Self Controlled Hyperthermia & MRI Contrast Enhancement via Iron

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Self Controlled Hyperthermia & MRI Contrast Enhancement via Iron Oxide Embedded Hydroxyapatite Superparamagnetic particles for Theranostic Application Ansar Ereath Beeran, Francis Boniface Fernandez, and P. R. Harikrishna Varma ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00244 • Publication Date (Web): 11 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018

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ACS Biomaterials Science & Engineering

Self Controlled Hyperthermia & MRI Contrast Enhancement via Iron Oxide Embedded Hydroxyapatite Superparamagnetic particles for Theranostic Application Ansar Ereath Beeran1, Francis Boniface Fernandez1 P. R. Harikrishna Varma1* 1

Division of Bioceramics, Department of Biomaterial Science and Technology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences & Technology, Poojappura, Kerala, India-695012 *Corresponding author: E-mail: [email protected]

ABSTRACT Increasing effectiveness of cancer therapeutics requires a multipronged approach. Delivery of controlled hyperthermia in the ranges of 43oC to 45oC on site aided by superparamagnetic particles ensures cell death via the apoptosis pathway.We demonstrated the use of iron-oxide embedded hydroxyapatite (HAIO) superparamagnetic particles for delivery of controlled hyperthermia and contrast enhancement in MRI. To determine optimal hyperthermia delivery, 5 mg/ml and 10 mg/ml concentrations of HAIO on various magnetic fields were used in alternating magnetic field (AMF) study. Time – temperature profile and specific loss power (SLP) data revealed that HAIO delivered precisely controlled temperature in contrast to superparamagnetic iron oxide nanoparticles (SPIONs). Earlier studies had demonstrated that HAIO concentrations of 0.5mg/ml to 3mg/ml are cytocompatible. Exposure of HeLa cells to HAIO at a concentration of 2mg/ml and applied field of 33.8 mT for a period of 30 min resulted in apoptosis induction in 75% of population. Significant cellular disruption was affirmed via FACS, ESEM and cLSM techniques. An aqueous phantom study and in-vitro cell culture study evaluation indicated relaxivity of 50.92 mM-1s-1and good pixel intensity variation in MRI. The current study assesses the potential of HAIO to deliver controlled hyperthermia and act as a negative MRI contrast agent. Repeated experiments have confirmed enhanced utility of the technique in the burgeoning field of theranostics.

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KEYWORDS: Diagnosis, therapy, alternating magnetic field, Magnetic hyperthermia, Apoptosis, Necrosis INTRODUCTION Multifunctional biocompatible nanoparticles have been developed to address combinatorial demands of drug delivery and imaging. Iron oxide based candidates due to their magnetic nature have received considerable attention for application in theranostics. Theranostic nanoparticles are tasked with discovery of disease states and delivery of therapy.1 Surface engineered superparamagnetic iron oxide nanoparticles (SPIONs) have been studied for theranostics applications in great detail.2, 3 Anti – cancer activity is based on hyperthermia generation via application of alternating magnetic field (AMF) on SPIONs. Thermal activity thus generated disrupts tumor cells via their inability to activate required protein responses. Normal cells show better resistance to temperature than the tumor ones via activation of heat shock proteins.4 This ensures selective removal of cancer cells with low collateral damage. Current treatments for cancer are radiation therapy, chemotherapy, laser therapy, immune therapy, hormone therapy etc.5,6 The major disadvantages of these techniques are poor targeting, severe side effects and induction of cell death via necrosispathways.7,

8

Conventionally, two nanoparticle-assisted hyperthermia systems are being used; (1) ultrasound heating of small bubbles9 and (2) optical heating using lasers.10 Among these techniques, ultrasound heating is promising as the energy is focused to a select location. However, it suffers from variation in the speed of sound in composite tissue groups.11 Optical methods are limited by the attenuation of the laser light by tissue.12 In this context, Magnetic hyperthermia assumes greater relevance as it can be focused at any depth for tumor treatment, with fewer side effects.13, 14


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SPIONs assisted hyperthermia for brain tumors has been clinically approved by the European drug control system.15 When SPIONs are exposed to an alternating magnetic field (AMF), they generate heat through Néel and Brownian relaxation losses. Studies indicate that cell injury leading to apoptosis or non – recoverable damage in cancer cells can be induced via thermal energy. Modification of essential molecules within cells leads to induction of cell death. SPIONs are the most common material tested until date due to their biocompatibility, enhanced specific loss power, low synthesis cost and easy functionalization.16 However, precise control of temperature distribution in SPIONs assisted hyperthermia is not possible. Control depends on particle parameters such as size, concentration, external applied field and treatment duration. Higher temperatures up to 60°C or rapid temperature rise can cause serious damage to normal tissues and coagulative necrosis. Hence, for apoptotic pathway initiation in cancer a regulated application of thermal treatment is critical. New combinations of materials are being investigated to fine – tune thermal delivery.16,

17

The new materials

should be biocompatible, with high thermal efficiency, stable in aqueous solution and high accumulation capacity inside tumour cells. This facilitates increment of temperature to induce cellular death under AMF. Superparamagentic particles with controlled temperature elevation will maintain therapeutic temperature, avoiding the use of any local temperature control system.18, 19 SPIONsare also used as an MRI contrast agent in clinical diagnosis.20 Some of the other contemporary non-invasive diagnostic techniques in clinical use are

therapeutic laser light

with plasmonic particles, near infrared imaging, computed tomography scanning, positron emission tomography and single-photon emission computed tomography. Limitations include poor penetration and collateral damage to high energy sources. SPIONs-mediated MRI is considered as an optimal system currently.21, 22



Thepresent study deals with use of superparamagnetic nanoparticle for the generation of controlled hyperthermia and contrast efficiency in MRI, for the first time. Recently we have developed a novel iron oxide-embedded hydroxyapatite (HAIO) superparamagnetic nanoparticle.23 In the current attempt, the HAIO assisted self-controlled hyperthermia and MRI contrast were investigated. The field strength of AMF and concentration of HAIO were varied and compared with SPIONs to assess the optimum conditions. The parameters for theranostic application of HAIO were assessed in vitro. The mechanism of HAIO assisted hyperthermia induced cellular damage or cell death was demonstrated and the appropriate criterion window for potential therapeutic protocols arrived. MATERIALS AND METHODS Materials FeCl2·4H2O (Merck, Darmstadt, Germany), FeCl3 (Merck),Ca(NO3)2·4H2O (Rankem, New Delhi, India), (NH4) H2PO4 (Rankem), 25%aqueous NH4OH (SD Fine Chemicals, Mumbai, India) and 35% HCl (SD FineChemicals) were used for the preparation of SPIONs and HAIO nanoparticles.Allchemicals used for the experiments other than those mentioned in the materialssection were obtained from Sigma-Aldrich, USA. Synthesis of HAIOand SPIONs HAIO was synthesised by co-precipitating iron salt and calcium phosphate precursors in alkaline medium at elevated temperature, as described in the previous study.23, 24 Similarly, SPIONs were synthesised as described in Ansar et al 2015.24 Detailed procedure and physicochemical characterizations of the prepared HAIO and SPIONs were described in supporting information.


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Preliminary cytotoxicity evaluation In vitro Biocompatibility - Cell Culture HeLa (human cervical carcinoma) cells were cultured in Dulbecco's Modified Eagle Medium-High Glucose (DMEM-HG) with 10% fetal bovine serum (FBS), 50 units per ml of penicillin and 50 mgml-1of streptomycin. All reagents were sourced from Invitrogen, India. The cell culture labware were purchased from NUNC, Denmark. Cells were seeded and maintained at 37°C and 5% CO2 atmosphere and experiments were performed at 80% confluence. Cytotoxicity measurement - Alamar blue assay The cytotoxicity of various concentrations of HAIO particles was evaluated via alamar blue (AB) assay. The AB assay was used to assess metabolically active cells via reduction potential studies based on vitality of mitochondrial reductase enzymes. Vitally active enzyme systems mediate transition of alamar blue to pink color. HeLa cells plated in 96 well plates were challenged with different concentrations [0.5mg/ml, 1mg/ml, 2mg/ml and 3mg/ml] of HAIO. After the exposure, 100 µl of alamar blue reagent (assay protocol) was added in each well and incubated for 4h at 37°C. The fluorescence was measured at 560 nm excitation and 590 nm emission wavelengths using a plate reader (HIDEX Chameleon) and expressed in percentage activity of live cells against control. The experiments werecarried out in six duplicates for each concentration. Magnetic Hyperthermia Evaluation and SLP Calculation In order to investigate the therapeutic hyperthermia potential of HAIO and SPIONs, the samples were subjected to AMF studies. For this analysis, the Ambrell EASYHEAT laboratory induction system was used. Itcontains induction coil with 4 cm diameter, 2.6 cm length and a total of 6 turns. The magnetic field frequency was set at 275 kHz for this



experiment. Temperature of induction coil was equilibrated with room condition using a chilled water re-circulator in closed loop mode. HAIO and SPIONs were subjected to an AMF through induction heating system and temperature of nanoparticles was measured with respect to time. Non – contact assessment of temperature using infrared thermometer (FLUKE 572) was carried out.To evaluated the specific loss power [SLP] of material, 5mg and 10mg of HAIO and SPIONs were dispersed in an aqueous solution in an insulated 1.5 mL eppendorf tube. Assembled unit was placed centrally within a copper coil integrated with a heat removal system and time temperature profile was obtained using AMF applications. The SLP was calculated by the equation (1) =

………. (1)

Where ‘C’ is the volume specific heat capacity of the sample (Cwater= 4185 J L−1K−1C), ‘Vs’ is the sample volume, and ‘m’is the mass of the magnetic particle present in the sample volume. dT/dt = initial slope of the change in temperature versus time curve (Ks-1). In vitro Hyperthermia Evaluation HeLa cells (1x106) and HAIO or SPIONs materials at a concentration of 2mg/ml were held in a 1.5 ml eppendorf tube with a final volume of 200µl. AMF of 33.8mT and 275 kHz were applied for 15min and 30min. Cell – material suspension was exposed to AMF hyperthermia in the centre of the coil with temperature monitoring via IR thermometry. Post – application, suspension was held at 37 °C for 1 hr. Cell suspension containing 2mg/ml HAIO was taken as control. The cell death was analyzed via quantitative and imaging methods using FACS, cLSM and ESEM techniques. FACS Analysis In this analysis, the control and test material-cell suspensions were washed twice in 1X phosphate buffered saline and re-suspended in the same. 200 µl (50 µg/ml stock solution) ethidium bromide solution was applied for half an hour. Further, the cell suspension was


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washed in 1X PBS solution to remove the excess stain and analysed via Fluorescence Activated Cell Sorting [FACS ARIA, BD Biosciences, San Jose, CA, USA]. The percentage of dead cells was calculated by using BD FACS Diva software (BD Biosciences, San Jose, CA, USA). Quantitative Estimation of Mechanism ofCell Death - FACS Analysis After 15 min and 30 minof AMF hyperthermia, the control and test material-cell suspensions were washed twice in 1X phosphate buffered saline and re-suspended in the same. Further staining was done with live-dead staining kit [Invitrogen FITC AnnexinV/Dead Cell Apoptosis cat no:V13242]. Analysis was via flow cytometric analysis and percentage of dead cells was calculated by using BD FACS Diva software. Hyperthermia-Treated Cells Morphology Evaluation – ESEM Technique To understand the morphology of hyperthermia-treated HeLa cells, the cells were seeded on round glass coverslips and grown to confluence (Blue Star, India). Cells were challenged with 2 mg/ml of HAIO particles, with AMF exposure for 30min. Post exposure incubation for 1h at 37°C was carried out. Fixation was by a 1% glutaraldehyde solution for 2 hrs and dehydrated via ascending alcohol series and evaluated by Environmental scanning electron microscopy (FEI QUANTA 200). In vitro MRI Analysis HeLa cells (1x106) were incubated with HAIO in a DMEM medium for 24h at Fe concentrations of 0.05, 0.1, 0.15, 0.2 and 0.25mM. After exposure time, the cells were washed twice with 1X PBS and re-suspended in1mL PBS in an eppendorf tube. MRI was performed a 1.5T MRI. T2-weighted images were acquired using the following parameters: temperature = 22°C, FOV = 8 cm x 8cm andslicethickness =3mm. The T2 signal intensities were measured the region of interest.



RESULTS AND DISCUSSION We previously reported the development of iron oxide-embedded hydroxyapatite (HAIO) nanocrystals via aqueous co-precipitation method.23 Magnetic nanoparticles thus obtained of 1:2 molar ratio of ferrous and ferric ionic concentration along with 1.67 Ca/P ratio of precursors at a higher pH of 12 and temperature at70°C. The developed particles’ size, morphology, composition, phase purity and magnetic measurement have been evaluated via TEM, ESEM, EDS, XRD and VSM analysis and are depicted in Figure1a-e respectively. The TEM and ESEM results revealed that HAIO showed polygonal faceted spherical morphology having uniform size distribution. Particles diameter was measured using Image J software from TEM image and obtained size range with ~25-35nm diameter. The EDS and XRD data demonstrated the phase pure existence of HAIO particles and crystallite size of iron oxide in HAIO observed at 17.04 ± 1.65nm from XRD patterns. Further the VSM analysis showed magnetic saturation values of 22 emu/g and 72 emu/g and coercivity values of Hc = 8.95 (Oe) and Hc = 0.37 (Oe) (at figure 1e inset) for HAIO and SPIONS respectively. The higher value of Hc in SPIONS might be due to the aggregation and while that of HAIO in negligible value as stable surface modification. Moreover the HAIO particles hydrodynamic diameter measured via dynamic light scattering (DLS) and is ~160nm (Figure S1). The preliminary cytotoxicity evaluation of concentrations 0.5mg/mL to 3mg/mL of HAIO was performed at in vitro condition and toxicity was examined via Alamar Blue assay and represented in Figure 1F. Lack of significant differences in viability of cells treated by HAIO at higher concentrations when compared with control for a period of 24 h was noted.


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Figure 1.(a) TEM (b) ESEM (c) EDS and (d) XRD (e) VSM measurement analysis of HAIO particles. (f) % activity of cells at 24h plotted to assess cytotoxicity effect of HAIOs (varying concentration) on HeLa analyzed viaalamar blue assay kit (p

Self Controlled Hyperthermia & MRI Contrast Enhancement via Iron

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