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Biodistribution and pharmacokinetic studies of SPION using particle electron paramagnetic resonance, MRI and ICP-MS

Aim: Superparamagnetic iron oxide nanoparticles (SPIONs) may play an important role in nanomedicine by serving as drug carriers and imaging agents. In this study, we present the biodistribution and pharmacokinetic properties of SPIONs using a new detection method, particle electron paramagnetic resonance (pEPR). Materials & methods: The pEPR technique is based on a low-field and low-frequency electron paramagnetic resonance. pEPR was compared with inductively coupled plasma mass spectrometry and MRI, in in vitro and in vivo. Results: The pEPR, inductively coupled plasma mass spectrometry and MRI results showed a good correlation between the techniques. Conclusion: The results indicate that pEPR can be used to detect SPIONs in both preclinical and clinical studies. Keywords: biodistribution • ICP-MS • MRI • nanomedicine • pEPR • pharmacokinetics • SPION

It is anticipated that nanostructures will play a crucial role in medicine by serving as drug carriers and as contrast agents for injured tissue [1] . The nanopharmaceutical aspect of nanomedicine involves the development of targeted delivery systems that carry drugs to the desired treatment location, hence sparing nontarget organs or tissues undesired side effects. Indeed, nanostructures not only efficiently deliver proteins, peptides and genes into the body, but these tend to be delivered safely with minimal adverse effects [2] . In addition, nanostructures also improve several conventional drugs limitations such as poor targeting, lack of water solubility, poor oral bioavailability and low therapeutic indices [3] . For example, in cancer treatment there are diverse types of nanoparticles (NPs) that have been synthesized to deliver therapeutic and diagnostic imaging contrast agents. These include chitosan NPs, liposomes, nanoemulsions to solid metal particles (gold, silver or iron oxide) and quantum dots [4] . Advances in biotechnology and biology are rapidly enabling the development of nanoparticles which combine both diag-

10.2217/NNM.15.22 © 2015 Future Medicine Ltd

nostic and therapeutic agents in a new area named theranostics. The main idea behind this combination is to visualize, diagnose and provide treatment of disease at its earliest stages [5] . While some NPs are loaded with drugs and contrast agents such as magnetic resonance imaging agents gadolinium [6] or manganese [7] , others are metal-based carriers with intrinsic contrast imaging properties (e.g., iron, nickel, silver, gold). Gadolinium and iron tracers or particles can both be tracked in real-time by using MRI, providing information on tumors and the distribution of NPs [6,8–10] . The MULTIFUN project [11] is pursuing a strategy based on the multifunctionalization of superparamagnetic iron oxide nanoparticles (SPIONs) or generically magnetic nanoparticles (MNPs), thus combining diagnostic and therapeutic techniques, to treat breast and pancreatic cancer. Due to their magnetism, SPIONs are not only used as efficient MRI contrast agents for cancer diagnosis, but are also used for magnetic hyperthermia [12] . SPIONs are generally nontoxic and well tolerated by organisms when properly synthesized and

Nanomedicine (Lond.) (2015) 10(11), 1751–1760

Oliviero L Gobbo*,1,2, Friedrich Wetterling2, Peter Vaes3, Stephanie Teughels3, Farouk Markos 4, Deirdre Edge4, Christine M Shortt4, Kieran Crosbie-Staunton5, Marek W Radomski1, Yuri Volkov‡,5,6 & Adriele Prina-Mello‡,5,6 1 School of Pharmacy & Pharmaceutical Sciences, Dublin, Ireland 2 Institute of Neuroscience, Trinity College Dublin, Dublin, Ireland 3 PEPRIC NV, Leuven, Belgium 4 Department of Physiology, University College Cork, Cork, Ireland 5 School of Medicine, Dublin, Ireland 6 CRANN, Trinity College Dublin, Dublin, Ireland *Author for correspondence: Tel.: +353 1896 3181 Fax.: +353 1896 2783 [email protected] ‡ Authors contributed equally

part of

ISSN 1743-5889

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Research Article Gobbo, Wetterling, Vaes et al. functionalized [13] . It is important to study the interactions that occur between animal/human organisms and each newly devised multifuntionalized nanoparticle at different stages of their synthesis [14] . Such preclinical pharmacological research includes biodistribution and pharmacokinetic studies. It is therefore essential to be able to detect these potential therapeutic agents. However, quantitative determination such as inductively coupled plasma-mass spectrometry can result in labor intensive and costly techniques that involve multidisciplinary setup and know-how. Therefore, the development and adoption of instrumentation that can rapidly provide information regarding nanostructure-based therapeutic agents would greatly improve the transferability of such nanotechnology into a preclinical or full clinical environment. In this study we present a newly introduced alternative analytical method [15] for detecting SPIONs in vitro and ex vivo that does not require complex sample preparation. Sampling errors often induced by the selection, manipulation and preparation, such as homogenization, dehydration or dilution, of tissue and blood samples can be avoided, resulting in a higher quality dataset. Ease of use is the major advantage against conventionally accepted pharmacological analytical techniques. The technique presented is based on a low-field and low-frequency electron paramagnetic resonance (EPR) [16] which selectively measures the magnetization of SPIONs. A radio frequency excitation is applied at the resonant frequency and the received signal is a measure of the amount of SPIONs present in the sample. The Pepric-developed instrument for particle electron paramagnetic resonance (pEPR) detection [15] constitutes a spectrometer (pepric particle spectrometer [PPS]) which was developed to measure the particle content in blood and tissue in a reliable and straightforward manner. Applicability of such instrumentation ranges from the laboratory analytical activity to the clinical environment. Previous work [17] has shown the advantages of the electron spin resonance (ESR) spectroscopy compared with inductively coupled plasma optical emission spectroscopy (e.g., ESR can distinguish between endogenous and exogenous sources of iron, ESR shows greater sensitivity for MNPs than for endogenous iron species but only in a biodistribution study of SPIONs). As opposed to pEPR method which results in a direct measurement of the SPIONs not requiring further data analysis, the ESR data were deduced from absorption ESR spectra, this is the first derivative of absorbed microwave power with respect to the applied field, from which the double integral was calculated to obtain a number proportional to the resonating electronic spins in the sample. Moreover a limitation imposed by the sample

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Nanomedicine (Lond.) (2015) 10(11)

preparation for these ESR measurements is the necessity to section the tissue samples in to 2 mm3 cubes to fit in the thin ESR glass tubes, in addition samples have to be precooled to prevent preparation errors due to tissue loss or cross contamination [17] . By contrast, pEPR is performed at low magnetic fields (10 millitesla) and low RF frequencies (300 MHz), offering the advantage that a much larger sample volume can be inserted and measured at room temperature. For the actual pEPR setup, samples of 150 μl can be inserted in commonly available plastic PCR tubes, so that larger parts of organ tissue or small organs like spleen can be measured and analyzed at once avoiding preparation errors. No further preparation is required. The pEPR method is a direct and selective method measuring the resonating MNP electron spins, and pEPR signal amplitude is proportional to the particle electron spins. pEPR is a nondestructive method, samples are stored or used for further ‘destructive’ analysis or confirmation tests like for immunochemistry or mass spectroscopy analysis. These advantages of pEPR compared with the commonly applied ESR techniques, do not require any sacrifice on sensitivity or accuracy of this method for the analysis of the magnetic particle content in blood or tissue. It is, however, important to point out that both techniques are limited to analyzing only superparamagnetic nanoparticles. The aim of the present study was to assess and validate the analysis, based on the pEPR technique, in suitably designed biodistribution and pharmacokinetic studies of SPIONs by means of in vivo MRI scans, and inductively coupled plasma mass spectrometry (ICP-MS), since the latter is a pharmaceutical analytical gold standard technique for such measurements. Materials & methods Animal husbandry

The experimental protocols employed in this study were approved by the Animal Research Ethics Committee, Trinity College Dublin and the Department of Health and Children (Ireland), in accordance with the European Community Directive (86/609). Pathogenfree adult male Wistar rats (283 ± 27 g) were supplied by the Trinity College Dublin BioResources Unit. At the same time, pathogen-free male and female BALBc mice (23 ± 2 g) were sourced from Harlan Laboratories, UK. The animals were housed in a thermo-regulated environment with a 12 h light/dark cycle. Food and water were available ad libitum. Animal handling was carried out by fully trained and licensed personnel whom at all stages assessed and recorded the well-being of the animals. Animals were humanely euthanized by injection of a very high dose of pentobarbital in accordance with the Irish Medicines Board standards.

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Biodistribution & PK studies of SPION using particle electron paramagnetic resonance, MRI & ICP-MS

Synthesis of superparamagnetic nanoparticles

Iron oxide nanoparticles were synthetized by using two different techniques within MULTIFUN project according to previously published work. First, the synthesis of the nanoparticles was done via an organic route according the protocol of Salas et al. [18] before to switch to an aqueous protocol, which is a more environmentally friendly production technique [14,18] . Magnetite (Fe3O4) SPIONs with size ranging between 12 and 15 nm in diameter were then coated with dimercaptosuccinic acid to provide colloidal stability. SPIONs were characterized by a transmission electron microscope, vibrating sample magnetometer, dynamic light scattering and electrophoretic zeta potential during preparation [14,18] .

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function of the diluted MNP content in the sample. Tissue, blood and calibration samples were analyzed on the PPS2 instrument (Pepric, Leuven, Belgium) at room temperature. The amplitude of the pEPR signal of MNPs containing tissue and blood samples was background corrected by subtracting the pEPR amplitude of corresponding control tissues or blood samples obtained from animals not exposed to MNPs or just before injection of MNPs for the blood samples. Tissue and blood MNP concentrations (mg (Fe)/kg and mg (Fe)/l) were recalculated from weight and volume normalized background-corrected pEPR amplitudes using calibration curves constructed with the MNP dilution standards. In vitro study

The pEPR technique & instrumentation

The PPS is an instrument for detection and quantification of magnetic iron oxide nanoparticles and is based on a direct and selective detection method pEPR [15] . In this study, all pEPR measurements were carried out with Pepric PPS2 instrument (Pepric, Leuven, Belgium). To perform the EPR on the iron oxide nanoparticles, the sample is immersed in a 10 millitesla magnetic field and low RF frequencies applied in a continuous wave mode (300 MHz). At these low fields only the magnetization of the nanoparticles aligns with this field as described by the Langevin function. The magnetization of the endogenous iron molecules present in the biological matrix of the sample however is oriented randomly. This makes the EPR method selective for the nanoparticles. The sample is further irradiated with a RF frequency of 300 MHz to induce the resonant tilting of the magnetic spin. The detected electromagnetic RF signal induced by the precessing of the tilted spins in excitation is proportional to the amount of particles, implying that the pEPR is a quantitative detection method. Whole blood or tissue samples are put directly in sample tubes. Blood or tissue samples can be treated with heparin or fixation liquids for a better preservation of the samples. The sample tubes are commonly available PCR tubes of 200 μl volume, with a sample content of maximum 150 μl, to be inserted into a sample holder. This sample holder is moved into the measurement module. The amount of particles contained into the sample is displayed within a few seconds on the read out and controlling computer. In the present study, the core diameter of the MNPs is approximately 10–15 nm and therefore the particles are treated as superparamagnetic, previously referred to as SPIONs. Calibration standards were made from the same MNP stock solution as used for the in vivo studies and dilution curves were measured demonstrating the linear decrease of the pEPR signal amplitude as a


In vitro work was carried out with the main objective to verify whether the pEPR technique was accurate in detecting a known quantity of SPIONs dispersed into biological samples. This therefore constituted the preliminary ‘biological’ validation of the analytical detectability in SPION-spiked blood. A ladder of SPION concentration samples (0, 0.001, 0.01, 0.1 and 1.0 g/l) were prepared starting from the supplied stock solution of 5 g/l. For each sample tested with the PPS2, three measures were taken and an average value was considered for analysis and graphical representation. The quantity of SPIONs was therefore measured using PPS2 and then confirmed by ICP-MS techniques in biological environments. The experimental design accounted for three biological solutions: rat whole blood, plasma and saline. Each sample (1 ml) was divided in two subgroups (0.5 ml); a half was analyzed by pEPR and the other half by ICP-MS, considered to be the gold standard technique for analytical quantification in the low concentration range (sensitivity range at part per billion). It is noteworthy that the pEPR samples did not require any preparation and measurements were carried out with the PPS2 at Pepric headquarter, Leuven, Belgium. By contrast, the ICP-MS samples were mineralized at 200°C for 30 min with 10 ml of nitric acid in a microwave oven and digested samples were diluted to 40 ml with ultrapure water (18 MΩ) and analyzed with an Agilent 7700x ICP-MS instrument (Agilent Technology, CA, USA). ICP-MS measurements were carried out by the Centre for Microscopy and Analysis, Trinity College Dublin, Ireland. Both systems were calibrated with internal controls and calibration curves were established with a set of standard samples of known concentration. The mean and standard deviation of the measured concentrations was plotted into separate graphs for ICP-MS and pEPR in order to validate the precision

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Research Article Gobbo, Wetterling, Vaes et al. in measuring known MNP concentrations. A linear regression model was used to fit the five concentration values measured for saline, blood and plasma. The linear slope of the regression lines was analyzed. In vivo study: pharmacokinetic study

Rats were anesthetized by an intraperitoneal (ip.) injection of a mixture of ketamine (60 mg/kg; Vetalar; Pfizer, Ireland) and medetomidine hydrochloride (0.5 mg/kg; Domitor, Pfizer, Ireland). The left femoral artery was cannulated with heparinized permanent polyurethane intravascular tubing for blood sampling. Rats (n = 7) were injected with 1 ml of 3.3 mg Fe/l MF66 solution (∼10 mg Fe/kg) through the tail vein; the injected 0.0033 mg of Fe is within the calibration range assessed during the in vitro measurements. The anesthetic reversal agent, atipamezole hydrochloride solution (Antisedan, Pfizer, Ireland), was injected ip. at 5 mg/kg. Blood samples (200 μl) were collected in heparinized tubes at the following times: 0; 3; 10; 20; 30; 60; 90; 120 and 180 min after SPION administration. In each case, blood was replaced by an equal volume of isotonic solution. The samples were analyzed by pEPR. The simultaneous circulation time depends on the surface coating, hydrodynamic size and the core. At the end of the experiment, the animals were euthanized using a very high dose of pentobarbital (Sodium Pentobarbital 100 mg/kg, ip.; Euthatal, Merial Animal Health Ltd, Harlow, Essex, UK ) and the organs were harvested for further investigation. Animal’s wellbeing was continuously monitored and did not show any sign of distress. Pharmacokinetic parameters were determined for each individual rat using a one-compartment model. Nanoparticle plasma concentrations (Cp) versus time profiles were fitted with Equation 1 using the least-squares method (Excel, Microsoft, USA).

Cp = Ae- at The distribution and elimination phases of Cp versus time profiles were characterized by the halflife times t1/2 _α = ln 2/α. Area under Cp versus time profiles were calculated using the linear trapezoidal rule [19] . Biodistribution

In vivo qualitative and quantitative biodistribution of SPIONs were respectively carried out by MRI and pERP techniques using a mouse model. Mice were injected with 100 μl of SPIONs at 10 mg Fe/ml or saline (control) through the tail vein. Mice were anesthetized using 5% isoflurane (Piramal Healthcare Ltd, Northumberland, UK) and maintained with 1.5%

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during the imaging period in the 7 T MRI Bruker system (Bruker Biospec, Coventry, UK) (respiration rate and temperature were continuously monitored). To acquire reference scans, all mice were imaged before injection. After injection, mice were imaged at 3 h, 24 h, 48 h and 96 h time points. T2-weighted images acquired using a spin echo sequence (Turbo RARE, TR/TE 4200/23.5 ms, FA 180°, FOV 6 cm, 5 averages, 35 slices, pixel resolution: 306 μm × 306 μm, slice thickness = 0.5 mm) were analyzed for nanoparticle uptake in different organs. The signal changes related to local SPION accumulation was evaluated after careful slice coregistration for each organ of interest (i.e., the heart, the liver, the spleen, the lungs and the kidneys). The baseline and SPION images were coregistered in the 2D plane using a control point method and an affine transformation in a script written in MATLAB® (The Mathworks, Natick, MA, USA). The intensity of the measured T2-weighted MR signal in a given pixel was normalized to the signal measured in a water tube which was located beside the animal and which appeared in each of the analyzed image slices. Signal changes (%) were computed by dividing the normalized pixel intensities of the SPION images by the baseline images [20] . The mean and standard deviation were extracted for further statistical analysis by manually drawing region of interests on the between-subject mean squares. This procedure was repeated for each organ in order to compensate for organ misalignment within slices which could have occurred in between the baseline and the SPION scans. Following the scans, mice were euthanized (Sodium Pentobarbital 100 mg/kg, ip.) and their organs (heart, liver, spleen, kidneys, brain and lungs) were harvested. Animal’s well-being was continuously monitored and they did not show any sign of distress. The quantity of nanoparticles were measured in the different organs at the four time points (3 h, 24 h, 48 h and 96 h) using the PPS model PPS2. Small tissue sample biopsies were taken, weighed and measured with pEPR without any preparation. The concentration of SPIONs was expressed in mg of iron per kg of tissue, and each organ was compared with controls across the different time points. The difference measured between organ controls and organ injected animals represents the iron amount from SPIONs. Statistics

T test was employed to examine differences between control and SPION-treated animals. The values are expressed as mean and standard deviation (mean ± SD). Statistical significance was assumed when p < 0.05.



Results In vitro evaluation of iron concentration in samples

The quantity of inorganic iron measured by pEPR and ICP-MS matched the known concentration of A

1.6

Slope (saline) = 1.4 ± 0.27 Slope (blood) = 1.4 ± 0.41 Slope (plasma) = 0.89 ± 0.03 Validation line

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Slope (saline) = 1 ± 0.022 Slope (blood) = 1 ± -0.015 Slope (plasma) = 1.1 ± 0.049 Validation line

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inorganic iron from the SPIONs (Figure 1) . From the results obtained, we were able to fully detect the total amount of SPIONs spiked into the three different biological environments as shown in Figure 1. It is interesting to note that both analytical techniques led B

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Figure 1. Superparamagnetic iron oxide nanoparticle detection in saline, plasma and blood by particle particle electron paramagnetic resonance and inductively coupled plasma mass spectrometry of samples containing known quantities of superparamagnetic iron oxide nanoparticles (n = 5). Data are plotted as concentration of inorganic iron subtracted from the iron value of their respective environmental controls. The linear slope fitted in each biological liquid is an indicator for how well the measured concentrations match the set-up concentrations. (A) Iron concentrations measured by ICP-MS. (B) Iron concentrations measured by pEPR. The graphs at the bottom of the figure correspond to magnification of the dotted frames of the graphs at the top. ICP-MS: Inductively coupled plasma mass spectrometry; pEPR: Particle electron paramagnetic resonance.



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SPION blood concentration (mg/l)

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Figure 2. Main pharmacokinetic parameters. (A) SPION blood concentration (in mg/l) versus time profile after intravenous administration of SPION suspension at a dose of 10 mg/kg. (B) Table of pharmacokinetic parameters after one-compartment analysis of the SPION blood concentration vs time profile (n = 7, mean ± standard deviation). AUC: Area under the curve; CL: Clearance; Cmax: Maximum concentration; SPION: Superparamagnetic iron oxide nanoparticle; t1/2: Half-life time.

to the same results. Limitations were imposed by the chosen biological environments (0.1 g/l in blood and plasma and at 0.01 g/l in saline) and by instrumentation error (part per billion). The linear slope fitted in each biological liquid is an indicator for how well the measured concentrations match the set-up concentrations with slope values near one indicating excellent correlation. Data presented in Figure 1 are plotted as concentration of inorganic iron subtracted from the iron value of their respective environmental controls (saline, plasma or blood). The saline and plasma controls contained very low quantities of iron (saline: 0.0014 ± 0.0007 g/l [ICP-MS] and 0.002 ± 0.002 g/l [pEPR]; in plasma: 0.0036 ± 0.0007 g/l [ICP-MS] and 0.001 ± 0.001 g/l [pEPR]). While ICP-MS measurements (0.44 ± 0.02g/l) showed higher levels of iron in blood controls than pEPR measurements (0.020 ± 0.007 g/l), due to iron naturally present in blood. Indeed, ICP-MS assesses the total quantity of iron in samples then pEPR measures only the amount of iron oxide nanoparticles. In vivo study: pharmacokinetics

Time dependence concentration of SPIONs in animal blood was measured by pEPR technique over 120 min

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for each animal (n = 7), the average curve is plotted in Figure 2. The SPION blood level decreased rapidly over the 120 min period, reaching 10 mg/l. The pharmacokinetic behavior of the SPION fits a mono-compartmental model, indicating that these nanoparticles are rapidly removed from the bloodstream. Indeed, the halflife of some iron oxide nanoparticles could be up to 182 min [21] . SPIONs reached the liver, kidneys, lungs and the spleen which are the organs primarily responsible for the distribution, metabolism and excretion of exogenous materials (see biodistribution). The main pharmacokinetic parameters are summarized in Figure 2. An important finding is that the half-life of SPIONs in blood was 32 min which agrees with a previous work [20] . Biodistribution

Magnetic resonance images of the abdomen were acquired using a T2-weighted sequence. Both SPIONinjected and saline-injected animals were monitored with respect to local signal changes. For the salineinjected control, the MRI signal change corresponding to saline administration was constant for all organs at every time point (data not shown). For the SPIONinjected animal, the MRI signal changes (relative to baseline level) were different for each organ (Figure 3A) .


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In particular, for the liver and spleen, the maximum MRI signal change (100%) was reached 3 h postinjection. For the lungs, MRI signal changes remained undetected pre- and post-SPION injection. Indeed, pulmonary tissue contains mostly air, resulting in large susceptibility to artefacts, cancelling out the low-water density which in turn translates into weak to nondetectable MRI signals [22] . For the heart, signal change decreased over the 96 h. Finally and interestingly, for the most sensitive organ, the kidneys, signal change reached a plateau for the cortex (+ 20% at 48 h and + 18.68% at 96 h) and for the medulla (+ 11.99% at 48 h and + 8.61% at 96 h) as shown in Figure 3B. After harvesting the organs, pEPR iron assay showed high concentrations of SPIONs in the liver, spleen, lungs and a small amount of iron was found in the heart and brain (Figure 4A) . As expected, the SPIONs were concentrated in the liver and spleen [23] , but also in the lungs (these SPION entrapments are probably due to their aggregation in pulmonary tissues). In the liver and spleen, the quantities remained constant over the 96-h period. A decrease of the iron concentration in the lungs and the heart was recorded over the same period; whereas for the kidneys (Figure 4A), the concentration of iron increased slowly, reaching a peak at 48 h postinjection before declining at 96 h. In Figure 4B the pEPR measurements (medulla + cortex) and the MRI signals (medulla and cortex) for the kidneys are plotted as a function of time after injection. It has shown that in both techniques the quantity of SPIONs changed over the 96-h period. Although we compare quantities of NPs measured A

Before injection

24 h after injection

B

by pEPR versus MRI signals induced by these NPs in two renal regions, we observe a similar pattern of changes in the same organ (kidney) with, however, some differences depending of the sampling areas. Indeed, for example, at the 24-h point, the pEPR measurement matches the MRI signal from the renal medulla, indicating that the tissue samples used for pEPR measurements were probably mainly from the medullary region. Overall, this simple comparison indicates a similar trend between the detected changes using pEPR and MRI techniques (Figure 4B) . In the first 24 h, NPs did not accumulate in kidneys. While at 48 and 96 h, there were increases of the quantity of NPs into kidney which were detected by pEPR and MRI. It is worth mentioning that the net result is a nonlinear relationship between signal intensity and the concentration of the nanoparticles [24] . Discussion In this study we present an analytical study of MULTIFUN-tailored nanoparticles developed for theragnostic treatment for cancer, with multiple techniques (i.e., ICP-MS, MRI and pEPR). A comparative assessment is presented between in vitro and in vivo of the pEPR technique with ICP-MS and MRI results, respectively. The results show good agreement between the various techniques used. In fact, the linear slope fitted for the pEPR technique was close to one within the error margins for all three sample types (saline, plasma and blood) while the slope for the ICP-MS technique showed larger deviation for saline samples. Also, ICPMS gave large variations of the measured values at 0.1g/l

40 Control medulla Medulla Control cortex Cortex

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Figure 3. Biodistribution in vivo of superparamagnetic iron oxide nanoparticles: MRI study. (A) T2-weighted MRIs showed distinctive changes of the signal in the different organs 24 h postinjection of superparamagnetic iron oxide nanoparticles compared with the signal before injection. (% reference vial) MR signal was normalized to signal measured in a water tube (reference vial) which was located beside the animal. (B) MRI signal change as a function of time after injection for medulla and cortex regions in mouse kidneys, mean ± standard deviation, t-test, *p < 0.05, **p < 0.01 vs control (n = between 4 and 8).



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Figure 4. Biodistribution in vivo of superparamagnetic iron oxide nanoparticles: MRI and particle electron paramagnetic resonance. (A) Quantity of nanoparticles in organs harvested after MRI scans measured by pEPR in mg Fe/kg of tissue. Mean ± standard deviation, n = between 4 and 8. (B) Comparison between quantity of nanoparticles measured by pEPR for kidneys (cortex + medulla) and absolute values of the MRI signal in renal cortex and renal medulla. Mean ± standard deviation, n = between 4 and 8. pEPR: Particle electron paramagnetic resonance.

MNP concentration, indicating that the detection threshold for ICP-MS was near this value while pEPR results were still capable of resolving this low concentration. Overall, the results for both techniques demonstrated that set-up MNP concentration can be quantified up to a range of 0.1g/l. The in vitro study showed that pEPR was a reliable technique for quantifying the nanoparticles in three biological environments and the limitations are related to the chosen biological model rather than the analytical method. For instance, the measurement in plasma and blood showed a slightly lower detection of SPIONs compared with the saline solution. In vitro the sensitivity for detection of SPIONs is the same for the particle EPR and the ICP-MS technique. Danhier et al. [25] have already shown that EPR is a highly sensitive technique for iron quantification for cell labeling studies when compared with ICPMS. The pEPR technique exhibits the following major advantages, first it measures the magnetization of the SPION directly and selectively and not of the natural iron molecules present in biological environments; second it requires no preparation of the samples eliminating manipulation errors and resulting in a higher quality sample set. pEPR is also a nondestructive method so that tissues are stored and preserved and postanalysis or confirmation tests are still possible, for example histological and biochemical analysis. As mentioned by Chertok et al. [17] , inductively coupled plasma optical emission spectrometry (ICP-OES) (and ICP-MS) provides a measure of total sample iron. The endogenous iron-containing species present in samples contribute to the total iron content, generating relatively high ICP-OES (or ICP-MS) background. This could reduce

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the detection of SPION when present at a low concentration. ICP-MS (or EOS) remains, however, the ‘gold standard’ technique for analyzing nanoparticles. Its major advantage compared with pEPR, is the evaluation of a myriad of metallic nanoparticles other than IONP in various situations, from environmental safety to biodistribution [26–28] . Further, the sample preparation used by Chertok et al. [17] for the ESR spectroscopy was more straightforward than for ICP-OES and ICPMS. However, as they have mentioned in their publication (even if they improve the technique), the ESR sample preparation is technically challenging [17] . Here, with the PPS, the samples (biological liquids or tissues) are directly introduced into 200 μl PCR tubes without any preparation. Tubes are only weighted before and after the samples were introduced into it, to assess the quantity of tissue or liquid. For the in vivo studies, rats were injected with SPIONs (10 mg Fe/kg) and they were monitored up to 120 min postinjection. The half-life of SPION described in Figure 2 is in good agreement with the previous literature as shown by Park et al. [20] . The pEPR technique was also utilized post mortem for the measurement of SPIONs in tissue samples after scanning the animals. The main advantage of the pEPR technique is that the samples are measured directly following harvesting, no preparation is required. Strikingly, the qualitative variation of SPION accumulation in vivo seen in MRI (e.g., for the kidneys) was consistent with the quantitative variation of SPION measured with pEPR, as shown in Figure 4B. Although there is a nonlinear relationship between signal intensity and the concentration of the nanoparticles [24] . MRI signal intensity changes



were very important in the liver and the spleen after SPION administration and the concentration of iron in these organs was higher compared with other organs as described previously by Park et al. [20] . Conclusion These preliminary results indicate that the biodistribution and pharmacokinetics of SPIONs can be assessed using pEPR. It is the first time a full in vivo study (biodistribution and pharmacokinetics) of SPIONs has been presented using this pEPR technique [15] with a specific instrument (PPS). To the best of our knowledge this is novel, and has great potential to become an easy ‘single step’ process to distinguish between SPION and naturally present iron, this is important since the natural-biological iron could mask the SPION iron if its concentration is too low. This problem is evident when measuring iron oxide nanoparticles in blood or tissues by ICP-MS technique. MRI technique presents the same advantage than pEPR by distinguishing SPION from the natural iron forms. This in vivo technique could, even, give a more sensitive evaluation of SPION accumulation in organs than pEPR technique. However, MRI data could only give qualitative indications of the SPION accumulation and remain an expensive technique. While pEPR technique presents numerous advantages as an ex vivo technique, i.e. has no sample preparation and the tissue samples are conserved allowing postanalysis and confirmation tests with biochemical and histological methods [29] . Moreover, the ease of application is even more important when translated to healthcare. Indeed, the growing use of SPION in nanomedicine will require analytical systems which can deliver pharmacological results efficiently. This is especially true for testing iron oxide-based agents for drug delivery/imaging in phase trials.

Research Article

Future perspective In the next decade, nanomedicine will become an important area in medicine. SPION research and application deserve a particular attention as they can be used for diagnostics (contrast agents in MRI) and therapy (magnetic drug targeting or hyperthermia). However, many effects of these nanoparticles on animals and humans are not known. Further preclinical and clinical investigations are, therefore, important to study their effects and toxicity. In this context, tools as the Pepric Particle Spectrometer based on the pEPR technique will be indispensable to facilitate this research. Acknowledgements The authors thank Dr Christian Kerskens for his assistance with MRI experiments and Rustam Rakhmatullin for his technical assistance.

Financial & competing interests disclosure This work was supported in part by the European Commission under MULTIFUN project (FP7 NMP-Large ref. 262943). P Vaes is shareholder and former CTO of Pepric. S Teughels is shareholder and CEO of Pepric. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Executive summary Background • The most used techniques to quantify superparamagnetic iron oxide nanoparticles (SPIONs) in biological fluids or tissues are inductively coupled plasma mass spectrometry (ICP-MS) or inductively coupled plasma optical emission spectrometry. MRI could also be used to study SPIONs in vivo. • We used a new technique called particle electron paramagnetic resonance (pEPR) to quantify SPIONs without any sample preparation.

Materials & methods • We compared pEPR technique with ICP-MS in vitro on biological samples containing SPIONs and conducted an in vivo study of SPIONs in mice and rats using pEPR and MRI.

Results • The quantities of inorganic iron measured by pEPR and ICP-MS led to the same results. • Pharmacokinetic parameters assessed by pEPR are comparable with previous publications. • There is a good relationship between the quantities of SPIONs detected using pEPR and MRI.

Conclusion • pEPR technique can be used to assess biodistribution and PK parameters of SPIONs.



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