Article pubs.acs.org/crt
Reduced Astrocyte Viability at Physiological Temperatures from Magnetically Activated Iron Oxide Nanoparticles Nicholas J. Schaub,†,‡,# Deniz Rende,§,∥,# Yuan Yuan,⊥ Ryan J. Gilbert,*,†,‡,§ and Diana-Andra Borca-Tasciuc*,§,⊥ †
Center for Biotechnology and Interdisciplinary Studies, ‡Department of Biomedical Engineering, §Rensselaer Nanotechnology Center, ∥Department of Materials Science and Engineering, ⊥Department of Mechanical, Aerospace and Nuclear Engineering, Rensselaer Polytechnic Institute, 110 8th Street, Troy, New York 12180-3590, United States ABSTRACT: Superparamagnetic iron oxide nanoparticles (SPIONs) can generate heat when subjected to an alternating magnetic field (AMF). In the European Union, SPIONs actuated by AMF are used in hyperthermia treatment of glioblastoma multiforme, an aggressive form of brain cancer. Current data from clinical trials suggest that this therapy improves patient life expectancy, but their effect on healthy brain cells is virtually unknown. Thus, a viability study involving SPIONs subjected to an AMF was carried out on healthy cortical rat astrocytes, the most abundant cell type in the mammalian brain. The cells were cultured with aminosilane- or starch-coated SPIONs with or without application of an AMF. Significant cell death (p < 0.05) was observed only when SPIONs were added to astrocyte cultures and subjected to an AMF. Unexpectedly, the decrease in astrocyte viability was observed at physiological temperatures (34−40 °C) with AMF. A further decrease in astrocyte viability was found only when bulk temperatures exceeded 45 °C. To discern differences in the astrocyte structure when astrocytes were cultured with particles with or without AMF, scanning electron microscopy (SEM) was performed. SEM images revealed a change in the structure of the astrocyte cell membrane only when astrocytes were cultured with SPIONs and actuated with an AMF. This study is the first to report that astrocyte death occurs at physiological temperatures in the presence of magnetic particles and AMF, suggesting that other mechanisms are responsible for inducing astrocyte death in addition to heat.
1. INTRODUCTION
Glioblastoma brain tumors are resistant to most forms of treatment,20 and magnetic nanoparticle hyperthermia has been successfully used to improve treatment outcomes in conjunction with chemotherapy or radiation. In this approach, magnetic nanoparticles are directly injected into the brain tumor. Upon application of AMF, the temperature of the tumor is elevated to a temperature of 51 °C (median tumor temperature in a large-scale clinical trial).21 Depending upon the temperature reached, the tumor is destroyed or becomes more responsive to chemotherapy or radiation, increasing life expectancy by several months.21,22 Currently, MagForce AG holds European Union approval for using magnetic nanoparticles for hyperthermia treatment of glioblastoma multiforme.21 Since patients in the previously mentioned study showed no long-term functional deficits as a result of SPION hyperthermia treatment, it is assumed to be relatively safe for healthy tissue.21 Given the aggressiveness of glioblastoma multiforme, treatment effects on healthy tissue may be of secondary importance, especially when no major side effects have been reported; however, it is still important to understand how healthy cells respond to this type of therapy to enable further treatment
Hyperthermia is a form of cancer therapy in which cancerous tissue is heated above physiological temperature to induce damage and subsequent death of cancerous cells.1−5 These effects are usually observed when the tissue’s temperature is above 42 °C for more than 30 min.2,3 Although there are multiple methods to produce hyperthermia,3,6−8 one promising approach involves the use of superparamagnetic iron oxide nanoparticles (SPIONs). When SPIONs are exposed to an AMF of rapidly changing polarity (in the range of 100s of kHz), heat is dissipated due to their inability to instantaneously follow the field. There are several approaches used to estimate the heat dissipation rate, which depends on parameters such as the applied magnetic field strength and the presence of particle agglomerates.9−13 An advantage of using magnetic nanoparticles in cancer hyperthermia is that the particles are easily injectable, so delivery of the particles to the tumor is done by either direct or intravenous injection of particles.14−17 Moreover, they are actuated by electromagnetic waves in the radiofrequency range (not harmful to nor significantly absorbed by the human body) and low field intensities, even when located in deep tissue. Compared to other therapies, such as regional hyperthermia, magnetic nanoparticles create a local temperature rise, producing limited patient discomfort.18,19 © 2014 American Chemical Society
Received: June 11, 2014 Published: October 27, 2014 2023
dx.doi.org/10.1021/tx500231f | Chem. Res. Toxicol. 2014, 27, 2023−2035
Chemical Research in Toxicology
Article
suspensions of SPIONs received from the manufacturer diluted to a concentration of 0.03 mg/mL, and zeta potential measurements used SPIONs diluted to concentrations of 0.06 mg/mL and 0.5 mg/L. SPIONs were diluted with HPLC grade water or astrocyte culture medium filtered through a 0.2 μm PTFE filter, and after filtering, the SPION suspensions were ultrasonicated for 1 min before measurements. The surface chemistry of the SPIONs was confirmed by FT-IR measurements (PerkinElmer, Spectrum One). SPIONs, as received from the manufacturer, were placed between ZnSe discs in a liquid cell, where water was measured as the background. For each sample, the average of 8 scans was taken in the region from 450 to 4000 cm−1 with a resolution of 4 cm−1. 2.3. Primary Astrocyte Isolation and Culture. Primary astrocytes cultures from the cerebral cortex of newborn Sprague− Dawley rats were isolated as previously described.38 Briefly, postnatal day 1 rat pups were euthanized by rapid decapitation in accordance with procedures approved by the Institutional Animal Care and Use Committee (IACUC) at Rensselaer Polytechnic Institute. The cerebral cortices were then separated from the meninges, hippocampi, and basal ganglia. The cortical tissue from four animals was minced and transferred to a solution containing TrypLE and OptiMEM at a 1:1 dilution. Cells were extracted using three 10 min incubations with TrypLE/OptiMEM additionally supplemented with DNase 1. The second and third extractions were combined with DMEM containing 10% HIHS and 1% P/S. Cells were pelleted using centrifugation (0.5 RCF for 5 min) and resuspended in astrocyte culture media. T75 culture flasks were coated with poly-D-lysine (20 μg/mL) for 1 h and then allowed to dry overnight. The dissociated cells were then plated on poly-D-lysine-coated T75 culture flasks at a density of 200 000 cells/flask. Astrocytes were cultured for 2−4 weeks in an incubator at 37 °C with 5% CO2 before they were used in the subsequent experiments. Astrocyte purity was determined by labeling for GFAP (DAKO, Carpinteria, CA) followed by a Alexa-488 conjugated rabbit secondary antibody (Sigma, St. Louis, MO), and all cell cultures were found to be >90% pure. 2.4. Measurement of Astrocyte Adhesion in the Presence of SPIONs. Before examining astrocyte behavior in the presence of nanoparticles and an AMF, it was important to understand how well astrocytes adhered to glass coverslips in the presence of SPIONs. In this adhesion experiment, astrocytes were mixed with nanoparticles in suspension before placing the cell−SPION mixture onto glass coverslips. The glass coverslips (12 mm diameter, #1 round coverslips, Long Island City, NY) used for all experiments were acid-etched in a 1:3 solution of 30% H2O2 and sulfuric acid for 1 h followed by washing three times in dH2O before storing in 100% ethanol. Before using the acid-etched glass coverslips for astrocyte culture, the coverslips were air-dried, sterilized using ultraviolet (UV) treatment (XL-1000 UV Cross-linker, Spectroline, Westbury, NY), plasma-cleaned (Harrick Plasma, Ithaca, NY), coated with poly-D-lysine (1 μg/mL) for 2 h at room temperature, and then washed twice with sterile water. To determine the ability of SPIONs to alter astrocyte adhesion to PDLcoated glass, 50 000 astrocytes in 1 mL of culture media were placed onto glass coverslips without nanoparticles, with FluidMAG-Amine nanoparticles (50 nm diameter), or with FluidMAG-D nanoparticles (50 nm diameter). The cell density was kept constant at 50 000 cells/ mL for all experiments. Both types of nanoparticles were added to different astrocyte suspensions at varying concentrations to determine if astrocyte adhesion was dependent on the amount of SPIONs within the liquid media. The concentrations of nanoparticles used for these adhesion studies are the same concentrations utilized in a previous study assessing neuronal response to nanoparticles: 0.25, 1.25, or 2.5 mg/mL.35 SPIONs were vortexed for 1 min to create a homogeneous suspension of nanoparticles before their addition to astrocyte suspensions or adherent astrocytes. After 24 h of culture, the culture medium was replaced with phosphate buffered saline (PBS, Gibco, Green Island, NY) to remove the majority of the nanoparticles. The PBS was subsequently replaced with calcein-AM (2 μg/mL in PBS, live cell label) for 30 min and incubated at 37 °C. Next, PBS containing calcein-AM was replaced
optimization. Therefore, this study focuses on healthy astrocytes, the most abundant cell type in mammalian brain. Astrocytes have critically important functions, including regulation of neuronal plasticity and maintaining neurotransmitter levels for efficient neuronal communication,23 and are involved in creating and sustaining the blood−brain barrier.24,25 Astrocytes also deliver nutrients to neurons,26,27 neutralize reactive oxygen species,28 and metabolize iron and other metals in the brain.29−31 Mutant astrocytes can also give rise to glioblastoma multiforme,32 the cancerous cells that SPION-mediated hyperthermia is used to treat, which is another reason that they are the subject of interest in this study. Since glioblastoma multiforme cells are susceptible to SPIONmediated hyperthermia, it is possible that the same mechanisms that cause cell death in glioblastoma multiforme cells could cause cell death in astrocytes. In the absence of any magnetic field, SPIONs are known to alter astrocyte adhesion24 and induce the formation of reactive oxygenating species (although not at cytotoxic levels).33 However, to the best of our knowledge, there are no reports on astrocyte response when they are cultured with SPIONs in an AMF, which is the subject of the investigation reported in this article. This study was carried out on healthy cortical rat astrocytes and employed commercially available SPIONs with two types of coatings: aminosilane (FluidMAG-Amine), because aminecoated particles are used in clinical applications,21,22 and starch (FluidMAG-D), because they are not cytotoxic to neurons.34−36 Thus, this study determined how SPION hyperthermia treatment affected astrocyte viability with respect to different particle coatings, AMF field strengths, and the bulk media temperature during heating. These results help to understand how native cells react to SPION-induced hyperthermia in order to develop improved methods of treatment.
2. EXPERIMENTAL PROCEDURES 2.1. Materials. Astrocyte culture media consisting of Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Green Island, NY) was supplemented with 10% heat-inactivated horse serum (HIHS, Gibco, Green Island, NY) and 1% penicillin/streptomycin (P/S, Invitrogen, Green Island, NY). Solutions used for cell culture and analysis include TrypLE (Invitrogen, Green Island, NY), OptiMEM (Invitrogen, Green Island, NY), DNase 1 (Sigma, St. Louis, MO), trypan blue (Sigma, St. Louis, MO), calcein-AM (Sigma, St. Louis, MO), propidium iodide (Sigma, St. Louis, MO), and poly-D-lysine (Sigma, St. Louis, MO). The cells were fixed with paraformaldehyde (Sigma, St. Louis, MO). Biofunctional iron oxide nanoparticles, FluidMAGAmine (aminosilane, 50 and 100 nm) and FluidMAG-D (starch, 50 nm), were purchased from Chemicell (Berlin, Germany). 2.2. Characterization of Nanoparticle Size and Agglomeration. Before the nanoparticles were placed in the astrocyte culture media, it was necessary to verify the size of the individual particles and determine if the media altered nanoparticle agglomeration. All three SPION types used in this study (aminosilane, 50 and 100 nm; starch, 50 nm) were measured to determine single-particle size by transmission electron microscopy (TEM) using a JEM-2011 (JEOL, Peadbody, MA) at 200 kV. To determine particle agglomerate size, dynamic light scattering (DLS) was performed on each type of SPION in water or astrocyte culture medium (Malvern Instruments, NanoZS) at a temperature of 25 °C with Mark−Houwink equation (a = 0.428 and κ = 7.67 × 10−5). The surface charge of the nanoparticles was measured using a Zetasizer (Malvern Instruments, NanoZS) at 25 °C, and the data was interpreted with the Smoluchowski equation. Measurements were performed in triplicate with 10−100 automated runs. The refractive index and absorbance used were 2.42 and 0.2 for iron oxide nanoparticles, respectively.37 DLS measurements were conducted on 2024
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Figure 1. Characterization of aminosilane- and starch-coated SPIONs. TEM images and their corresponding size distributions of (A, C) FluidMAGAmine (50 nm) and (B, D) FluidMAG-D (50 nm) (scale bars = 20 nm). with PBS containing propidium iodide (2 μg/mL, dead cell label) and incubated at 37 °C for 15 min. Live and dead cell images were collected using an Olympus DSU microscope (Center Valley, PA) with constant gain and exposure settings from six unique locations and analyzed employing a cell counting algorithm developed in-house using Python. The same algorithm was used for both the propidium iodide and calcein channels. To describe the cell counting algorithm procedure, each image was convolved with a low-pass filter to remove noise, and the background was removed using a median filter. Next, a k-means algorithm with five clusters was used to determine a threshold to generate a binary image. Then, small objects (assumed to be noise) in the binary image were removed using a binary opening filter. Finally, the number of objects in the image was counted to determine the number of living (calcein) or dead (propidium iodide) cells. 2.5. Measurement of Astrocyte Viability in the Presence of SPIONs. In the viability experiment, astrocytes were allowed to adhere to glass coverslips for 24 h before the addition of nanoparticles. Glass coverslips were prepared exactly as described in Section 2.4 (acidetched, PDL-coated, etc.). Next, 50 000 astrocytes were placed onto each glass coverslip (1 mL of astrocyte medium containing 50 000 cells/mL). Astrocytes were allowed to adhere for 24 h, and then astrocyte media was replaced with astrocyte media containing FluidMAG-Amine or FluidMAG-D SPIONs at the same concentrations as described in Section 2.4. Astrocytes were incubated in SPION containing media for 24 or 72 h, after which each sample was treated with calcein and propidium iodide as described in Section 2.4. Each sample was then imaged and a cell count was obtained using the methods described in Section 2.4. 2.6. Analysis of Astrocyte Viability in the Presence of SPIONs and AMF. After completing the experiments conducted in Section 2.4, there was no decrease in cell viability in experiments where astrocytes were allowed to adhere to the coverslips for 72 h before addition of SPIONs without AMF. Hence, astrocytes were allowed to adhere to coverslips for 72 h prior to all subsequent experiments, regardless of experimental conditions. With all heated samples as well as associated controls, SPIONs were added to the culture media at a final concentration of 2.5 mg/mL, and the particles were allowed to interact with the cells for a period of 30 min at 37 °C, 5% CO2 incubator before the cultures were placed within the alternating magnetic field (AMF) or heated water bath.
An AMF was generated using an induction heating system from RDO Induction (Model HFI 3-135/400, Washington, NJ). The magnetic field of the coil was adjusted by setting the output power of the system to pre-established values, determined in heating of SPIONs added to cell media initially at room temperature. In these preliminary experiments, the power of the system was adjusted to reach 37 or 42 °C in the test samples after 60 min of heating. These two temperature regimes were selected to mimic physiological temperatures (37 °C) or to provide a temperature used frequently in hyperthermia treatments (42 °C). Heating duration (1 h) was comparable to the heating duration used in reported clinical and animal studies.22,39 The order in which cells were placed in the heating coil were changed for each replicate of the experiment since heat dissipation from the coil was significant enough to alter the bulk temperature. To ensure that all cultures received the same magnitude of AMF, glass vials containing the astrocyte cultures were placed in the same location within the middle of the coil. The coil used in this study and the method of characterizing the magnetic field using 2D Flux software was previously described.36 From 2D Flux software simulations, the magnetic field strength varied less than 10% between the center of the glass coverlip compared to the edge of the glass coverslip. The temperature of the culture media within the coil was recorded over time with a fiber optic sensor (Luxtron Corporation, Santa Clara, CA). The fiber optic sensor was placed in the center of the heated glass vial containing media, 1 mm from the surface of the coverslip containing adherent astrocytes. To determine astrocyte response to SPIONs in the presence of heat alone without AMF, astrocytes cultures were placed in heated water baths (set at 40 °C). Following AMF heating or water bath heating, the vials were returned back to 37 °C, 5% CO2 incubator. After 24 h, the cells were stained with calcein-AM/ propidium iodide as described in Section 2.4 and imaged. Six images were randomly collected from the sides and center of the glass coverslip and analyzed further with the cell counting algorithm as described in Section 2.4. 2.7. Scanning Electron Microscopy of Astrocytes. Astrocytes were analyzed by scanning electron microscopy to investigate potential alteration of the cell membrane in the presence of SPIONS. Astrocytes were allowed to adhere to glass coverslips for 72 h before addition of 2.5 mg/mL of either FluidMAG-D or FluidMAG-Amine SPIONs. Thirty minutes after the addition of SPIONs, astrocytes were either 2025
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Figure 2. DLS measurements of SPIONs in water and astrocyte medium. The results are presented as (A) Z-average and (B) intensity mean distributions, indicating that the SPIONs form agglomerates in culture medium. Measurements were performed in triplicate with 10−100 automated runs. Bars are the mean, with error bars representing the standard deviation.
Table 1. Summary of the Hydrodynamic Diameter, Polydispersity Index, and Zeta-Potential Measurements of SPIONs Suspended in Water and Astrocyte Medium astrocyte mediuma
water FluidMAG-Amine (50 nm) FluidMAG-Amine (100 nm) FluidMAG-D (50 nm) a
dDLS (nm)
PDI
zeta potential (mV)
dDLS (nm)
PDI
zeta potential (mV)
79 ± 0 83 ± 1 55 ± 5
0.23 0.15 0.28
24.3 ± 2.4 34.5 ± 0.7 2.9 ± 0.1
78 ± 1 99 ± 1 28 ± 0
0.34 0.33 0.32
−7.8 ± 0.5 −6.3 ± 0.3 −5.4 ± 0.5
The zeta potential of the astrocyte medium is −5.6 ± 1.2 mV.
exposed to an AMF of 10.82 kA/m magnitude for 1 h or were placed in the cell culture incubator without being exposed to the AMF. Immediately after heating astrocytes, cells were fixed with a 4% paraformaldehyde solution (Sigma, St. Louis, MO) for 30 min and subsequently washed three times with PBS. Astrocytes were then exposed to a 1% solution of osmium tetroxide (Sigma, St. Louis, MO) for 1 h, followed by a serial dehydration of the sample with 50, 60, 70, 80, 90, and 100% ethanol (v/v). Astrocytes were then CO2 critical point dried (Tousimis, Rockville, MD), sputter-coated with platinum (Denton Desk IV, Moorestown, NJ) and imaged with a Carl Zeiss Supra55 SEM (Thornwood, NY). 2.8. Statistics. Adhesion, viability, and astrocyte culture heating experiments were performed in triplicate, with each replicate containing astrocytes isolated from three different rats (n = 3). However, when the magnetic field was lowered to 7.31 kA/m to determine how AMF field strength altered cell viability, only one trial was carried out. Since the results were comparable to previous experiments, additional replicates were not carried out. JMP statistical software (SAS, Cary, NC) was used for all statistical analysis. A oneway ANOVA was used to compare live and dead cell counts data between different experimental and control groups. Subsequently, a posthoc Tukey−Kramer HSD was used to determine differences between groups. Results were considered statistically significant if the statistical test resulted in p < 0.05.
100 particles. Analysis of TEM images of FluidMAG-Amine particles revealed the mean single-particle diameter to be 9.4 ± 2.7 nm (Figure 1A,C), and the FluidMAG-D mean singleparticle diameter was found to be 9.3 ± 2.2 nm (Figure 1B,D). These values are in agreement with the manufacturer’s specifications of 10 nm particle diameters. The agglomeration diameter of SPIONs in water or astrocyte medium was determined using DLS. The Z-average diameter (Figure 2A) and the intensity mean distribution (Figure 2B) revealed SPION agglomerates. In the intensity mean distribution, we observed that there was a unimodal distribution of hydrodynamic diameters for all SPIONs in water, but there was a bimodal distribution of hydrodynamic diameters for all SPIONs in astrocyte medium. In water, the Z-average agglomeration diameter of SPIONs was 79 ± 0 nm (FluidMAG-Amine, 50 nm), 83 ± 1 nm (FluidMAG-Amine, 100 nm), or 55 ± 5 nm (FluidMAG-D, 50 nm). In astrocyte medium, the Z-average agglomeration diameter of the particles was 78 ± 1 nm (FluidMAG-Amine, 50 nm), 99 ± 1 nm (FluidMAG-Amine, 100 nm), or 28 ± 0 nm (FluidMAG-D, 50 nm). The intensity mean distributions for FluidMAG-Amine SPIONs in astrocyte medium (50 and 100 nm, Figure 2B) revealed one peak centered around 10 nm and another peak centered around 100 nm. For both sizes of FluidMAG-Amine SPIONs, the peak centered around 10 nm had a much smaller intensity than the peak centered around 100 nm. Additionally, the 100 nm peak of FluidMAG-Amine SPIONs in astrocyte medium was always shifted toward a larger diameter than a similar peak observed around 100 nm for SPIONs in water. The bimodal distribution observed in the intensity mean distribution of FluidMAG-D SPIONs in astrocyte medium indicates the coexistence of small and large agglomerates in
3. RESULTS 3.1. Characterization of SPIONs in Different Aqueous Media. SPIONs agglomerate within different aqueous media,12,13 and the agglomeration affects the heat generation rate. Agglomeration may also influence the ability of cells to uptake SPIONs. Due to agglomeration influencing heat generation and cellular uptake, it was important to determine agglomeration size in the presence of astrocyte media. Individual SPION diameters were determined using TEM for 2026
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astrocyte medium. The two peaks in the intensity mean distribution for FluidMAG-D SPIONs had similar intensities, which likely led to a decrease in Z-average agglomerate diameter in astrocyte culture media (Figure 2A). The polydispersity indexes (PDI) of the samples suspended in water were low,
Reduced Astrocyte Viability at Physiological Temperatures from Magnetically Activated Iron Oxide Nanoparticles Nicholas J. Schaub,†,‡,# Deniz Rende,§,∥,# Yuan Yuan,⊥ Ryan J. Gilbert,*,†,‡,§ and Diana-Andra Borca-Tasciuc*,§,⊥ †
Center for Biotechnology and Interdisciplinary Studies, ‡Department of Biomedical Engineering, §Rensselaer Nanotechnology Center, ∥Department of Materials Science and Engineering, ⊥Department of Mechanical, Aerospace and Nuclear Engineering, Rensselaer Polytechnic Institute, 110 8th Street, Troy, New York 12180-3590, United States ABSTRACT: Superparamagnetic iron oxide nanoparticles (SPIONs) can generate heat when subjected to an alternating magnetic field (AMF). In the European Union, SPIONs actuated by AMF are used in hyperthermia treatment of glioblastoma multiforme, an aggressive form of brain cancer. Current data from clinical trials suggest that this therapy improves patient life expectancy, but their effect on healthy brain cells is virtually unknown. Thus, a viability study involving SPIONs subjected to an AMF was carried out on healthy cortical rat astrocytes, the most abundant cell type in the mammalian brain. The cells were cultured with aminosilane- or starch-coated SPIONs with or without application of an AMF. Significant cell death (p < 0.05) was observed only when SPIONs were added to astrocyte cultures and subjected to an AMF. Unexpectedly, the decrease in astrocyte viability was observed at physiological temperatures (34−40 °C) with AMF. A further decrease in astrocyte viability was found only when bulk temperatures exceeded 45 °C. To discern differences in the astrocyte structure when astrocytes were cultured with particles with or without AMF, scanning electron microscopy (SEM) was performed. SEM images revealed a change in the structure of the astrocyte cell membrane only when astrocytes were cultured with SPIONs and actuated with an AMF. This study is the first to report that astrocyte death occurs at physiological temperatures in the presence of magnetic particles and AMF, suggesting that other mechanisms are responsible for inducing astrocyte death in addition to heat.
1. INTRODUCTION
Glioblastoma brain tumors are resistant to most forms of treatment,20 and magnetic nanoparticle hyperthermia has been successfully used to improve treatment outcomes in conjunction with chemotherapy or radiation. In this approach, magnetic nanoparticles are directly injected into the brain tumor. Upon application of AMF, the temperature of the tumor is elevated to a temperature of 51 °C (median tumor temperature in a large-scale clinical trial).21 Depending upon the temperature reached, the tumor is destroyed or becomes more responsive to chemotherapy or radiation, increasing life expectancy by several months.21,22 Currently, MagForce AG holds European Union approval for using magnetic nanoparticles for hyperthermia treatment of glioblastoma multiforme.21 Since patients in the previously mentioned study showed no long-term functional deficits as a result of SPION hyperthermia treatment, it is assumed to be relatively safe for healthy tissue.21 Given the aggressiveness of glioblastoma multiforme, treatment effects on healthy tissue may be of secondary importance, especially when no major side effects have been reported; however, it is still important to understand how healthy cells respond to this type of therapy to enable further treatment
Hyperthermia is a form of cancer therapy in which cancerous tissue is heated above physiological temperature to induce damage and subsequent death of cancerous cells.1−5 These effects are usually observed when the tissue’s temperature is above 42 °C for more than 30 min.2,3 Although there are multiple methods to produce hyperthermia,3,6−8 one promising approach involves the use of superparamagnetic iron oxide nanoparticles (SPIONs). When SPIONs are exposed to an AMF of rapidly changing polarity (in the range of 100s of kHz), heat is dissipated due to their inability to instantaneously follow the field. There are several approaches used to estimate the heat dissipation rate, which depends on parameters such as the applied magnetic field strength and the presence of particle agglomerates.9−13 An advantage of using magnetic nanoparticles in cancer hyperthermia is that the particles are easily injectable, so delivery of the particles to the tumor is done by either direct or intravenous injection of particles.14−17 Moreover, they are actuated by electromagnetic waves in the radiofrequency range (not harmful to nor significantly absorbed by the human body) and low field intensities, even when located in deep tissue. Compared to other therapies, such as regional hyperthermia, magnetic nanoparticles create a local temperature rise, producing limited patient discomfort.18,19 © 2014 American Chemical Society
Received: June 11, 2014 Published: October 27, 2014 2023
dx.doi.org/10.1021/tx500231f | Chem. Res. Toxicol. 2014, 27, 2023−2035
Chemical Research in Toxicology
Article
suspensions of SPIONs received from the manufacturer diluted to a concentration of 0.03 mg/mL, and zeta potential measurements used SPIONs diluted to concentrations of 0.06 mg/mL and 0.5 mg/L. SPIONs were diluted with HPLC grade water or astrocyte culture medium filtered through a 0.2 μm PTFE filter, and after filtering, the SPION suspensions were ultrasonicated for 1 min before measurements. The surface chemistry of the SPIONs was confirmed by FT-IR measurements (PerkinElmer, Spectrum One). SPIONs, as received from the manufacturer, were placed between ZnSe discs in a liquid cell, where water was measured as the background. For each sample, the average of 8 scans was taken in the region from 450 to 4000 cm−1 with a resolution of 4 cm−1. 2.3. Primary Astrocyte Isolation and Culture. Primary astrocytes cultures from the cerebral cortex of newborn Sprague− Dawley rats were isolated as previously described.38 Briefly, postnatal day 1 rat pups were euthanized by rapid decapitation in accordance with procedures approved by the Institutional Animal Care and Use Committee (IACUC) at Rensselaer Polytechnic Institute. The cerebral cortices were then separated from the meninges, hippocampi, and basal ganglia. The cortical tissue from four animals was minced and transferred to a solution containing TrypLE and OptiMEM at a 1:1 dilution. Cells were extracted using three 10 min incubations with TrypLE/OptiMEM additionally supplemented with DNase 1. The second and third extractions were combined with DMEM containing 10% HIHS and 1% P/S. Cells were pelleted using centrifugation (0.5 RCF for 5 min) and resuspended in astrocyte culture media. T75 culture flasks were coated with poly-D-lysine (20 μg/mL) for 1 h and then allowed to dry overnight. The dissociated cells were then plated on poly-D-lysine-coated T75 culture flasks at a density of 200 000 cells/flask. Astrocytes were cultured for 2−4 weeks in an incubator at 37 °C with 5% CO2 before they were used in the subsequent experiments. Astrocyte purity was determined by labeling for GFAP (DAKO, Carpinteria, CA) followed by a Alexa-488 conjugated rabbit secondary antibody (Sigma, St. Louis, MO), and all cell cultures were found to be >90% pure. 2.4. Measurement of Astrocyte Adhesion in the Presence of SPIONs. Before examining astrocyte behavior in the presence of nanoparticles and an AMF, it was important to understand how well astrocytes adhered to glass coverslips in the presence of SPIONs. In this adhesion experiment, astrocytes were mixed with nanoparticles in suspension before placing the cell−SPION mixture onto glass coverslips. The glass coverslips (12 mm diameter, #1 round coverslips, Long Island City, NY) used for all experiments were acid-etched in a 1:3 solution of 30% H2O2 and sulfuric acid for 1 h followed by washing three times in dH2O before storing in 100% ethanol. Before using the acid-etched glass coverslips for astrocyte culture, the coverslips were air-dried, sterilized using ultraviolet (UV) treatment (XL-1000 UV Cross-linker, Spectroline, Westbury, NY), plasma-cleaned (Harrick Plasma, Ithaca, NY), coated with poly-D-lysine (1 μg/mL) for 2 h at room temperature, and then washed twice with sterile water. To determine the ability of SPIONs to alter astrocyte adhesion to PDLcoated glass, 50 000 astrocytes in 1 mL of culture media were placed onto glass coverslips without nanoparticles, with FluidMAG-Amine nanoparticles (50 nm diameter), or with FluidMAG-D nanoparticles (50 nm diameter). The cell density was kept constant at 50 000 cells/ mL for all experiments. Both types of nanoparticles were added to different astrocyte suspensions at varying concentrations to determine if astrocyte adhesion was dependent on the amount of SPIONs within the liquid media. The concentrations of nanoparticles used for these adhesion studies are the same concentrations utilized in a previous study assessing neuronal response to nanoparticles: 0.25, 1.25, or 2.5 mg/mL.35 SPIONs were vortexed for 1 min to create a homogeneous suspension of nanoparticles before their addition to astrocyte suspensions or adherent astrocytes. After 24 h of culture, the culture medium was replaced with phosphate buffered saline (PBS, Gibco, Green Island, NY) to remove the majority of the nanoparticles. The PBS was subsequently replaced with calcein-AM (2 μg/mL in PBS, live cell label) for 30 min and incubated at 37 °C. Next, PBS containing calcein-AM was replaced
optimization. Therefore, this study focuses on healthy astrocytes, the most abundant cell type in mammalian brain. Astrocytes have critically important functions, including regulation of neuronal plasticity and maintaining neurotransmitter levels for efficient neuronal communication,23 and are involved in creating and sustaining the blood−brain barrier.24,25 Astrocytes also deliver nutrients to neurons,26,27 neutralize reactive oxygen species,28 and metabolize iron and other metals in the brain.29−31 Mutant astrocytes can also give rise to glioblastoma multiforme,32 the cancerous cells that SPION-mediated hyperthermia is used to treat, which is another reason that they are the subject of interest in this study. Since glioblastoma multiforme cells are susceptible to SPIONmediated hyperthermia, it is possible that the same mechanisms that cause cell death in glioblastoma multiforme cells could cause cell death in astrocytes. In the absence of any magnetic field, SPIONs are known to alter astrocyte adhesion24 and induce the formation of reactive oxygenating species (although not at cytotoxic levels).33 However, to the best of our knowledge, there are no reports on astrocyte response when they are cultured with SPIONs in an AMF, which is the subject of the investigation reported in this article. This study was carried out on healthy cortical rat astrocytes and employed commercially available SPIONs with two types of coatings: aminosilane (FluidMAG-Amine), because aminecoated particles are used in clinical applications,21,22 and starch (FluidMAG-D), because they are not cytotoxic to neurons.34−36 Thus, this study determined how SPION hyperthermia treatment affected astrocyte viability with respect to different particle coatings, AMF field strengths, and the bulk media temperature during heating. These results help to understand how native cells react to SPION-induced hyperthermia in order to develop improved methods of treatment.
2. EXPERIMENTAL PROCEDURES 2.1. Materials. Astrocyte culture media consisting of Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Green Island, NY) was supplemented with 10% heat-inactivated horse serum (HIHS, Gibco, Green Island, NY) and 1% penicillin/streptomycin (P/S, Invitrogen, Green Island, NY). Solutions used for cell culture and analysis include TrypLE (Invitrogen, Green Island, NY), OptiMEM (Invitrogen, Green Island, NY), DNase 1 (Sigma, St. Louis, MO), trypan blue (Sigma, St. Louis, MO), calcein-AM (Sigma, St. Louis, MO), propidium iodide (Sigma, St. Louis, MO), and poly-D-lysine (Sigma, St. Louis, MO). The cells were fixed with paraformaldehyde (Sigma, St. Louis, MO). Biofunctional iron oxide nanoparticles, FluidMAGAmine (aminosilane, 50 and 100 nm) and FluidMAG-D (starch, 50 nm), were purchased from Chemicell (Berlin, Germany). 2.2. Characterization of Nanoparticle Size and Agglomeration. Before the nanoparticles were placed in the astrocyte culture media, it was necessary to verify the size of the individual particles and determine if the media altered nanoparticle agglomeration. All three SPION types used in this study (aminosilane, 50 and 100 nm; starch, 50 nm) were measured to determine single-particle size by transmission electron microscopy (TEM) using a JEM-2011 (JEOL, Peadbody, MA) at 200 kV. To determine particle agglomerate size, dynamic light scattering (DLS) was performed on each type of SPION in water or astrocyte culture medium (Malvern Instruments, NanoZS) at a temperature of 25 °C with Mark−Houwink equation (a = 0.428 and κ = 7.67 × 10−5). The surface charge of the nanoparticles was measured using a Zetasizer (Malvern Instruments, NanoZS) at 25 °C, and the data was interpreted with the Smoluchowski equation. Measurements were performed in triplicate with 10−100 automated runs. The refractive index and absorbance used were 2.42 and 0.2 for iron oxide nanoparticles, respectively.37 DLS measurements were conducted on 2024
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Figure 1. Characterization of aminosilane- and starch-coated SPIONs. TEM images and their corresponding size distributions of (A, C) FluidMAGAmine (50 nm) and (B, D) FluidMAG-D (50 nm) (scale bars = 20 nm). with PBS containing propidium iodide (2 μg/mL, dead cell label) and incubated at 37 °C for 15 min. Live and dead cell images were collected using an Olympus DSU microscope (Center Valley, PA) with constant gain and exposure settings from six unique locations and analyzed employing a cell counting algorithm developed in-house using Python. The same algorithm was used for both the propidium iodide and calcein channels. To describe the cell counting algorithm procedure, each image was convolved with a low-pass filter to remove noise, and the background was removed using a median filter. Next, a k-means algorithm with five clusters was used to determine a threshold to generate a binary image. Then, small objects (assumed to be noise) in the binary image were removed using a binary opening filter. Finally, the number of objects in the image was counted to determine the number of living (calcein) or dead (propidium iodide) cells. 2.5. Measurement of Astrocyte Viability in the Presence of SPIONs. In the viability experiment, astrocytes were allowed to adhere to glass coverslips for 24 h before the addition of nanoparticles. Glass coverslips were prepared exactly as described in Section 2.4 (acidetched, PDL-coated, etc.). Next, 50 000 astrocytes were placed onto each glass coverslip (1 mL of astrocyte medium containing 50 000 cells/mL). Astrocytes were allowed to adhere for 24 h, and then astrocyte media was replaced with astrocyte media containing FluidMAG-Amine or FluidMAG-D SPIONs at the same concentrations as described in Section 2.4. Astrocytes were incubated in SPION containing media for 24 or 72 h, after which each sample was treated with calcein and propidium iodide as described in Section 2.4. Each sample was then imaged and a cell count was obtained using the methods described in Section 2.4. 2.6. Analysis of Astrocyte Viability in the Presence of SPIONs and AMF. After completing the experiments conducted in Section 2.4, there was no decrease in cell viability in experiments where astrocytes were allowed to adhere to the coverslips for 72 h before addition of SPIONs without AMF. Hence, astrocytes were allowed to adhere to coverslips for 72 h prior to all subsequent experiments, regardless of experimental conditions. With all heated samples as well as associated controls, SPIONs were added to the culture media at a final concentration of 2.5 mg/mL, and the particles were allowed to interact with the cells for a period of 30 min at 37 °C, 5% CO2 incubator before the cultures were placed within the alternating magnetic field (AMF) or heated water bath.
An AMF was generated using an induction heating system from RDO Induction (Model HFI 3-135/400, Washington, NJ). The magnetic field of the coil was adjusted by setting the output power of the system to pre-established values, determined in heating of SPIONs added to cell media initially at room temperature. In these preliminary experiments, the power of the system was adjusted to reach 37 or 42 °C in the test samples after 60 min of heating. These two temperature regimes were selected to mimic physiological temperatures (37 °C) or to provide a temperature used frequently in hyperthermia treatments (42 °C). Heating duration (1 h) was comparable to the heating duration used in reported clinical and animal studies.22,39 The order in which cells were placed in the heating coil were changed for each replicate of the experiment since heat dissipation from the coil was significant enough to alter the bulk temperature. To ensure that all cultures received the same magnitude of AMF, glass vials containing the astrocyte cultures were placed in the same location within the middle of the coil. The coil used in this study and the method of characterizing the magnetic field using 2D Flux software was previously described.36 From 2D Flux software simulations, the magnetic field strength varied less than 10% between the center of the glass coverlip compared to the edge of the glass coverslip. The temperature of the culture media within the coil was recorded over time with a fiber optic sensor (Luxtron Corporation, Santa Clara, CA). The fiber optic sensor was placed in the center of the heated glass vial containing media, 1 mm from the surface of the coverslip containing adherent astrocytes. To determine astrocyte response to SPIONs in the presence of heat alone without AMF, astrocytes cultures were placed in heated water baths (set at 40 °C). Following AMF heating or water bath heating, the vials were returned back to 37 °C, 5% CO2 incubator. After 24 h, the cells were stained with calcein-AM/ propidium iodide as described in Section 2.4 and imaged. Six images were randomly collected from the sides and center of the glass coverslip and analyzed further with the cell counting algorithm as described in Section 2.4. 2.7. Scanning Electron Microscopy of Astrocytes. Astrocytes were analyzed by scanning electron microscopy to investigate potential alteration of the cell membrane in the presence of SPIONS. Astrocytes were allowed to adhere to glass coverslips for 72 h before addition of 2.5 mg/mL of either FluidMAG-D or FluidMAG-Amine SPIONs. Thirty minutes after the addition of SPIONs, astrocytes were either 2025
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Figure 2. DLS measurements of SPIONs in water and astrocyte medium. The results are presented as (A) Z-average and (B) intensity mean distributions, indicating that the SPIONs form agglomerates in culture medium. Measurements were performed in triplicate with 10−100 automated runs. Bars are the mean, with error bars representing the standard deviation.
Table 1. Summary of the Hydrodynamic Diameter, Polydispersity Index, and Zeta-Potential Measurements of SPIONs Suspended in Water and Astrocyte Medium astrocyte mediuma
water FluidMAG-Amine (50 nm) FluidMAG-Amine (100 nm) FluidMAG-D (50 nm) a
dDLS (nm)
PDI
zeta potential (mV)
dDLS (nm)
PDI
zeta potential (mV)
79 ± 0 83 ± 1 55 ± 5
0.23 0.15 0.28
24.3 ± 2.4 34.5 ± 0.7 2.9 ± 0.1
78 ± 1 99 ± 1 28 ± 0
0.34 0.33 0.32
−7.8 ± 0.5 −6.3 ± 0.3 −5.4 ± 0.5
The zeta potential of the astrocyte medium is −5.6 ± 1.2 mV.
exposed to an AMF of 10.82 kA/m magnitude for 1 h or were placed in the cell culture incubator without being exposed to the AMF. Immediately after heating astrocytes, cells were fixed with a 4% paraformaldehyde solution (Sigma, St. Louis, MO) for 30 min and subsequently washed three times with PBS. Astrocytes were then exposed to a 1% solution of osmium tetroxide (Sigma, St. Louis, MO) for 1 h, followed by a serial dehydration of the sample with 50, 60, 70, 80, 90, and 100% ethanol (v/v). Astrocytes were then CO2 critical point dried (Tousimis, Rockville, MD), sputter-coated with platinum (Denton Desk IV, Moorestown, NJ) and imaged with a Carl Zeiss Supra55 SEM (Thornwood, NY). 2.8. Statistics. Adhesion, viability, and astrocyte culture heating experiments were performed in triplicate, with each replicate containing astrocytes isolated from three different rats (n = 3). However, when the magnetic field was lowered to 7.31 kA/m to determine how AMF field strength altered cell viability, only one trial was carried out. Since the results were comparable to previous experiments, additional replicates were not carried out. JMP statistical software (SAS, Cary, NC) was used for all statistical analysis. A oneway ANOVA was used to compare live and dead cell counts data between different experimental and control groups. Subsequently, a posthoc Tukey−Kramer HSD was used to determine differences between groups. Results were considered statistically significant if the statistical test resulted in p < 0.05.
100 particles. Analysis of TEM images of FluidMAG-Amine particles revealed the mean single-particle diameter to be 9.4 ± 2.7 nm (Figure 1A,C), and the FluidMAG-D mean singleparticle diameter was found to be 9.3 ± 2.2 nm (Figure 1B,D). These values are in agreement with the manufacturer’s specifications of 10 nm particle diameters. The agglomeration diameter of SPIONs in water or astrocyte medium was determined using DLS. The Z-average diameter (Figure 2A) and the intensity mean distribution (Figure 2B) revealed SPION agglomerates. In the intensity mean distribution, we observed that there was a unimodal distribution of hydrodynamic diameters for all SPIONs in water, but there was a bimodal distribution of hydrodynamic diameters for all SPIONs in astrocyte medium. In water, the Z-average agglomeration diameter of SPIONs was 79 ± 0 nm (FluidMAG-Amine, 50 nm), 83 ± 1 nm (FluidMAG-Amine, 100 nm), or 55 ± 5 nm (FluidMAG-D, 50 nm). In astrocyte medium, the Z-average agglomeration diameter of the particles was 78 ± 1 nm (FluidMAG-Amine, 50 nm), 99 ± 1 nm (FluidMAG-Amine, 100 nm), or 28 ± 0 nm (FluidMAG-D, 50 nm). The intensity mean distributions for FluidMAG-Amine SPIONs in astrocyte medium (50 and 100 nm, Figure 2B) revealed one peak centered around 10 nm and another peak centered around 100 nm. For both sizes of FluidMAG-Amine SPIONs, the peak centered around 10 nm had a much smaller intensity than the peak centered around 100 nm. Additionally, the 100 nm peak of FluidMAG-Amine SPIONs in astrocyte medium was always shifted toward a larger diameter than a similar peak observed around 100 nm for SPIONs in water. The bimodal distribution observed in the intensity mean distribution of FluidMAG-D SPIONs in astrocyte medium indicates the coexistence of small and large agglomerates in
3. RESULTS 3.1. Characterization of SPIONs in Different Aqueous Media. SPIONs agglomerate within different aqueous media,12,13 and the agglomeration affects the heat generation rate. Agglomeration may also influence the ability of cells to uptake SPIONs. Due to agglomeration influencing heat generation and cellular uptake, it was important to determine agglomeration size in the presence of astrocyte media. Individual SPION diameters were determined using TEM for 2026
dx.doi.org/10.1021/tx500231f | Chem. Res. Toxicol. 2014, 27, 2023−2035
Chemical Research in Toxicology
Article
astrocyte medium. The two peaks in the intensity mean distribution for FluidMAG-D SPIONs had similar intensities, which likely led to a decrease in Z-average agglomerate diameter in astrocyte culture media (Figure 2A). The polydispersity indexes (PDI) of the samples suspended in water were low,
