (å-Fe2O3), with α-Fe2O3 and γ-Fe2O3 as the most familiar motifs. In particular, α-Fe2O3 has been synthesized as different morphologies, including as particles, cubes, and rods, and has been incorporated as Toxicol Res (Camb). Author manuscript; available in PMC 2017 May 01.
Lewis et al.
Page 3
Author Manuscript
functional components of gas sensors, CO oxidation catalysts, lithium-ion batteries, and 22 26 colloidal mediators for hyperthermia treatment. – In a number of these aforementioned 27 applications, the α-Fe2O3 nanoparticles have been employed as particulate, aerosolized motifs. Therefore, it is imperative to understand the potential toxicological effect of exposure to nanoscale hematite, as manifested by different intake routes such as inhalation, ingestion, and injection.
Author Manuscript
Nevertheless, the intrinsic toxicity of Fe2O3 nanostructures still remains a matter of considerable controversy. For example, in vitro studies have shown that α-Fe2O3 nanoparticles larger than 90 nm in diameter (i.e. ~250 nm and ~1.2 µm) gave rise to little if any toxicity with respect to human lung epithelial cells (A549) and murine alveolar 28 macrophages (MH-S). Moreover, α-Fe2O3 nanotubes, characterized by ~200 nm diameters, were found to be compatible with rat adrenal medulla cells (PC12), and in fact served as a potential delivery vehicle for nerve growth factor (NGF) in order to convert these 29 cells into neurons. By contrast, animal studies using Fe2O3 nanoparticles have revealed that these nanostructures may detrimentally induce either airway inflammation in healthy 30 mice or cellular reduction in alveolus and lymph nodes in allergic mice.
Author Manuscript
According to Brunauer-Emmett-Teller (BET) analysis, nanoscale rhombohedra, normalized for geometric considerations, possess a higher surface area (~45 m2/g) than either nanocubes 22 31 (~13.5 m2/g) or nanorods (~39 m2/g), depending on their size. , Therefore, since the surface area of α-Fe2O3 N-Rhomb is second only to that of spherical nanoparticles (~133 m2/g), which have already been extensively explored in cytotoxic analysis, this observation provides us with a rationale to fully understand the shape dependence of α-Fe2O3 N32 Rhomb’s interaction with cells, especially when engulfed. Moreover, with various reports on the shape-dependent cytotoxic behavior of nanowires versus nanoparticles under various 33 cellular conditions, it is therefore necessary to gain a similar insight into the analogous effects of rhombohedral α-Fe2O3 in a biological context. In terms of a prior report with comparable objectives to our own, it is worth noting that studies involving LiNbO3 nanorhombohedra have suggested that these nanostructures maintained cell viabilities of 34 ~80% after 48 hours of incubation within mouse macrophage cells.
Author Manuscript
Prior size and morphology-specific studies of various nanoparticles have indicated the ability of these nanoscale sized motifs to cross the blood brain barrier and thereby enter the 35 36 central nervous system (CNS) of higher order biological organisms, such as mammals. , In light of this result, many metal oxides such as Fe2O3 and TiO2 have been previously 37 39 probed for possible neurotoxic effects upon exposure. – With α-Fe2O3 N-Rhomb’s small size (
Toxicol Res (Camb). Author manuscript; available in PMC 2017 May 01. Published in final edited form as: Toxicol Res (Camb). 2016 May 1; 5(3): 836–847. doi:10.1039/C5TX00421G.
Absence of Cytotoxicity towards Microglia of Iron Oxide (αFe2O3) Nanorhombohedra Crystal S. Lewis1,#, Luisa Torres2,#, Jeremy T. Miyauchi2,#, Cyrus Rastegar2, Jonathan M. Patete1, Jacqueline M. Smith1, Stanislaus S. Wong1,3,*, and Stella E. Tsirka2,* 1Department
of Chemistry, State University of New York at Stony Brook, Stony Brook,New York 11794-3400, USA
Author Manuscript
2Department
of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651, USA
3Condensed
Matter Physics and Materials Science Department, Building 480, Brookhaven National Laboratory, Upton, New York 11973, USA
Abstract
Author Manuscript
Understanding the nature of interactions between nanomaterials, such as commercially ubiquitous hematite (α-Fe2O3) Nanorhombohedra (N-Rhomb) and biological systems is of critical importance for gaining insight into the practical applicability of nanomaterials. Microglia represent the first line of defense in the central nervous system (CNS) during severe injury or disease such as Parkinson’s and Alzheimer’s disease as illustrative examples. Hence, to analyze the potential cytotoxic effect of nanorhombohedra exposure in the presence of microglia, we have synthesized Rhodamine B (RhB) labeled-α-Fe2O3 N-Rhomb, with lengths of 47 ± 10 nm and widths of 35 ± 8 nm. Internalization of RhB labeled-α-Fe2O3 N-Rhomb by microglia in the mouse brain was observed, and a dose-dependent increase in the cellular iron content as probed by cellular fluorescence was detected in cultured microglia after nanoparticle exposure. The cells maintained clear functional viability, exhibiting little to no cytotoxic effects after 24 and 48 hours at acceptable, physiological concentrations. Importantly, the nanoparticle exposure did not induce microglial cells to produce either tumor necrosis factor alpha (TNFα) or interleukin 1-beta (IL1β), two pro-inflammatory cytokines, nor did exposure induce the production of nitrites and reactive oxygen species (ROS), which are common indicators for the onset of inflammation. Finally, we propose that under the conditions of our experiments, i.e. in the presence of RhB labeled-α-Fe2O3 N-Rhomb maintaining concentrations of up to 100 µg/mL after 48 hours of incubation, the in vitro and in vivo internalization of RhB labeled-α-Fe2O3 N-Rhomb are likely to be clathrin-dependent, which represents a conventional mechanistic uptake route for most cells. Given the crucial role that microglia play in many neurological disorders, understanding the potential cytotoxic effects of these nanostructures is of fundamental importance if they are to be used in a therapeutic setting.
Author Manuscript *
; Email: [email protected]; ; Email: [email protected] #These authors contributed equally to the work.
Lewis et al.
Page 2
Author Manuscript
Keywords α-Fe2O3 nanostructures; Microglia; Cytotoxicity; Neurotoxicity
1. Introduction Nanomaterials, comprising nanoscale structures measuring between 1 and 100 nm in size, have attracted significant research interest due to their unique structure-dependent physical properties. Recently, concerns have been raised over the potentially deleterious effects of 1 3 these nanomaterials on human health and the environment. – From a toxicological perspective, nanoscale materials can induce different types of cellular responses, characterized by a variety of distinctive uptake mechanisms, such as endocytosis, mediated 4 6 for example by receptor-specific target sites. –
Author Manuscript Author Manuscript
For a given nanomaterial, morphology (e.g. in terms of its size and shape) is thought to be one of the key factors that can decisively determine the observed degree of its cytotoxicity and cellular uptake. Indeed, significant effort, including from one of our groups in particular, has been involved with systematically synthesizing novel motifs of diverse classes of nanomaterials, such as but not limited to derivatized carbon nanotubes (CNTs), rare earth ion-doped cerium phosphate (CePO4) nanowires, silicon dioxide (SiO2) nanotubes, titanium dioxide (TiO2) nanostructures, and zinc oxide (ZnO) nanowires and nanoparticles, to analyze their potential for biomedical applications. The objective of that prior body of work had been to correlate size, shape, morphology, and chemical composition of nanomaterials with their corresponding uptake mechanisms in an effort to probe and understand their 7 14 individual and collective impact upon cellular toxicity, in general. – In effect, we had been interested in determining the specific factors that control nanoscale toxicity.
Author Manuscript
The model system we study herein is related to a family of magnetic iron oxide (Fe3O4) nanostructures that has already been well studied. Indeed, nanoparticulate magnetite have previously been extensively investigated for incorporation into diverse applications, including for biological fluids, tissue-specific release of therapeutic agents, anti-cancer drug delivery systems, hyperthermia, and contrast enhancement for magnetic resonance imaging 15 18 (MRI). – In this context, the study of their potential toxicology to cells has served as a valuable means of gauging the viability, biocompatibility, and overall practicality of this 19 magnetic iron oxide platform for ubiquitous use in these assorted contexts. Nevertheless, the use of Fe3O4 for biomedical applications has been limited by issues associated not only with particle inhomogeneity and cost concerns but also with its inability to effectively differentiate between tumors and artifacts arising from bleeding, metal deposits, and/or 20 calcification in T2-weighted MRI images. A common, companion material to Fe3O4, i.e. hematite (Fe2O3), possesses a rhombohedral 21 crystal structure with a R3c space group. However, unlike Fe3O4, hematite can exist in different crystallographic forms such as alpha-hematite (α-Fe2O3), beta-hematite (β-Fe2O3), gamma-hematite (γ-Fe2O3), and epsilon-hematite (å-Fe2O3), with α-Fe2O3 and γ-Fe2O3 as the most familiar motifs. In particular, α-Fe2O3 has been synthesized as different morphologies, including as particles, cubes, and rods, and has been incorporated as Toxicol Res (Camb). Author manuscript; available in PMC 2017 May 01.
Lewis et al.
Page 3
Author Manuscript
functional components of gas sensors, CO oxidation catalysts, lithium-ion batteries, and 22 26 colloidal mediators for hyperthermia treatment. – In a number of these aforementioned 27 applications, the α-Fe2O3 nanoparticles have been employed as particulate, aerosolized motifs. Therefore, it is imperative to understand the potential toxicological effect of exposure to nanoscale hematite, as manifested by different intake routes such as inhalation, ingestion, and injection.
Author Manuscript
Nevertheless, the intrinsic toxicity of Fe2O3 nanostructures still remains a matter of considerable controversy. For example, in vitro studies have shown that α-Fe2O3 nanoparticles larger than 90 nm in diameter (i.e. ~250 nm and ~1.2 µm) gave rise to little if any toxicity with respect to human lung epithelial cells (A549) and murine alveolar 28 macrophages (MH-S). Moreover, α-Fe2O3 nanotubes, characterized by ~200 nm diameters, were found to be compatible with rat adrenal medulla cells (PC12), and in fact served as a potential delivery vehicle for nerve growth factor (NGF) in order to convert these 29 cells into neurons. By contrast, animal studies using Fe2O3 nanoparticles have revealed that these nanostructures may detrimentally induce either airway inflammation in healthy 30 mice or cellular reduction in alveolus and lymph nodes in allergic mice.
Author Manuscript
According to Brunauer-Emmett-Teller (BET) analysis, nanoscale rhombohedra, normalized for geometric considerations, possess a higher surface area (~45 m2/g) than either nanocubes 22 31 (~13.5 m2/g) or nanorods (~39 m2/g), depending on their size. , Therefore, since the surface area of α-Fe2O3 N-Rhomb is second only to that of spherical nanoparticles (~133 m2/g), which have already been extensively explored in cytotoxic analysis, this observation provides us with a rationale to fully understand the shape dependence of α-Fe2O3 N32 Rhomb’s interaction with cells, especially when engulfed. Moreover, with various reports on the shape-dependent cytotoxic behavior of nanowires versus nanoparticles under various 33 cellular conditions, it is therefore necessary to gain a similar insight into the analogous effects of rhombohedral α-Fe2O3 in a biological context. In terms of a prior report with comparable objectives to our own, it is worth noting that studies involving LiNbO3 nanorhombohedra have suggested that these nanostructures maintained cell viabilities of 34 ~80% after 48 hours of incubation within mouse macrophage cells.
Author Manuscript
Prior size and morphology-specific studies of various nanoparticles have indicated the ability of these nanoscale sized motifs to cross the blood brain barrier and thereby enter the 35 36 central nervous system (CNS) of higher order biological organisms, such as mammals. , In light of this result, many metal oxides such as Fe2O3 and TiO2 have been previously 37 39 probed for possible neurotoxic effects upon exposure. – With α-Fe2O3 N-Rhomb’s small size (
