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BY-NC-ND 3.0 license Open Access Published by De Gruyter March 29, 2014

Targeted hyperthermia-induced cancer cell death by superparamagnetic iron oxide nanoparticles conjugated to luteinizing hormone-releasing hormone

  • Faruq Mohammad

    Faruq Mohammad obtained his PhD in Environmental Toxicology from Southern University and A&M College (Baton Rouge, LA, USA) in May 2011. Prior to this, in 2005, Mohammad obtained his Master’s degree in Synthetic Organic Chemistry from Acharya Nagarjuna University (Namburu, India). He is currently working as a Postdoctoral Researcher in the Pharmacy Department at the North-West University (Potchefstroom campus; South Africa). His area of research is in the development of polymeric nano drug delivery systems for malaria and cancer in addition to understanding the toxicity of nanomaterials. He has several publications in these areas.

    , Achuthan C. Raghavamenon

    Achuthan C. Raghavamenon is an Assistant Professor at the Amala Cancer Research Center in Thrissur (India). He is a recognized research advisor by the University of Calicut (Malappuram, India) and Mahathma Gandhi University (Kottayam, India). After obtaining his PhD in Biochemistry from Mahathma Gandhi University, Raghavamenon performed postdoctoral research in the areas of oxidative biology and cell signaling at the LSU Health Science Center (New Orleans, LA, USA), the Ohio State University Medical Centre (Columbus, OH, USA), and the Southern University Environmental Toxicology PhD program (Baton Rouge, LA, USA). He has numerous peer-reviewed publications to his credit, and his current research interest is in the drug development for various degenerative diseases.

    , Michelle O. Claville

    Michelle O. Fletcher Claville is the Assistant Dean for the School of Science and an Associate Professor of Chemistry at Hampton University. She received a PhD in Chemistry, BS in Chemistry, and BA in English from the University of Florida, Gainesville, FL, USA. Formerly, she served as an Associate Professor and Chair in the Department of Chemistry at Southern University and A&M College, Baton Rouge, LA, USA. Claville is a recipient of the Faculty Early Development Career (CAREER, 2009) Award and the Achieving Competitive Excellence (ACE) Implementation Award (2012), both funded by the National Science Foundation. Claville has mentored scores of undergraduate students through physical organic chemistry research on the reactive intermediates derived from biomolecules and nanomaterials.

    , Challa S.S.R. Kumar

    Challa S.S.R. Kumar is the Director of Nanofabrication and Nanomaterials at the Center for Advanced Microstructures and Devices at the Louisiana State University in Baton Rouge and is also associated with the DOE-supported Energy Frontier Research Center (EFRC) – Center for Atomic-Level Catalyst Design. He is a winner of the 2006 Nano 50 Technology Award for his work on magnetic-based nanoparticles for cancer imaging and treatment. His research interests are in developing novel synthetic methods, including those based on microfluidic reactors for multifunctional nanomaterials. He has 8 years of industrial R&D experience working for Imperial Chemical Industries and United Breweries. He is the Editor of two online series on Nanotechnologies for the Life Sciences (NtLS) and Nanomaterials for the Life Sciences (NmLS) and a book series on the characterization of nanomaterials. He is also currently the Editor-in-Chief of the journal Nanotechnology Reviews and founding editor of the Journal of Biomedical Nanotechnology. Numerous books and original research papers are part of his extensive publication record.

    and Rao M. Uppu

    Rao M. Uppu is a Professor and Director of the Environmental Toxicology PhD Program at Southern University at Baton Rouge (SUBR). He obtained his PhD in Biochemistry in 1988 and carried out his postdoctoral research at the Eppley Institute for Research in Cancer and Allied Diseases (Omaha, NE, USA) and the Biodynamics Institute at Louisiana State University (Baton Rouge, LA, USA). His research interests include biological reactive intermediates, cell signalling by “ozone-specific” oxysterols and biomedical applications of core/shell nanoparticles. Uppu has mentored numerous graduate students (MS and PhD) and postdoctoral fellows and has published over 60 articles in peer-reviewed journals and book series. He is an elected Fellow of the Academy of Toxicological Sciences (Reston, VA, USA) and has been honored with numerous awards including the University-wide Outstanding Research Investigator Award (2007), Telugu Association of North America Excellence in Science Award (2011), and SUBR Chancellor’s Award for Excellence in Teaching (2013).

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From the journal Nanotechnology Reviews

Abstract

The hyperthermia-induced cytotoxicity of gold-coated SPIONs conjugated to LHRH (SPIONs@Au-LHRH) has been studied in LHRH-receptor overexpressing murine GT1-7 hypothalamic neurons (noncancerous) and human LNCaP cells (cancerous). In the absence of an external magnetic field, SPIONs@Au-LHRH were least cytotoxic to either cell type. When cells were pretreated with SPIONs@Au-LHRH and then exposed to a magnetic field (465 Oe for 15 or 2×15 min), both cell types showed marked decreases in viability and proliferation. The cell death in GT1-7 neurons was found to be late apoptosis or early necrosis, while necrosis was prominent in LNCaP cells. The LNCaP cells exposed to the magnetic field for 15 min showed a significant drop in the mitochondrial transmembrane potential; however, no such change was evident in GT1-7 neurons for the first 15 min. The cell death in LNCaP cells was found to be mediated through the caspase-3-dependent pathway. There was an increased expression of heat shock protein 70 in GT1-7 neurons, and no such increase was seen in LNCaP cells. It is suggested that noncancerous GT1-7 neurons are more resistant to the heat-induced cytotoxicity of SPIONs@Au-LHRH than are LNCaP cells due to increased expression of HSP70. The results show promise toward the selective tumoricidal actions of targeted magnetic nanoparticles.

1 Introduction

In recent years, research on hyperthermia-based treatment of malignant tumors has gained much attention due to the specific use of magnetic nanoparticles (MNPs) for heat release applications. For centuries, certain Egyptian and Indian cultures have utilized hyperthermia for treating cancer and other diseases [1]. In current medical research, however, “magnetic fluid hyperthermia”, a form of hyperthermia, which utilizes magnetic materials, has gained prominence, mainly due to the advent of technologies that allowed optimization of heat release through external magnetic fields and monitoring of prognosis during the course of treatment [2]. The ever-increasing availability of advanced instrumental facilities has also contributed to the successful applications of MNPs not only as therapeutic agents but also as better contrast agents for early detection of cancers.

Several factors make MNP-based hyperthermia a promising approach, including: (i) a direct injection of MNPs into the tumor bed to accomplish localized heat release, (ii) selective enrichment of MNPs in tumor cells through use of tumor-specific binding agents, (iii) easy passage of MNPs through various biological membranes and barriers, (iv) control of heat release by external means, and (v) likelihood of antitumor immunity that generally develops against heat-denatured cellular proteins [2–6]. It has been reported that hyperthermia-based therapies are far more superior when applied in combination with chemotherapy and/or radiation. Some of the chemotherapeutic agents tested for such applications include hydroxycamptothecin [7], 5-fluorouracil [8], and docetaxel [9]. In addition to combined use of MNPs with anticancer agents, a number of superparamagnetic materials have also been tested for their hyperthermia-based therapy, hyperthermia-based controlled drug delivery, or a combination of both [3].

Luteinizing hormone-releasing hormone (LHRH) is a neurohormone produced by the hypothalamus and several extrapituitary tissues, normal as well as tumoral, where it serves an autocrine and/or paracrine function [10–14]. Most cancers of the reproductive organs such as the breast, ovaries, and the prostate overexpress the receptors for LHRH, and accordingly, delivery of contrast or therapeutic agents through LHRH tagging has become a favored approach for targeted delivery [15, 16]. Leuschner and coworkers have recently shown that superparamagnetic iron oxide nanoparticles (SPIONs; Fe3O4) bound to LHRH accumulate in metastatic tumor cells up to 72 pg/cell, and this could provide an easy means of identification by magnetic resonance imaging (MRI) [17]. Based on the Leuschner’s study, which showed a practical application of LHRH-SPIONs for targeted delivery, we have reasoned that MNPs bound to LHRH would also serve as ideal candidates for targeted hyperthermia when applied in conjunction with external magnetic fields. Toward realizing this goal, we have previously reported a synthetic strategy to obtain gold-coated SPIONs (SPIONs@Au) and subsequently bound them to LHRH through cysteamine crosslinker [18]. The SPIONs@Au-LHRH thus synthesized were found to have a mean diameter of 6.7±0.8 nm with saturation magnetization (Ms) of 32 emu/g (at 27°C) and a blocking temperature (TB) of ∼300 K [18, 19].

In this paper, we have studied the cytotoxic effects of SPIONs@Au-LHRH in LHRH-receptor overexpressing cancerous and noncancerous cells under the influence of a low-frequency oscillating magnetic field. We chose to study the normal cells to reflect on how SPIONs@Au-LHRH-induced hyperthermia could affect normal tissues or organs, particularly those that express receptors for LHRH. Also, the exposure of normal cells to SPIONs@Au-LHRH represents a more likely scenario in almost all cases of diagnosis-based imaging as well as hyperthermia-mediated cancer therapy. The studies were performed by exposing cell cultures to SPIONs@Au-LHRH for specific periods of time. Following removal of extracellular SPIONs@Au-LHRH, the cells were subjected to a magnetic field strength (465 Oe) operating at 44 Hz for a period of 15 min each time. These parameters were chosen based on our previous studies in which heat release by SPIONs@Au was studied in water and other solvents at various low oscillatory magnetic fields [19]. The results showed that murine GT1-7 hypothalamic neurons (noncancerous) respond differently from those of human prostate carcinoma cells (LNCaP; cancerous) in terms of cell viability and proliferation, release of heat shock protein 70 (HSP70), decrease in mitochondrial transmembrane potential (ΔΨm), activity of caspases, and eventual cell death by apoptosis or necrosis, all of which indicate a step forward for promising applications of SPIONs@Au-LHRH for therapeutic and/or imaging purposes.

2 Materials and methods

2.1 Chemicals, cell culture supplies, and cells

Murine GT1-7 hypothalamic neurons were a gift from Dr. Pam Mellon, University of California, San Diego, CA, USA. Human LNCaP prostate carcinoma cells and RPMI-1640 medium were purchased from ATCC (Manassas, VA, USA); Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin (stabilized solution containing 10,000 units/ml penicillin and 10 mg/ml streptomycin), phosphate-buffered saline (PBS), trypsin-EDTA (2.5 g trypsin and 0.2 g EDTA-Na4 per liter of HBSS) from Sigma (St. Louis, MO, USA); fetal bovine serum (FBS) from Atlanta Biological (Lawrenceville, GA, USA); acridine orange (AO) and ethidium bromide (EB) from Invitrogen (Eugene, OR, USA); pancaspase inhibitor (PCI) and inhibitors of caspase-3 (C3I), caspase-8 (C8I), and caspase-9 (C9I) from Calbiochem (La Jolla, CA, USA); and tissue culture flasks (T25) and 6-, 24-, and 96-well plates from Corning (Acton, MA, USA). Kits for cell viability (CellTiter-Blue®), mitochondrial ΔΨm (MitoCapture®), and heat shock protein 70 (HSP70) were from Promega (Madison, WI, USA), Calbiochem (San Diego, CA, USA) and Assay Designs (Ann Arbor, MI, USA), respectively.

SPIONs@Au-LHRH were synthesized as described in our previous publications [18, 19]. Briefly, following synthesis of SPIONs of Fe3O4 (average diameter: 5.1 nm) by reduction of Fe(II) acetylacetonate at 210°C using 1,2 hexadecanediol in the presence of surfactants (oleic acid and oleylamine), they were coated with Au in situ through reduction of Au(III) acetylacetonate. The purified SPIONs@Au (average diameter: 6.1 nm) were then linked to LHRH using cysteamine linker.

2.2 Cell culture and maintenance

GT1-7 neurons were maintained in DMEM containing 4 mml-glutamine, 1.5 g/l sodium bicarbonate (pH adjustments), 4.5 g/l glucose, and 10% FBS in 5% CO2/95% humidified air incubator at 37°C. All experiments requiring exposure to SPIONs@Au-LHRH were performed using a medium that contained 2% FBS.

LNCaP cells were maintained in RPMI-1640 containing 2 mml-glutamine, 10 mm HEPES, 1 mm sodium pyruvate, 4.5 g/l glucose, and 1.5 g/l sodium bicarbonate, and 10% FBS in 5% CO2/95% humidified air incubator at 37°C. Similar to neuronal cells, all treatments with SPIONs@Au-LHRH were performed in RPMI-1640 containing 2% FBS.

2.3 Application of AC magnetic field

The in vitro applications of magnetic field were conducted as described by Kim et al (2010) using a custom-made Behlman AC magnetic field setup (Figure 1) [20]. This setup contained a coil with outer and inner diameters of 6.75″ and 0.69″, respectively. The thickness of the coil was 0.5″, and it contained a total of 258 turns. The magnetic field was generated by passage of electric current through the coil. It was found that the instrument so constructed could generate magnetic fields in the frequency range of 44–430 Hz.

Figure 1 A custom-made Behlman AC magnetic field setup used for treating GT1-7 neurons and LNCaP cells in vitro. The instrument was sterilized each time it was used. The culture plates, placed onto the top of copper coil, were exposed to a magnetic field strength of 465 Oe applied at a frequency of 44 Hz for 15 min.
Figure 1

A custom-made Behlman AC magnetic field setup used for treating GT1-7 neurons and LNCaP cells in vitro. The instrument was sterilized each time it was used. The culture plates, placed onto the top of copper coil, were exposed to a magnetic field strength of 465 Oe applied at a frequency of 44 Hz for 15 min.

GT1-7 neurons and LNCaP cells, seeded in six-well plates at 1×106/well, were exposed to SPIONs@Au-LHRH (25–500 μg/ml) for 24 h in DMEM or RPMI-1640 that also contained 2% FBS. At the end of the incubation period, to remove SPIONs@Au-LHRH that were not taken up by cells, the medium was removed, and the cells were washed three times with PBS. Following addition of fresh medium containing 2% FBS, the cells were normalized for 3 h and subjected to a magnetic field strength of 465 Oe operated at 44 Hz frequency for 15 min. Following exposure to the magnetic field, the culture plates were again normalized for 3 or 4 h at 37°C. Thereafter, the cells were isolated and analyzed for viability and proliferation, AO-EB dual staining, mitochondrial ΔΨm, HSP70 expression, and caspase-3 activity. For studies that did not involve exposure to the magnetic field, the cells were analyzed for the above parameters directly after the necessary incubations.

2.4 Measurement of cell viability and proliferation

Cell viability was assessed based on metabolic activity of cells using the CellTiter-Blue® reagent from Promega (Madison, WI, USA). The assay involved reduction of resazurin, a nonfluorescent blue dye, into a pink-colored, red-fluorescent resorufin. GT1-7 neurons (1×106/well; volume 0.5 ml) seeded in six-well plates were exposed to SPIONs@Au-LHRH (25–500 μg/ml) in 2% FBS for 24 h. The cells, with or without prior exposure to the magnetic field as described above, were incubated with the CellTiter-Blue® reagent (250 μl per well) for 3–4 h. The amount of resorufin formed during this time was measured at excitation and emission wavelengths of 560 and 590 nm (respectively) using a Spectramax EM Gemini spectrofluorimeter (Molecular Devices, Sunnyvale, CA, USA). Background fluorescence from wells that contained SPIONs@Au-LHRH in DMEM but no added cells was subtracted. The viability of cells not exposed to SPIONs@Au-LHRH and magnetic field was set at 100%, and the viability in SPIONs@Au-LHRH-treated cultures was expressed relative to the controls. Control cultures that were set up concurrently did not contain either SPIONs@Au-LHRH or subsequent exposure to the magnetic field. Positive controls were established using phorbol myristate acetate (PMA; 10 μg/ml).

Similar to GT1-7 neurons, the cell viability for LNCaP cells was measured following the treatment with SPIONs@Au-LHRH (25–500 μg/ml) in 2% FBS for 24 h.

2.5 Analysis of cell morphology

LNCaP cells and GT1-7 neurons were exposed to 100 and 250 μg/ml SPIONs@Au-LHRH (respectively) for 24 h. Following application of magnetic field (44 Hz for 15 min), the cells were harvested by trypsinization and centrifugation at 300×g and resuspended in a small volume (50 μl each) of the medium containing 10% FBS. An aliquot (4 μl each) of the AO-EB reagent (AO: 100 μg/ml; and EB: 100 μg/ml) in saline was added to cells and visualized under a Nikon Optiphot fluorescence microscope with blue filter. Images were captured using a CoolSNAP camera (Photometrics, Tuscon, AZ, USA) and analyzed using MetaMorph software (Molecular Devices). The cell populations were enumerated for the presence of viable-non-apoptotic (uniformly stained green nucleus), viable-apoptotic (intact cell membrane but condensed chromatin with irregular green nucleus), nonviable-apoptotic (orange nucleus with condensed or fragmented chromatin), and necrotic cells (uniformly stained orange-red colored nucleus) [21, 22].

2.6 Measurement of mitochondrial transmembrane potential

Mitochondrial ΔΨm was determined based on the relative distribution of 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), a cationic dye, in mitochondrial and cytoplasmic fractions (MitoCapture kit) [21]. Briefly, LNCaP cells and GT1-7 neurons seeded in 24-well plates were allowed to grow in the complete medium (10% FBS) up to 80% confluence. The cells were exposed to 100 or 250 μg/ml of SPIONs@Au-LHRH in 2% FBS for 24 h. (The IC50 values of SPIONs@Au-LHRH, determined based on metabolic activity are 100 and 250 μg/ml for LNCaP cells and GT1-7 neurons, respectively). At the end, the medium was removed, and the cells were washed twice with PBS. Following addition of fresh medium, the cells were normalized for 3 h and then subjected to an AC magnetic field operating at 44 Hz for 15 min. Again, after the application of the magnetic field, the plates were incubated at 37°C for 3 h to normalize the cells. Control cultures were set up without being exposed to SPIONs@Au-LHRH and/or the magnetic field. Control cells and cells previously exposed to SPIONs@Au-LHRH (± magnetic field) were incubated with JC-1 for 30 min at 37°C. The unbound dye was removed by washing with PBS-G buffer (provided as part of the kit). A freshly prepared PBS-G (500 μl) was added to each well, and the fluorescence was measured at excitation and emission wavelengths of 488 and 590 nm, respectively, using a Spectramax EM Gemini spectrofluorimeter. The results are expressed as % decrease in ΔΨm (fluorescence) compared to the untreated controls. Cells exposed to rotenone (10 μm) were used as the positive control [21].

2.7 Role of caspase in cell viability

The measurements of cell viability, performed based on resazurin reduction were repeated with additional inclusion of PCI and other caspase inhibitors such as C3I, C8I, and C9I in the culture medium. Briefly, in these experiments, GT1-7 neurons and LNCaP cells were incubated, either individually or in combination, with C3I, C8I, C9I and PCI for 1 h (the final concentration caspase inhibitor(s) was 20 μm in each case). At the end of the incubation period, the cells were washed free of caspase inhibitors, and a fresh medium containing SPIONs@Au-LHRH (0, 100, or 250 μg/ml) was added and incubated for 24 h. After removal of SPIONs@Au-LHRH (that were not taken up by cells) and subsequent exposure to the magnetic field (465 Oe; 15 min), resazurin reduction by viable cells was measured as described earlier.

2.8 Expression of heat shock protein 70

The HSP70 levels in GT1-7 neurons and LNCaP cells were determined using a commercially available sandwich ELISA kit from Assay Designs (Ann Arbor, MI, USA). HSP70 standards (provided by the manufacturer) and lysates of GT1-7 neurons and LNCaP cells (100 μl, each) were added to a 96-well plate precoated with rat monoclonal antibody specific for HSP70. The HSP70 captured by the excess mouse monoclonal antibody was detected using rabbit polyclonal detection antibody (conjugated to horseradish peroxidase, HRP). After removal of the unbound HRP-conjugated rabbit polyclonal antibody, solutions of tetramethylbenzidine and H2O2 were added to the wells. The reaction was terminated by acidification with HCl, and the extent of chromophore formation was quantified at 450 nm using a BioTek ELx800 microplate reader (Winooski, VT, USA). The amount of HSP70 present in the samples was determined by reading against the standard curve (concentration range: 0.2–12.5 ng/ml; r2=0.975). Results are expressed as fold-increase relative to the control, untreated GT1-7 neurons or LNCaP cells.

2.9 Statistical analysis

Data are presented as mean±SD of three to five separate experiments. Statistical analyses were performed using a one-way analysis of variance (ANOVA) and Bonferroni’s method for multiple comparisons. Probability of p<0.05 was considered as statistically significant and p<0.01 as highly statistical significant.

3 Results

3.1 Heat release by SPIONs@Au-LHRH under the influence of AC magnetic field

The heat release by SPIONs@Au-LHRH (1.0 mg), dispersed in DMEM (0.5 ml), was measured in the presence of a magnetic field strength of 465 Oe (frequency: 44 Hz) over a period of 60 min. The extent of heat release as indicated by the increase in temperature of the medium was highest in the first 15 min (Figure 2). An essentially similar type of profile of increase in temperature was observed in our previous studies where SPIONs@Au were exposed to the magnetic field in ethanol, toluene, and water [19]. When matched for field strength, the increase in temperature in DMEM was nearly the same as that observed in water, consistent with the fact that the two solvents have similar viscosities and therefore have similar heat release.

Figure 2 (A) High-resolution transmission electron microscopy (HRTEM) image of SPIONs@Au-LHRH (size=6.7±0.8 nm) and its schematic representation. (B) Heat release by SPIONs@Au-LHRH in DMEM over a period of 60 min when exposed to a magnetic field strength of 465 Oe (frequency: 44 Hz) and inset showing the magnetic hysteresis curve with an Ms of 32 emu/g.
Figure 2

(A) High-resolution transmission electron microscopy (HRTEM) image of SPIONs@Au-LHRH (size=6.7±0.8 nm) and its schematic representation. (B) Heat release by SPIONs@Au-LHRH in DMEM over a period of 60 min when exposed to a magnetic field strength of 465 Oe (frequency: 44 Hz) and inset showing the magnetic hysteresis curve with an Ms of 32 emu/g.

3.2 SPIONs@Au-LHRH are noncytotoxic to LNCaP cells and GT1-7 neurons

The viability of GT1-7 neurons and LNCaP cells was measured following exposure to various concentrations of SPIONs@Au-LHRH (25–500 μg/ml) for 24 h. In the absence of an external magnetic field, neither GT1-7 neurons nor LNCaP cells showed significant changes in cell viability (Figure 3). These results of cytotoxicity are similar to those reported with SPIONs@Au in MCF-7 breast carcinoma cells and H9c2 cardiomyoblasts [19]. The minimal cytotoxicity of SPIONs@Au and SPIONs@Au-LHRH was thought to be a consequence of the presence of a biocompatible gold monolayer, which understandably prevented the Fe3O4 core from coming in direct contact with either the cells or the medium. Also, the gold layer might have suppressed the erosion of Fe3O4 and thereby contributed to little or no increase in the concentration of free Fe(II) or Fe(III) in cells and in the culture medium. While both these processes would have minimized the pro-oxidant effects of the Fe3O4 core or the products derived therefrom, there is probably some added protection due either to LHRH or other ligands such as oleylamine and oleic acid, all of which display some antioxidant properties.

Figure 3 Measurement of cell viability of (A) GT1-7 neurons and (B) LNCaP cells pretreated with SPIONs@Au-LHRH (25–500 μg/ml) for 24 h. The viability was measured based on metabolic activity (reduction of resazurin to resorufin) as described in the Methods section. The viability of cells not exposed to SPIONs@Au-LHRH was set at 100%. Exposure to PMA (10 μg/ml) represented a positive control. Each data point indicates mean±SD of three to five independent observations.
Figure 3

Measurement of cell viability of (A) GT1-7 neurons and (B) LNCaP cells pretreated with SPIONs@Au-LHRH (25–500 μg/ml) for 24 h. The viability was measured based on metabolic activity (reduction of resazurin to resorufin) as described in the Methods section. The viability of cells not exposed to SPIONs@Au-LHRH was set at 100%. Exposure to PMA (10 μg/ml) represented a positive control. Each data point indicates mean±SD of three to five independent observations.

3.3 SPIONs@Au-LHRH exhibit cytotoxicity upon exposure to magnetic field

Figure 4 shows the changes in viability of GT1-7 neurons and LNCaP cells pretreated with SPIONs@Au-LHRH (25–100 μg/ml; 24 h) and subsequently exposed to the magnetic field (465 Oe; 15 min). There were significant decreases in the viability of GT1-7 neurons and LNCaP cells pretreated with SPIONs@Au-LHRH and then exposed to the magnetic field. When matched for SPIONs@Au-LHRH concentration and magnetic field strength, the loss of viability was more pronounced in LNCaP cells than in GT1-7 neurons. For instance, at a SPIONs@Au-LHRH concentration of 100 μg/ml and a magnetic field exposure of 465 Oe, GT1-7 neurons showed a 35% decrease in viability as against 60% loss of viability observed in LNCaP cells under identical conditions. A second time exposure to the magnetic field (465 Oe; 15 min) caused a further loss of viability in GT1-7 neurons by 80% at 500 μg/ml (Figure 5). However, even with this second application of magnetic field, the concentration of SPIONs@Au-LHRH required for 50% decrease in GT1-7 neuronal cell viability was 250 μg/ml. This value is much higher than 100 μg/ml SPIONs@Au-LHRH that was required for 50% decrease in viability of LNCaP cells with a single application of magnetic field (465 Oe; 15 min). In all these cases, the exposure to magnetic field per se (i.e., without SPIONs@Au-LHRH pretreatment) did not decrease in viability of either GT1-7 neurons or LNCaP cells, indicating the safe use of low-frequency magnetic fields for therapeutic purposes.

Figure 4 Changes in viability of (A) GT1-7 neurons and (B) LNCaP cells pretreated with SPIONs@Au-LHRH (25–500 μg/ml) for 24 h and then exposed to a magnetic field strength of 465 Oe for 15 min. The cell viability was measured based on reduction of resazurin to resorufin as described in Figure 3. Data shown are the % change in viability relative to the untreated controls (i.e., without SPIONs@Au-LHRH pretreatment and exposure to the magnetic field). The yields of resazurin in these controls were at 100%. GT1-7 neurons and LNCaP cells treated with PMA (10 μg/ml) were used as positive controls. Data presented are mean±SD of three to five independent experiments (* and ** indicate significance at p<0.05 and p<0.01 versus untreated controls).
Figure 4

Changes in viability of (A) GT1-7 neurons and (B) LNCaP cells pretreated with SPIONs@Au-LHRH (25–500 μg/ml) for 24 h and then exposed to a magnetic field strength of 465 Oe for 15 min. The cell viability was measured based on reduction of resazurin to resorufin as described in Figure 3. Data shown are the % change in viability relative to the untreated controls (i.e., without SPIONs@Au-LHRH pretreatment and exposure to the magnetic field). The yields of resazurin in these controls were at 100%. GT1-7 neurons and LNCaP cells treated with PMA (10 μg/ml) were used as positive controls. Data presented are mean±SD of three to five independent experiments (* and ** indicate significance at p<0.05 and p<0.01 versus untreated controls).

Figure 5 Viability of GT1-7 neurons following the second time exposure to magnetic field. GT1-7 neurons were pretreated with SPIONs@Au-LHRH (25–500 μg/ml) for 24 h and then exposed to a magnetic field strength of 465 Oe (2×15 min). The metabolic activity of cells was measured using a resazurin reagent, as described in the text and legend to Figure 3. Data presented are the change of % viability with respect to the untreated controls (i.e., without SPIONs@Au-LHRH pretreatment and exposure to the magnetic field). The yields of resorufin in these controls were at 100%. Data presented are mean±SD of three to five independent experiments  (* and ** indicate significance at p<0.05 and p<0.01 versus untreated controls).
Figure 5

Viability of GT1-7 neurons following the second time exposure to magnetic field. GT1-7 neurons were pretreated with SPIONs@Au-LHRH (25–500 μg/ml) for 24 h and then exposed to a magnetic field strength of 465 Oe (2×15 min). The metabolic activity of cells was measured using a resazurin reagent, as described in the text and legend to Figure 3. Data presented are the change of % viability with respect to the untreated controls (i.e., without SPIONs@Au-LHRH pretreatment and exposure to the magnetic field). The yields of resorufin in these controls were at 100%. Data presented are mean±SD of three to five independent experiments (* and ** indicate significance at p<0.05 and p<0.01 versus untreated controls).

3.4 Heat stress induced by SPIONs@Au-LHRH causes characteristic changes in cell morphology

To understand the mode of cell death, a detailed analysis of cell morphology was performed following dual staining with AO-EB (Figure 6A–C). The dual staining allowed visualization of (i) live cells, (ii) cells in early apoptosis, (iii) cells in late apoptosis, and finally (iv) cells undergoing necrosis [15]. As shown in Figure 6A, in the case of LNCaP cell-pretreated SPIONs@Au-LHRH (100 μg/ml; 24 h), a single exposure to magnetic field (465 Oe; 1×15 min) caused a marked increase in the population of necrotic cells (82%) as evidenced by the occurrence of cells with uniformly stained orange-red nuclei. In the case of GT1-7 neurons that were pretreated with SPIONs@Au-LHRH (250 μg/ml; 24 h) and then exposed to 465 Oe magnetic field (15 min), we see cells had an orange-red color nucleus with irregular green fluorescence indicative of cells in their apoptotic stage (Figure 6B). About 55% of cells were found to be apoptotic, while the rest confirmed to necrotic cell death. As the healthy normal cells are known to have morphology that after dual staining presents a uniformly stained green nucleus, the observation of cells with irregular green and red fluorescence was thought to indicate the destruction due to heat release by oscillation of SPIONs@Au-LHRH in these cells. Figure 6C shows the changes in morphology of GT1-7 neurons exposed twice to the magnetic field (465 Oe; 2×15 min), i.e., following the pretreatment with SPIONs@Au-LHRH (250 μg/ml; 24 h). The GT1-7 neurons in this case exhibited orange (or dark red)-stained nuclei indicative of necrotic cell death (86%). Also, in these GT1-7 neurons, there was membrane damage as indicated by the leakage of intracellular components (Figure 6C) [21, 22].

Figure 6 AO-EB dual staining of (A) LNCaP cells and (B, C) GT1-7 neurons pretreated with SPIONs@Au-LHRH for 24 h and then exposed to a magnetic field strength of 465 Oe: (A, B) applied at a frequency of 44 Hz for 1x15 min and (C) applied at a frequency of 44 Hz for 2×15 min. The concentrations of SPIONs@Au-LHRH employed in the case of LNCaP cells and GT1-7 neurons were 100 and 250 μg/ml, respectively. Within A–C, representative images of (a) phase contrast microscopy and cells stained with (b) AO and (c) EB are presented along with (d) combined images of b and c (magnification: 400×). The corresponding % apoptotic and necrotic cells in LNCaP cells and GT1-7 neurons are shown in (e).
Figure 6

AO-EB dual staining of (A) LNCaP cells and (B, C) GT1-7 neurons pretreated with SPIONs@Au-LHRH for 24 h and then exposed to a magnetic field strength of 465 Oe: (A, B) applied at a frequency of 44 Hz for 1x15 min and (C) applied at a frequency of 44 Hz for 2×15 min. The concentrations of SPIONs@Au-LHRH employed in the case of LNCaP cells and GT1-7 neurons were 100 and 250 μg/ml, respectively. Within A–C, representative images of (a) phase contrast microscopy and cells stained with (b) AO and (c) EB are presented along with (d) combined images of b and c (magnification: 400×). The corresponding % apoptotic and necrotic cells in LNCaP cells and GT1-7 neurons are shown in (e).

3.5 Mitochondrial mediation of cytotoxicity by SPIONs@Au-LHRH

Figure 7 shows changes in mitochondrial ΔΨm in LNCaP cells and GT1-7 neurons pretreated with 0, 100, or 250 μg/ml SPIONs@Au-LHRH for 24 h and then exposed to magnetic field (465 Oe; 15 min). LNCaP and GT1-7 cells exposed to rotenone (10 μm) were used as positive controls. As can be seen, there was a 70% decrease in mitochondrial ΔΨm in LNCaP cells that were pretreated with SPIONs@Au-LHRH (100 μg/ml) and then exposed to the magnetic field as against the control cells that were neither pretreated with SPIONs@Au-LHRH nor exposed to the magnetic field. Also, cells that were not pretreated with SPIONs@Au-LHRH but exposed to the magnetic field were found to show little or no change in ΔΨm. The decrease in ΔΨm in GT1-7 neurons in response to pretreatment with SPIONs@Au-LHRH (250 μg/ml) and the subsequent exposure to the magnetic field was only about 30% (i.e., compared to the control cells that were neither pretreated with SPIONs@Au-LHRH nor exposed to the magnetic field).

Figure 7 Changes in mitochondrial ΔΨm in GT1-7 neurons and LNCaP cells pretreated with 100 or 250 μg/ml SPIONs@Au-LHRH for 24 h and then exposed to a magnetic field (465 Oe; 15 min). GT1-7 neurons and LNCaP cells that were neither pretreated with SPIONs@Au-LHRH nor exposed to the magnetic field were used as controls, while cells exposed to rotenone (10 μm) served as positive controls. Data shown are the % change in ΔΨm relative to the respective controls. Values presented are mean±SD of three to five independent observations (* and ** indicate significance at p<0.05 and p<0.01 versus untreated controls).
Figure 7

Changes in mitochondrial ΔΨm in GT1-7 neurons and LNCaP cells pretreated with 100 or 250 μg/ml SPIONs@Au-LHRH for 24 h and then exposed to a magnetic field (465 Oe; 15 min). GT1-7 neurons and LNCaP cells that were neither pretreated with SPIONs@Au-LHRH nor exposed to the magnetic field were used as controls, while cells exposed to rotenone (10 μm) served as positive controls. Data shown are the % change in ΔΨm relative to the respective controls. Values presented are mean±SD of three to five independent observations (* and ** indicate significance at p<0.05 and p<0.01 versus untreated controls).

3.6 Caspases play a role in the cytotoxicity induced by SPIONs@Au-LHRH

As shown in Figure 8, C3I and PCI offered significant protection against the cytotoxicity induced by SPIONs@Au-LHRH (100 μg/ml; 24 h) and subsequent exposure to the magnetic field (465 Oe; 15 min) in LNCaP cells. In these cells, C8I and C9I, both individually and together, did not offer significant protection. The observations indicate that under conditions of moderate increase in the heat release, the cell death in LNCaP cells is mediated mainly through a caspase-3-dependent pathway. Unlike the case of LNCaP cells, exposure to SPIONs@Au-LHRH and the magnetic field did not cause significant loss of viability in GT1-7 neurons (Figure 8), an observation that is consistent with the fact that noncancerous cells such as GT1-7 neurons are more resistant to the heat stress or heat-induced cytotoxicity. It was difficult to assess the protective effects of C3I, C8I, C9I, C8I+C9I, and PCI in GT1-7 neurons exposed to SPIONs@Au-LHRH and the magnetic field, as the cell viability itself was not significantly different from that observed with the control, untreated cells. This could be one of the reasons for finding a similar protective effect by caspase inhibitors in the case of GT1-7 neurons (Figure 8).

Figure 8 Effect of caspase inhibitors C3I, C8I, C9I, and PCI on the viability of GT1-7 neurons and LNCaP cells pretreated SPIONs@Au-LHRH and then exposed to a magnetic field (465 Oe; 15 min). Both GT1-7 neurons and LNCaP cells after 1 h preincubation with caspase inhibitors (final concentration: 20 μm) were exposed to 0, 100, or 250 μg/ml SPIONs@Au-LHRH for 24 h. Following the incubation, the cells were exposed to the magnetic field, and the viability of cells were measured based on the reduction of resazurin to resorufin. Cells exposed to PMA (10 μg/ml) were used as positive controls. Values presented indicate % change in viability compared to the respective untreated controls. Data presented are mean±SD of three to five independent experiments (* and ** indicate significance at p<0.05 and p<0.01 versus untreated controls).
Figure 8

Effect of caspase inhibitors C3I, C8I, C9I, and PCI on the viability of GT1-7 neurons and LNCaP cells pretreated SPIONs@Au-LHRH and then exposed to a magnetic field (465 Oe; 15 min). Both GT1-7 neurons and LNCaP cells after 1 h preincubation with caspase inhibitors (final concentration: 20 μm) were exposed to 0, 100, or 250 μg/ml SPIONs@Au-LHRH for 24 h. Following the incubation, the cells were exposed to the magnetic field, and the viability of cells were measured based on the reduction of resazurin to resorufin. Cells exposed to PMA (10 μg/ml) were used as positive controls. Values presented indicate % change in viability compared to the respective untreated controls. Data presented are mean±SD of three to five independent experiments (* and ** indicate significance at p<0.05 and p<0.01 versus untreated controls).

3.7 Expression of HSP70 in LNCaP cells and GT1-7 neurons exposed to SPIONs@Au-LHRH

The results of HSP70 protein expression in GT1-7 neurons and LNCaP cells subjected to heat stress are shown in Figure 9. There was nearly a threefold increase in the expression of HSP70 in GT1-7 neurons pretreated with 250 μg/ml SPIONs@Au-LHRH for 24 h and then exposed to the magnetic field (465 Oe; 15 min). The extent of increase in the expression of HSP70 following the pretreatment with SPIONs@Au-LHRH (100 μg/ml; 24 h) and then exposed to the magnetic field (465 Oe; 15 min) was much smaller in LNCaP cells (Figure 9).

Figure 9 Expression of HSP70 in GT1-7 neurons and LNCaP cells following pretreatment with SPIONs@Au-LHRH and subsequent exposure to the magnetic field (465 Oe; 15 min). GT1-7 neurons and LNCaP cells were pretreated with 100 or 250 μg/ml of SPIONs@Au-LHRH for 24 h and then exposed to the magnetic field. The levels of HSP70 were measured based on a sandwich ELISA as described in the Methods section. The control assays used cells that were not exposed to SPIONs@Au-LHRH and subsequent exposure to the magnetic field. Data shown are the fold increase in the expression of HSP70 relative to the respective control(s). Values presented are mean±SD of three to five independent experiments (* and ** indicate significance at p<0.05 and p<0.01 versus untreated controls).
Figure 9

Expression of HSP70 in GT1-7 neurons and LNCaP cells following pretreatment with SPIONs@Au-LHRH and subsequent exposure to the magnetic field (465 Oe; 15 min). GT1-7 neurons and LNCaP cells were pretreated with 100 or 250 μg/ml of SPIONs@Au-LHRH for 24 h and then exposed to the magnetic field. The levels of HSP70 were measured based on a sandwich ELISA as described in the Methods section. The control assays used cells that were not exposed to SPIONs@Au-LHRH and subsequent exposure to the magnetic field. Data shown are the fold increase in the expression of HSP70 relative to the respective control(s). Values presented are mean±SD of three to five independent experiments (* and ** indicate significance at p<0.05 and p<0.01 versus untreated controls).

4 Discussion

When subjected to an oscillating magnetic field, magnetic nanoparticles (MNPs) such as SPIONs@Au-LHRH have long been anticipated to afford destruction of cancerous but not noncancerous cells. For the first time, the present study, which employed two different immortalized cells, one noncancerous (GT1-7 neurons) and the other cancerous (LNCaP cells) but both overexpressing LHRH receptors, provide experimental proof of this concept [11–13]. In the absence of external magnetic fields, SPIONs@Au-LHRH are least cytotoxic to either cell type (Figure 3), an observation that is consistent with the results of our previous studies, which employed H9c2 cardiomyoblasts and MCF-7 breast carcinoma cells [19]. This type of nontoxic behavior of SPIONs@Au-LHRH, in addition to the observed increase in the temperature of the DMEM only after exposure to the external magnetic field (Figure 2B), confirms the safe use of targeted MNPs for hyperthermia-related applications.

Traditionally, hyperthermia treatment is classified into three different categories, namely, (a) thermo ablation where the temperature goes to 46°C or higher (up to 56°C) causing cells to undergo direct necrosis, (b) moderate hyperthermia (41°C<T<46°C) resulting in activation and/or inactivation of many cellular mechanisms like protein denaturation, protein folding, aggregation, induction and regulation of apoptosis, signal transduction, multidrug resistance and HSP expression, and (c) diathermia, which results in a very moderate raise in temperature (<41°C) as seen in physiotherapy of rheumatic disease and possibly other inflammatory conditions [3]. The results of nuclear morphology, which revealed that LNCaP cells mainly undergo necrotic cell death (Figure 6), suggest the possibility of thermo ablation as an underlying mechanism of cytotoxicity. The fact that GT1-7 neurons are resistant to cell death the first-time around (Figure 4) and only undergo significant cell death upon repeated exposures to oscillatory magnetic fields (Figure 5) suggests that the increase in temperature in these cells may not be as dramatic as in the case of LNCaP cells. The manifestation of apoptotic cell death in GT1-7 neurons, especially during the first 15 min of magnetic field exposure can be taken as an indication of moderate hyperthermia. The mechanism of cell death in these cells changes to necrosis during the second 15 min of magnetic field exposure, presumably as a result of thermo ablation.

Our analysis of HSP70 levels in LNCaP cells and GT1-7 neurons, both of which were pretreated with SPIONS@Au-LHRH and then subjected to magnetic field exposure, indicated the possibility of induction of thermo tolerance mainly in GT1-7 neurons. When the magnetic field exposure was limited to 15 min, we observed a threefold increase in the levels of HSP70 in GT1-7 neurons, but not in LNCaP cells, as against the respective untreated control cells (Figure 9). A number of studies have demonstrated that upregulation of cellular HSPs is part of a defense strategy against heat-induced stress [5, 6, 23–25] and that inhibition of HSP synthesis often is detrimental to cell survival [26]. Animal models of stroke have also indicated that expression of HSP70 significantly reduces the ischemic injury and thereby protects both neuronal and glial cells [25, 27]. Further, in experimental animals that overexpress HSP70, there is generally an improvement in the neurological scores, smaller infarcts, reduced protein aggregation, and decreased nuclear translocation of apoptosis-inducing factor [25].

In principle, the HSP family of proteins offer protection against the heat-induced stress through modulation of protein folding, faster enablement of protein re-naturation, prevention of binding of reactive electrophilic metabolites to crucial cellular proteins and enzymes, and control of cell cycle, growth, and differentiation [25, 28], all of which require active energy metabolism [23]. Exposure to magnetic field for 15 min resulted in a significant decrease in the mitochondrial ΔΨm in LNCaP cells with little or no changes observed in the case of GT1-7 neurons (Figure 7). A relatively high change in ΔΨm in LNCaP cells would mean a more profound loss of mitochondrial function that naturally could have led these cells to necrosis. The studies of caspase inhibitors, especially in LNCaP cells exposed to the first 15 min of magnetic field (Figure 8) do indicate that possible mediation of cell death through mitochondrial (caspase-3-dependent) pathway. In the case of GT1-7 neurons, because of much less change in ΔΨm, the mitochondrial function was presumably less compromised, with the cells undergoing a more orchestrated form of cell death, namely, apoptosis which requires cellular energy.

5 General summary and conclusions

In conclusion, we report a promising SPIONs@Au-LHRH that have suitable physical, chemical, biological, and targeting properties and can be useful for the selective killing of LHRH-receptor-expressing cancer cells through hyperthermia. We believe this is the first report of hyperthermia-mediated cell death tested using LHRH-SPIONs under the influence of oscillating magnetic fields with two different immortalized cells, one noncancerous (GT1-7 neurons) and the other cancerous (LNCaP cells) but both expressing LHRH receptors. The overall objective of this study was to examine the possible thermo tolerance in LHRH-receptor-expressing noncancerous as against cancerous cells in response to the heat stress induced by LHRH-SPIONs. Although we have very limited information on the differential expression of LHRH receptors between the two cell types, the differences in the extent of cell death that typically followed the pretreatment with LHRH-SPIONs suggest that the LHRH receptor expression could play a role in the uptake and the associated cell death during the magnetic field exposure. The cell death in GT1-7 neurons confirms to late apoptosis or early necrosis, while with the LNCaP cells, necrosis is prominent. There has been a significant decrease in the mitochondrial ΔΨm in LNCaP cancer cells after exposure to magnetic field. In these cells, associated with the loss of mitochondrial function, there could be release of executioner caspases (caspases-3/7). Analysis of heat-shock protein-70 indicated an increase in the expression level of HSP70 (∼threefold increase) in GT1-7 neurons, and no changes were observed for LNCaP cells exposed to magnetic field for the first 15 min. Therefore, the observed differences in cell viability between the two cell types (cancerous and noncancerous) appear to be due to differences in the release of HSP70 and possibly other members of HSP family. Although it is too early to comment on clinical use(s) of SPIONs@Au-LHRH, a promise along these lines is indicated based on the fact that GT1-7 neurons elaborate HSP70 and thus are resistant from the toxicity associated with the MNP-induced heat stress. Considering that our recent investigations demonstrate extremely sensitive in vivo detection of pulmonary micrometastases using LHRH-SPIONs as MRI contrast agents [29], we believe there is potential opportunity for application of SPIONs@Au-LHRH in diagnosis as well as therapy.


Corresponding author: Rao M. Uppu, Environmental Toxicology PhD Program and the Health Research Center, Southern University and A & M College, Baton Rouge, LA 70813, USA, e-mail:

About the authors

Faruq Mohammad

Faruq Mohammad obtained his PhD in Environmental Toxicology from Southern University and A&M College (Baton Rouge, LA, USA) in May 2011. Prior to this, in 2005, Mohammad obtained his Master’s degree in Synthetic Organic Chemistry from Acharya Nagarjuna University (Namburu, India). He is currently working as a Postdoctoral Researcher in the Pharmacy Department at the North-West University (Potchefstroom campus; South Africa). His area of research is in the development of polymeric nano drug delivery systems for malaria and cancer in addition to understanding the toxicity of nanomaterials. He has several publications in these areas.

Achuthan C. Raghavamenon

Achuthan C. Raghavamenon is an Assistant Professor at the Amala Cancer Research Center in Thrissur (India). He is a recognized research advisor by the University of Calicut (Malappuram, India) and Mahathma Gandhi University (Kottayam, India). After obtaining his PhD in Biochemistry from Mahathma Gandhi University, Raghavamenon performed postdoctoral research in the areas of oxidative biology and cell signaling at the LSU Health Science Center (New Orleans, LA, USA), the Ohio State University Medical Centre (Columbus, OH, USA), and the Southern University Environmental Toxicology PhD program (Baton Rouge, LA, USA). He has numerous peer-reviewed publications to his credit, and his current research interest is in the drug development for various degenerative diseases.

Michelle O. Claville

Michelle O. Fletcher Claville is the Assistant Dean for the School of Science and an Associate Professor of Chemistry at Hampton University. She received a PhD in Chemistry, BS in Chemistry, and BA in English from the University of Florida, Gainesville, FL, USA. Formerly, she served as an Associate Professor and Chair in the Department of Chemistry at Southern University and A&M College, Baton Rouge, LA, USA. Claville is a recipient of the Faculty Early Development Career (CAREER, 2009) Award and the Achieving Competitive Excellence (ACE) Implementation Award (2012), both funded by the National Science Foundation. Claville has mentored scores of undergraduate students through physical organic chemistry research on the reactive intermediates derived from biomolecules and nanomaterials.

Challa S.S.R. Kumar

Challa S.S.R. Kumar is the Director of Nanofabrication and Nanomaterials at the Center for Advanced Microstructures and Devices at the Louisiana State University in Baton Rouge and is also associated with the DOE-supported Energy Frontier Research Center (EFRC) – Center for Atomic-Level Catalyst Design. He is a winner of the 2006 Nano 50 Technology Award for his work on magnetic-based nanoparticles for cancer imaging and treatment. His research interests are in developing novel synthetic methods, including those based on microfluidic reactors for multifunctional nanomaterials. He has 8 years of industrial R&D experience working for Imperial Chemical Industries and United Breweries. He is the Editor of two online series on Nanotechnologies for the Life Sciences (NtLS) and Nanomaterials for the Life Sciences (NmLS) and a book series on the characterization of nanomaterials. He is also currently the Editor-in-Chief of the journal Nanotechnology Reviews and founding editor of the Journal of Biomedical Nanotechnology. Numerous books and original research papers are part of his extensive publication record.

Rao M. Uppu

Rao M. Uppu is a Professor and Director of the Environmental Toxicology PhD Program at Southern University at Baton Rouge (SUBR). He obtained his PhD in Biochemistry in 1988 and carried out his postdoctoral research at the Eppley Institute for Research in Cancer and Allied Diseases (Omaha, NE, USA) and the Biodynamics Institute at Louisiana State University (Baton Rouge, LA, USA). His research interests include biological reactive intermediates, cell signalling by “ozone-specific” oxysterols and biomedical applications of core/shell nanoparticles. Uppu has mentored numerous graduate students (MS and PhD) and postdoctoral fellows and has published over 60 articles in peer-reviewed journals and book series. He is an elected Fellow of the Academy of Toxicological Sciences (Reston, VA, USA) and has been honored with numerous awards including the University-wide Outstanding Research Investigator Award (2007), Telugu Association of North America Excellence in Science Award (2011), and SUBR Chancellor’s Award for Excellence in Teaching (2013).

Acknowledgments

This research was supported by NSF grants HRD0450375 (from the HBCU-RISE program) and HRD1043316 (from the HBCU-UP ACE Implementation program). The authors acknowledge NIH-supported INBRE program of NCRR (P20RR016456) for the summer- 2010 funding. Partial funding for this work from NIH (1RO1CA142-01A1) is gratefully acknowledged.

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Received: 2013-11-7
Accepted: 2013-12-18
Published Online: 2014-3-29
Published in Print: 2014-8-1

©2014 by De Gruyter

This article is distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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