Effect of magnetic nanoparticle heating on cortical neuron viability

Abstract

Purpose: Superparamagnetic iron oxide nanoparticles are currently approved for use as an adjunctive treatment to glioblastoma multiforme radiotherapy. Radio frequency stimulation of the nanoparticles generates localised hyperthermia, which sensitises the tumour to the effects of radiotherapy. Clinical trials reported thus far are promising, with an increase in patient survival rate; however, what are left unaddressed are the implications of this technology on the surrounding healthy tissue.

Methods and materials: Aminosilane-coated iron oxide nanoparticles suspended in culture medium were applied to chick embryonic cortical neuron cultures. Cultures were heated to 37 °C or 45 °C by an induction coil system for 2 h. The latter regime emulates the therapeutic conditions of the adjunctive therapy. Cellular viability and neurite retraction was quantified 24 h after exposure to the hyperthermic events.

Results: The hyperthermic load inflicted little damage to the neuron cultures, as determined by calcein-AM, propidium iodide, and alamarBlue® assays. Fluorescence imaging was used to assess the extent of neurite retraction which was found to be negligible.

Conclusions: Retention of chick, embryonic cortical neuron viability was confirmed under the thermal conditions produced by radiofrequency stimulation of iron oxide nanoparticles. While these results are not directly applicable to clinical applications of hyperthermia, the thermotolerance of chick embryonic cortical neurons is promising and calls for further studies employing human cultures of neurons and glial cells.

• Aminosilane
• cortical neurons
• iron oxide nanoparticles
• superparamagnetic

Introduction

Adjunctive, hyperthermic treatment of cancer employing superparamagnetic iron oxide nanoparticles (IONP) is gaining slow but sure acceptance [1]. In this therapeutic approach, IONP are first injected into the tumour and then exposed to an alternating magnetic field applied remotely, which produces a controllable rise in tumour temperature [2]. Hyperthermic cancer cells are increasingly sensitive to subsequent radiation or chemotherapy treatments, improving therapeutic efficacy and lowering the requisite dosage [3,4]. This is especially beneficial as chemo- and radiotherapies are highly toxic, and lowering the overall dosage, especially in the case of central nervous system (CNS) tumours, is of critical importance as they are shown to induce post-chemotherapy cognitive impairment, or ‘chemo brain’ [5]. However, heating in itself can induce physiological changes as well [6,7]. Thus, a balance must exist between increasing chemo- and radiotherapy efficacy while minimising perturbations associated with hyperthermia.

Induction heating of particulate iron oxide is reported as early as 1957, where Gilchrist et al. loaded lymph nodes with micron-sized particles to thermally ablate residual cancerous tissue after tumour resection [8]. Since then, IONP heating technologies matured on many facets through advanced particle fabrication and characterisation [9,10] and modelling of heat generation [11–14]. Recently, MagForce (Berlin, Germany) received European Union (EU) approval for electromagnetic stimulation of aminosilane-coated IONPs for glioblastoma multiforme therapy [15]. Clinical trials to validate IONP-based hyperthermia for enhancement of chemo- and radiotherapies in other forms of cancer are underway in the USA and EU [16–19]. However, what are often left unaddressed are the implications of this technology on the surrounding healthy tissue, especially in scenarios where IONP are applied to the CNS.

To the best of our knowledge, the viability of healthy neurons exposed to this type of treatment has not been reported yet, although understanding neurons’ thermo-tolerance is important to further optimisation of this method of treatment. As a starting point for addressing this issue we investigate here the thermotolerance of chick embryonic cortical neurons. Previously, we found that primary cortical neurons are susceptible to the effects of superparamagnetic IONP in vitro [20]. Specifically, IONP-induced cytotoxicity was surface coating dependent. Polydimethylamine-coated particles displayed rapid removal of the plasma membrane. Aminosilane-coated nanoparticles were relatively innocuous even at relatively high concentrations, in agreement with previous reports [21,22]. Building on these results, the present study investigates the thermo-tolerance of primary cortical neurons when subjected to heating via alternating magnetic field stimulation in the presence of aminosilane-coated IONP, which are currently employed in treatment of glioblastoma multiforme [15,23].

Materials and methods

Culture heating systems

An induction heating system (HFI 3-135/400, RDO Induction, Washington, NJ) was used with a 4.0 cm × 4.3 cm diameter coil to generate two distinct culture temperatures of 37 °C and 45 °C by using a current of 79.59 A (at 16% power output [PWR]) and 139.2 A (at 35% PRW) respectively, at 220 kHz. A schematic of the system is shown in Figure 1(A). Flux 2D simulation of the magnetic field distribution generated by the coil at these settings is shown in Figure 1(B). The culture chamber containing the IONP suspension, as shown in Figure 1(A), was placed in the region of the coil that produces the most uniform magnetic field as demarcated by the black rectangle in Figure 1(B). Water bath heating samples (used as a control heating system) were immersed 1 cm below the water surface to facilitate conduction-based heating. The sample temperatures for both heating systems were monitored in real time using a LumaSense fibre-optic probe with TrueTemp software (LumaSense Technologies, Santa Clara, CA) for temperature reading. Typically, the water bath achieved 45 °C within 2 min, whereas the induction coil system required between 5–15 min to arrive at the two temperature set points and remained stable thereafter as shown in Figure 1(C).

Figure 1. Heating of aminosilane-coated iron oxide nanoparticles by an alternating electromagnetic field. (A) Schematic of the induction coil system with cell culture chamber and fibre-optic probe for real-time temperature recording. (B) Flux 2D simulation of the magnetic field generated by the induction coil with 79.59A (16% PWR) and 139.2A (35% PWR) applied current (to produce 37 °C and 45 °C, respectively). The cell culture chamber is placed in the area of the coil that generates the most uniform magnetic field as demarcated by the black contour rectangle. (C) Example of sample temperature reading as a function of time. Each point is the average of three samples and the error bars indicate one standard deviation. For comparison, temperature data for water bath heating (45.5 °C bath) is also included.

Cell culture conditions

Primary cortical neurons were isolated from the frontal lobes of embryonic day 8 chicks in accordance to the procedure described in [20]. Neurons were seeded at a density of 750 000 cells per mL in each well of a 12-well plate onto piranha-cleaned coverslips coated with 10 μg/mL of laminin and 10 μg/mL poly-L-lysine in double distilled H₂O. Phenol red-free Neurobasal culture medium containing 2% B-27 supplement, 1% penicillin/streptomycin, and 0.5 mM L-glutamine was exchanged 24 h after the initial plating and then every 2 days for maintenance thereafter. Cultures used for the study were grown for 4 or 5 days in a standard incubating environment (i.e. 37 °C, 5% CO₂). Cultures from day 4 or day 5 were statistically similar in cell number and metabolic activity (data not shown). After this the media was exchanged for the IONP solution – 10% (v/v) aminosilane-coated particles (fluidMAG-Amine 50 mg/mL, 50 nm dia. from Chemicell, Berlin) in culture medium. Extensive characterisation of these particles, including magnetic core and hydrodynamic size as well as zeta potential in Neurobasal culture media was reported previously [20] and will not be discussed here.

Each sample was then incubated for 24 h prior to exposure to the prescribed heating conditions (i.e. coil, bath, or incubator) stated in the ‘Culture heating systems’ section above. The exposure to the prescribed heating regime (i.e. temperature and duration) was performed without rinsing excess IONP particles (particles not internalised by or attached to cell membranes as seen in Figure 7B in Rivet et al. [20]) from the suspension media, which tended to accumulate toward the bottom part of the culture chamber. Following heating, each sample was returned to an incubator for 24 h. The IONP rich media was then removed and analyses were performed.

Viability analysis

Each sample was imaged for live and dead cells using calcein-AM and propidium iodide, respectively, 24 h after exposure to heating. The IONP solution was removed and a 2 μg/mL solution of propidium iodide in PBS was added to each sample and incubated at 37 °C for 30 min. The solution was then replaced with a 2 μg/mL solution of calcein-AM in PBS and incubated for 15 min at 37 °C. This solution was removed and the sample was rinsed three times with PBS before imaging. Each sample was imaged at three random locations with a 20× objective lens. Image analysis was performed with ImageJ software to obtain the percentage area value of cells. This value was then normalised to the control sample (without particles).

Metabolic activity analysis

Metabolic activity was also assessed 24 h after exposure to the experimental conditions. For this purpose, the IONP solution was removed and each sample was washed three times with PBS. A 10% (v/v) solution of alamarBlue® in culture medium was then applied to each sample and incubated at 37 °C for 4 h. Upon completion a magnet was applied to each sample in the transfer process to sequester residual IONP. Duplicate 100-μL samples of each solution were then transferred to a 96-well plate for absorbance measurement (590 nm). Results from each sample were normalised to those obtained from the control sample without IONP.

Immunocytochemistry

The cultures used for alamarBlue® assay were fixed using a 37 °C, 4% paraformaldehyde solution in PBS immediately after collection of the metabolic activity sample. Immunocytochemistry for neurofilament was performed using an RT97 primary (Developmental Studies Hybridoma Bank, Iowa, 1:500) and Alexafluor 594 donkey anti-mouse (Invitrogen, Grand Island, NY, 1:1000) secondary. Staining procedures were performed as described previously [20]. All samples were imaged using a 40× objective lens at two random locations. The extent of neurofilament expression was quantified using the same method as employed for calcein-AM analysis.

Statistical analysis

All experiments were repeated in triplicate to produce three independent samples. Each calcein-AM/propidum iodide sample was imaged at three random locations (n = 9), and the alamarBlue® and neurofilament samples were measured in duplicate (n = 6). Error bars denote standard deviation. Statistical analysis was performed using Jmp software (SAS). The calcein-AM, propidium iodide, alamarBlue® and neurofilament results were evaluated using a one-way ANOVA with a Tukey-HSD test to compare all pairs of means. A p value of less than 0.05 was considered statistically significant.

Results

Figures 2(A–D) show live and dead cells stained with calcein-AM (green) and propidium iodide (red), respectively, 24 h after heating. Control samples (with and without IONP) were kept in an incubator at 37 °C and not exposed to the magnetic field or water bath. The percentage of area covered by live cells (calcein-AM) and dead nuclei (propidium iodide) was quantified by first converting each image to a binary format and subsequently measuring the percentage of area covered by both signals using ImageJ. These results are shown in Figures 2(E–F). All values are represented as a percentage of the incubator control without IONP. The percentage of area covered by live cells was statistically similar for all the groups with IONP, but significantly less than the control without IONP.

Figure 2. Viability assessment of primary cortical neurons exposed to heat generated by iron oxide nanoparticles in an alternating electromagnetic field or a water bath. Images of live neurons by calcein-AM (green) and dead nuclei by propidium iodide (red) 24 h after exposure to: control without nanoparticles (37 °C inc. w/o P) (A), and with nanoparticles (37 °C inc. w/P) stored in the incubator (B), heating to 45 °C by water bath immersion (45 °C bath) (C), and by magnetic stimulation (45 °C coil) for 2 h (D), both in the presence of nanoparticles. Quantification of the percentage area covered, of each image relative to the control for live neurons (E) and dead nuclei (F). For comparison, these graphs also show results for cells with nanoparticles heated to 37 °C for 2 h in a water bath (37 °C bath) and a magnetic field (37 °C coil). All samples with nanoparticles were significantly reduced in the percentage area covered as compared to the control, whereas only the 45 °C coil sample displayed a statistically significant increase in the number of dead nuclei (*p < 0.05 relative to control without particles). Error bars represent one standard deviation, n = 9 for all samples. All images are captured with a 20× objective lens. Scale bar represents 50 μm. For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article at www.informahealthcare.com.

The number of dead cells was significantly increased at the 2-h time point in the 45 °C coil heating group as compared to the control without IONP, warranting further investigation by use of a metabolic activity assay, alamarBlue®. For these experiments the samples were heated for three durations (0.5, 1.0, or 2.0 h). It was found that the metabolic activity was not significantly altered as a function of temperature or time, as shown in Figure 3. The extent of neurite outgrowth, and the potential for retraction thereof based on induction heating stimulation, were also assessed as described next. Samples that were used to generate the metabolic activity results were fixed and stained using the RT97 neurofilament antibody 24 h after exposure to the prescribed heating dosages (37 °C or 45 °C for 0.5, 1.0, and 2.0 h). Nuclei were counterstained with DAPI. Each sample was imaged at random locations using a 40× objective lens, Figures 4(A–D). The percentage area covered by neurofilament signal is shown in Figure 4(E). All samples were found to express statistically similar quantities of neurofilament as compared to the control without IONP.

Figure 3. Metabolic activity of cortical neurons exposed to hyperthermic conditions as determined by the alamarBlue® assay. Samples were analysed 24 h after exposure to 0.5 h, 1 h and 2 h heating at 37 ° or 45 °C in an alternating magnetic field. All samples were measured for absorbance at 570 nm using a multi-well plate reader. All values are expressed as a percentage of the control; error bars represent one standard deviation. All samples are statistically similar, indicating no influence of heating duration or temperature on the neuronal metabolic processes (n = 6).

Figure 4. Fluorescent images of cortical neurons stained for neurofilament (green, RT97) and nuclei (blue, DAPI) 24 h after exposure to the corresponding heating dosage. (A) Control without (37 °C inc. w/o P) and (B) with (37 °C inc. w/P) nanoparticles and cultures heated by magnetic stimulation for 2 h at (C) 37 °C (37 °C coil 2.0 h), and (D) 45 °C (45 °C coil 2.0 h). Each image is a composite of four images stitched together. All images were captured using a 40× objective lens. Scale bar represents 50 μm. (E) Neurofilament percentage area for cultures shown in A–B and other cultures heated to 37 °C or 45 °C for 0.5 h and 1 h. All images are statistically similar (n = 6, error bar represents one standard deviation), indicating no reduction in neurite processes as a function of time and temperature of heating process. For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article at www.informahealthcare.com.

Discussion

Clinical trials of adjunctive hyperthermia to radiotherapy display an increase in patient survival rates when glioblastoma are heated to 43 °C [24]. However, traditional, clinical heating modalities are not favourable for a repetitious hyperthermia strategy, as they require implantation prior to each use. Injection of IONP reduces implantation-associated complications and remote, induction-based stimulation minimises the impact on healthy tissue in the line of application [25].

This study explores the response of chick embryonic cortical neurons to magnetic field stimulation in the presence of aminosilane-coated IONPs. While this cell type is not directly relevant to application of hyperthermia in clinical settings, it provides a starting point for understanding the thermotolerance of neurons. A hyperthermic state of 45 °C was chosen based on the findings of Jordan et al. [23], where it was found to most effectively increase animal survival rates. This temperature falls within the moderate hyperthermia definition (41 °C to 46 °C) by Kumar et al. [26]. The prescribed temperature regimes (37 °C or 45 °C), produced by either magnetic field or water bath immersion, do not negatively influence cellular viability when compared to control samples incubated with IONP as seen from Figure 2(E). Cellular viability appears to be significantly reduced only by the addition of particles, as all groups with particles are approximately 65% of the control without particles. This reduction, however, could also be partially attributed to physical impediment of the signal as some IONP residue is always deposited on the culture surface, as seen from scanning electron microscopy images reported previously [20]. The layer of IONP is visible to the naked eye, is impossible to completely remove by multiple rinsing and may contribute to the observed reduction in fluorescent signal (compared to the control) presented in Figure 2(E). This issue is difficult to avoid and is expected to be encountered in any cell-counting method based on measurement of fluorescent signal.

In Figure 2(F) there is a statistically significant increase in dead nuclei as visualised by propidium iodide in the 45 °C coil group. This observed increase in dead cells prompted a follow-up study of metabolic activity as a function of heating duration. The results, shown in Figure 3, indicate there is no difference in metabolic activities among all groups, which appear to support the conclusion from live cell studies (Figure 2E), namely that heating regimes have minimal effect on these cultures. Moreover, minimal difference appears in metabolic activity between control samples with and without nanoparticles, which is in contrast to 65% reduction observed for calcein-AM-stained group. A possible explanation for this apparent discrepancy is the resolution of the metabolic assay, which is known to depend strongly on the cell number and could be somewhat limited at the high cell density used in these studies [27], especially if the changes in metabolic rate are subtle. Cell seeding was carried out with 750 000 cells per 22 mm diameter well, which was optimised to produce robust cultures and not necessarily the highest sensitivity of alamarBlue® assay. Despite all these potential shortcomings, from both cell survival and metabolic assay studies it is clear there is no catastrophic death event in any of the cortical neuron cultures subjected to heating.

As mentioned before, propidium iodine is the only test to indicate a slightly elevated death rate in one group, the 45 °C coil heated culture, as compared to the 37 °C incubated without particles (inc. w/o P) sample. This particular test is believed to be the most sensitive to subtle changes in cultures as it counts individually dead nuclei, rather than cell surface (note that percentage area was still used in reporting propidium iodine results to ensure consistency with other graphs). In contrast, small changes are more difficult to assess based on surface area since cortical neurons form multicellular, three-dimensional aggregates. However, although there is a statistically significant increase in the number of dead cells in the 45 °C coil group, the damage is not to such an extent to say that cytotoxic conditions are present. The absence of a large reduction in viability may be counterintuitive and could apparently contradict other literature reports, where cytotoxicity is shown to occur at temperatures as low as 42 °C [28]. However, it should be noted that the neuronal type may play a critical role in thermotolerance, and meaningful comparisons are not possible at this point as they could only be made between identical cells. In addition, developmental stage may also be a factor of importance.

It is well known that hyperthermia affects the cytoskeleton in various ways (e.g. blebbing, flattening, retraction) [29], and thus its influence on cytoskeletal filaments was assessed. The images are quantified for percentage area covered by fluorescent signal and the results normalised to the control sample (without IONP) are shown in Figure 4. Inspecting this figure, it can be concluded there is negligible difference among all cell groups in the area covered by neurites. For instance, neuron cultures without particles kept at 37 °C (Figure 4A) are not significantly different from cultures with particles (Figure 4B) when placed in the electromagnetic field for 2 h at either 37 °C (Figure 4C) or 45 °C (Figure 4D). Hence, retraction of neurites does not appear to occur 24 h after exposure to the various heating dosages. However, these results should be interpreted with more caution, as neurite formations are inherently three-dimensional and the analysis carried out here is two-dimensional. A more detailed analysis of neurite degeneration, such as that conducted by Ravikumar et al. [30], although not within the scope of this paper, may provide more information on the influence of heating by IONP on the cytoskeletal network.

Conclusion

Gaining insight into the neuronal response to heating by IONP in vivo is not trivial given the number of variables involved. However, identifying potential implications of this technology via in vitro studies may prove useful, especially given difficulties associated with CNS variability and complexity. Thus, using chick embryonic primary cortical neuron cultures that are relatively pure and absent of other cell types provides a window into the effects on IONP-based hyperthermia on neuronal health. The live cell and metabolic rate studies carried out here suggest these cells possess high thermo-tolerance in the presence of substantial heat. While the results cannot be extrapolated to human brain cells, they are promising and call for further studies of thermo-tolerance of cell types directly relevant to clinical applications of hyperthermia. Further examination of the molecular pathways activated during hyperthermia treatment will provide greater insight into the neuronal response while increasing the applications of IONP-based hyperthermia for therapeutic, diagnostic, and investigative studies.

Declaration of interest

This work was supported by the US National Science Foundation under awards CBET 0846433 to D.-A. B.-T., and CAREER: BMAT 1150125 to R.J.G. The National Institutes of Health, NINDS, provided additional support, R21NS62392, to R.J.G. The RT97 monoclonal antibody developed by J. Wood was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. The authors alone are responsible for the content and writing of the paper.