Michele Wabler

Magnetic resonance imaging contrast of iron oxide nanoparticles developed for hyperthermia is dominated by iron content

Abstract Purpose: Magnetic iron oxide nanoparticles (MNPs) are used as contrast agents for magnetic resonance imaging (MRI) and hyperthermia for cancer treatment. The relationship between MRI signal intensity and cellular iron concentration for many new formulations, particularly MNPs having magnetic properties designed for heating in hyperthermia, is lacking. In this study, we examine the correlation between MRI T2 relaxation time and iron content in cancer cells loaded with various MNP formulations. Materials and methods: Human prostate carcinoma DU-145 cells were loaded with starch-coated bionised nanoferrite (BNF), iron oxide (Nanomag® D-SPIO), Feridex™, and dextran-coated Johns Hopkins University (JHU) particles at a target concentration of 50 pg Fe/cell using poly-D-lysine transfection reagent. T2-weighted MRI of serial dilutions of these labelled cells was performed at 9.4 T and iron content quantification was performed using inductively coupled plasma mass spectrometry (ICP-MS). Clonogenic assay was used to characterise cytotoxicity. Results: No cytotoxicity was observed at twice the target intracellular iron concentration (∼100 pg Fe/cell). ICP-MS revealed highest iron uptake efficiency with BNF and JHU particles, followed by Feridex and Nanomag-D-SPIO, respectively. Imaging data showed a linear correlation between increased intracellular iron concentration and decreased T2 times, with no apparent correlation among MNP magnetic properties. Conclusions: This study demonstrates that for the range of nanoparticle concentrations internalised by cancer cells the signal intensity of T2-weighted MRI correlates closely with absolute iron concentration associated with the cells. This correlation may benefit applications for cell-based cancer imaging and therapy including nanoparticle-mediated drug delivery and hyperthermia.

Preliminary study of injury from heating systemically delivered, nontargeted dextran- superparamagnetic iron oxide nanoparticles in mice

AIM To assess the potential for injury to normal tissues in mice due to heating systemically delivered magnetic nanoparticles in an alternating magnetic field (AMF). MATERIALS & METHODS Twenty three male nude mice received intravenous injections of dextran-superparamagnetic iron oxide nanoparticles on days 1-3. On day 6, they were exposed to AMF. On day 7, blood, liver and spleen were harvested and analyzed. RESULTS Iron deposits were detected in the liver and spleen. Mice that had received a high-particle dose and a high AMF experienced increased mortality, elevated liver enzymes and significant liver and spleen necrosis. Mice treated with low-dose superparamagnetic iron oxide nanoparticles and a low AMF survived, but had elevated enzyme levels and local necrosis in the spleen. CONCLUSION Magnetic nanoparticles producing only modest heat output can cause damage, and even death, when sequestered in sufficient concentrations. Dextran-superparamagnetic iron oxide nanoparticles are deposited in the liver and spleen, making these the sites of potential toxicity. Original submitted 16 August 2011; Revised submitted 21 March 2012; Published online 26 July 2012.

The effect of cell-cluster size on intracellular nanoparticle-mediated hyperthermia: is it possible to treat microscopic tumors?

AIM To compare the measured surface temperature of variable size ensembles of cells heated by intracellular magnetic fluid hyperthermia with heat diffusion model predictions. MATERIALS & METHODS Starch-coated Bionized NanoFerrite (Micromod Partikeltechnologie GmbH, Rostock, Germany) iron oxide magnetic nanoparticles were loaded into cultured DU145 prostate cancer cells. Cell pellets of variable size were treated with alternating magnetic fields. The surface temperature of the pellets was measured in situ and the associated cytotoxicity was determined by clonogenic survival assay. RESULTS & CONCLUSION For a given intracellular nanoparticle concentration, a critical minimum number of cells was required for cytotoxic hyperthermia. Above this threshold, cytotoxicity increased with increasing cell number. The measured surface temperatures were consistent with those predicted by a heat diffusion model that ignores intercellular thermal barriers. These results suggest a minimum tumor volume threshold of approximately 1 mm(3), below which nanoparticle-mediated heating is unlikely to be effective as the sole cytotoxic agent.

Magnetic nanoparticle hyperthermia enhances radiation therapy: A study in mouse models of human prostate cancer

Abstract Purpose: We aimed to characterise magnetic nanoparticle hyperthermia (mNPH) with radiation therapy (RT) for prostate cancer. Methods: Human prostate cancer subcutaneous tumours, PC3 and LAPC-4, were grown in nude male mice. When tumours measured 150 mm3 magnetic iron oxide nanoparticles (MIONPs) were injected into tumours to a target dose of 5.5 mg Fe/cm3 tumour, and treated 24 h later by exposure to alternating magnetic field (AMF). Mice were randomly assigned to one of four cohorts to characterise (1) intratumour MIONP distribution, (2) effects of variable thermal dose mNPH (fixed AMF peak amplitude 24 kA/m at 160 ± 5 kHz) with/without RT (5 Gy), (3) effects of RT (RT5: 5 Gy; RT8: 8 Gy), and (4) fixed thermal dose mNPH (43 °C for 20 min) with/without RT (5 Gy). MIONP concentration and distribution were assessed following sacrifice and tissue harvest using inductively coupled plasma mass spectrometry (ICP-MS) and Prussian blue staining, respectively. Tumour growth was monitored and compared among treated groups. Results: LAPC-4 tumours retained higher MIONP concentration and more uniform distribution than did PC3 tumours. AMF power modulation provided similar thermal dose for mNPH and combination therapy groups (CEM43: LAPC-4: 33.6 ± 3.4 versus 25.9 ± 0.8, and PC3: 27.19 ± 0.7 versus 27.50 ± 0.6), thereby overcoming limitations of MIONP distribution and yielding statistically significant tumour growth delay. Conclusion: PC3 and LAPC-4 tumours represent two biological models that demonstrate different patterns of nanoparticle retention and distribution, offering a model to make comparisons of these effects for mNPH. Modulating power for mNPH offers potential to overcome limitations of MIONP distribution to enhance mNPH.

New iron-oxide particles for magnetic nanoparticle hyperthermia: an in-vitro and in-vivo pilot study

Magnetic nanoparticle hyperthermia (mNHP) is regarded as a promising minimally invasive procedure. These nanoparticles generate heat when exposed to alternating magnetic fields (AMFs) and thus have shown a potential for selective delivery of heat to a target such as a cancer cell. Despite the great promise however, successful clinical translation has been limited in part by technical challenges of selectively delivering heat only to the target tissue. Interaction of AMF with tissues also deposits heat through Joule heating via eddy currents. Considerations of patient safety thus constrain the choice of AMF power and frequency to values that are insufficient to produce desirable heating from available nanoparticle formulations. Therefore, considerable effort must be directed to the design of particles and the AMF device to maximize the specific delivery of heat to the intended target while minimizing the unintended and non-specific heating. We have recently developed new iron-oxide nanoparticles (IONPs) having much higher heating capability at the clinically relevant amplitudes and frequencies than other formulations. Here, we utilize a new rectangular coil designed for treating multi well tissue culture plate and show that these particles are superior to two commercially available IONPs for hyperthermia of DU145 prostate cancer cells in culture. We report results of pilot in-vivo experiments using the DU145 human prostate xenograft model in nude male mouse. AMF treatment yielded an intratumor temperature rise > 10 °C in <10 min heating (AMF amplitude 29 kA/m @160 kHz) with ~4 mg nanoparticle /g tumor while maintaining rectal (core) temperature well within physiological range.

Monitoring nanoparticle-mediated cellular hyperthermia with a high-sensitivity biosensor

AIM To develop and apply a heat-responsive and secreted reporter assay for comparing cellular response to nanoparticle (NP)- and macroscopic-mediated sublethal hyperthermia. MATERIALS & METHODS Reporter cells were heated by water bath (macroscopic heating) or iron oxide NPs activated by alternating magnetic fields (nanoscopic heating). Cellular responses to these thermal stresses were measured in the conditioned media by secreted luciferase assay. RESULTS & CONCLUSION Reporter activity was responsive to macroscopic and nanoparticle heating and activity correlated with measured macroscopic thermal dose. Significant cellular responses were observed with NP heating under doses that were insufficient to measurably change the temperature of the system. Under these conditions, the reporter response correlated with proximity to cells loaded with heated nanoparticles. These results suggest that NP and macroscopic hyperthermia may be distinctive under conditions of mild hyperthermia.

Characterization of intratumor magnetic nanoparticle distribution and heating in a rat model of metastatic spine disease: Laboratory investigation

OBJECT The goal of this study was to optimize local delivery of magnetic nanoparticles in a rat model of metastatic breast cancer in the spine for tumor hyperthermia while minimizing systemic exposure. METHODS A syngeneic mammary adenocarcinoma was implanted into the L-6 vertebral body of 69 female Fischer rats. Suspensions of 100-nm starch-coated iron oxide magnetic nanoparticles (micromod Partikeltechnologie GmbH) were injected into tumors 9 or 13 days after implantation. For nanoparticle distribution studies, tissues were harvested from a cohort of 36 rats, and inductively coupled plasma mass spectrometry and histopathological studies with Prussian blue staining were used to analyze the samples. Intratumor heating was tested in 4 anesthetized animals with a 20-minute exposure to an alternating magnetic field (AMF) at a frequency of 150 kHz and an amplitude of 48 kA/m or 63.3 kA/m. Intratumor and rectal temperatures were measured, and functional assessments of AMF-exposed animals and histopathological studies of heated tumor samples were examined. Rectal temperatures alone were tested in a cohort of 29 rats during AMF exposure with or without nanoparticle administration. Animal studies were completed in accordance with the protocols of the University Animal Care and Use Committee. RESULTS Nanoparticles remained within the tumor mass within 3 hours of injection and migrated into the bone at 6, 12, and 24 hours. Subarachnoid accumulation of nanoparticles was noted at 48 hours. No evidence of lymphoreticular nanoparticle exposure was found on histological investigation or via inductively coupled plasma mass spectrometry. The mean intratumor temperatures were 43.2°C and 40.6°C on exposure to 63.3 kA/m and 48 kA/m, respectively, with histological evidence of necrosis. All animals were ambulatory at 24 hours after treatment with no evidence of neurological dysfunction. CONCLUSIONS Locally delivered magnetic nanoparticles activated by an AMF can generate hyperthermia in spinal tumors without accumulating in the lymphoreticular system and without damaging the spinal cord, thereby limiting neurological dysfunction and minimizing systemic exposure. Magnetic nanoparticle hyperthermia may be a viable option for palliative therapy of spinal tumors.

Image-guided thermal therapy with a dual-contrast magnetic nanoparticle formulation: A feasibility study

Abstract Purpose/objective: The aim of this study was to develop and investigate the properties of a magnetic iron oxide nanoparticle–ethiodised oil formulation for image-guided thermal therapy of liver cancer. Materials and methods: The formulation comprises bionised nano-ferrite (BNF) nanoparticles suspended in ethiodised oil, emulsified with polysorbate 20 (BNF-lip). Nanoparticle size was measured via photon correlation spectroscopy and transmission electron microscopy. In vivo thermal therapy capability was tested in two groups of male Foxn1nu mice bearing subcutaneous HepG2 xenograft tumours. Group I (n = 12) was used to screen conditions for group II (n = 48). In group II, mice received one of BNF-lip (n = 18), BNF alone (n = 16), or PBS (n = 14), followed by alternating magnetic field (AMF) hyperthermia, with either varied duration (15 or 20 min) or amplitude (0, 16, 20, or 24 kA/m). Image-guided fluoroscopic intra-arterial injection of BNF-lip was tested in New Zealand white rabbits (n = 10), bearing liver VX2 tumours. The animals were subsequently imaged with CT and 3 T MRI, up to 7 days post-injection. The tumours were histopathologically evaluated for distribution of BNF-lip. Results: The BNF showed larger aggregate diameters when suspended in BNF-lip, compared to clear solution. The BNF-lip formulation produced maximum tumour temperatures with AMF >20 kA/m and showed positive X-ray visibility and substantial shortening of T1 and T2 relaxation time, with sustained intratumoural retention up to 7 days post-injection. On pathology, intratumoural BNF-lip distribution correlated well with CT imaging of intratumoural BNF-lip distribution. Conclusion: The BNF-lip formulation has favourable thermal and dual imaging capabilities for image-guided thermal therapy of liver cancer, suggesting further exploration for clinical applications.

Calibration of a Quasi-Adiabatic Magneto-Thermal Calorimeter Used to Characterize Magnetic Nanoparticle Heating

To assess and validate temperature measurement and data analysis techniques for a quasi-adiabatic calorimeter used to measure amplitude-dependent loss power of magnetic nanoparticles exposed to an alternating magnetic field (AMF) at radiofrequencies (160 65 kHz). The data collected and methods developed were used to measure the specific loss power (SLP) for two magnetic iron oxide nanoparticles (IONPs) suspensions, developed for magnetic nanoparticle hyperthermia. Calibration was performed by comparing measured against calculated values of specific absorption rate (SAR) of a copper wire subjected to AMF. Rate of temperature rise from induced eddy currents was measured (n ¼4) for a copper wire of radius 0.99mm and length of 3.38mm in an AMF at amplitudes (H) of 16, 20, 24, and 28kA/m. The AMF was generated by applying an alternating current using an 80-kW induction power supply to a capacitance network containing a 13.5-cm vertical solenoid that held the calorimeter. Samples were taped to an optical fiber temperature probe and inserted into a standard (polystyrene, 5ml) test tube which was suspended in the calorimeter. The sample was subjected to the AMF for 30s or until the temperature of the sample, increased by 30 � C, recorded at 0.3-s intervals. The SAR of the sample was normalized by H 2 f 1/2 , averaged, and compared to theoretical values. Iron (Fe) normalized SLPs of two IONPs (JHU-MION and bionized-nanoferrite (BNF) particles (Micromod Partikeltechnologie, GmbH)) in aqueous suspension were measured in the same setup. We report experimental SAR values for the copper of 2.4 60.1, 4.3 60.2, 6.2 60.1, and 8.5 60.1W/g compared to theoretical values 3.1 60.1, 4.5 60.2, 6.5 60.1, and 9.2 60.2W/g at AMF amplitudes of 16 60.1, 20 60.2, 24 60.1, and 28 60.1kA/m, respectively. Normalized experimental data followed a linear trend approximately parallel to theoretical values with an R 2 -value of 0.99. The measured SLPs of the JHU particles are higher than BNF particles within the tested AMF amplitude range of 15kA/m to 45kA/m. We demonstrated that copper can be used to calibrate magneto-thermal calorimetric systems used for SLP measurements of magnetic nanoparticles for a field range of 15‐28kA/m at 160 65kHz. We also note that the electrical conductivity, diameter of copper sample and accuracy, and response time of thermometry constrain calibration to lower amplitudes, highlighting the need for development of standard reference materials for such applications. [DOI: 10.1115/1.4024273]

An optimised spectrophotometric assay for convenient and accurate quantitation of intracellular iron from iron oxide nanoparticles

Abstract We report the development and optimisation of an assay for quantitating iron from iron oxide nanoparticles in biological matrices by using ferene-s, a chromogenic compound. The method is accurate, reliable and can be performed with basic equipment common to many laboratories making it convenient and inexpensive. The assay we have developed is suited for quantitation of iron in cell culture studies with iron oxide nanoparticles, which tend to manifest low levels of iron. The assay was validated with standard reference materials and with inductively coupled plasma-mass spectrometry (ICP-MS) to accurately measure iron concentrations ∼1 × 10−6 g in about 1 × 106 cells (∼1 × 10−12 g Fe per cell). The assay requires preparation and use of a working solution to which samples can be directly added without further processing. After overnight incubation, the absorbance can be measured with a standard UV/Vis spectrophotometer to provide iron concentration. Alternatively, for expedited processing, samples can be digested with concentrated nitric acid before addition to the working solution. Optimization studies demonstrated significant deviations accompany variable digestion times, highlighting the importance to ensure complete iron ion liberation from the nanoparticle or sample matrix to avoid underestimating iron concentration. When performed correctly, this method yields reliable iron ion concentration measurements to ∼2 × 10−6 M (1 × 10−7 g/ml sample).