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Effect of Anesthesia on Magnetic Nanoparticle Biodistribution After Intravenous Injection Lucía Gutiérrez
, Raquel Mejías , Francisco J. Lázaro , Carlos J. Serna , Domingo F. Barber , and M. Puerto Morales
Instituto de Ciencia de Materiales de Madrid, ICMM-CSIC, Cantoblanco Madrid, 28049, Spain School of Physics, M013, The University of Western Australia, Crawley, WA 6009, Australia Centro Nacional de Biotecnología,CNB-CSIC, Cantoblanco Madrid, 28049, Spain Departamento de Ciencia y Tecnología de Materiales y Fluidos, Universidad de Zaragoza, Zaragoza, 50018, Spain The role of anesthesia on magnetic nanoparticle biodistribution among different organs after intravenous injection has been studied in a murine model. Animals were anesthetized by inhalation with isoflurane (0.5% in oxygen) or by intraperitoneal injection with a mixture of ketamine and xylazine. Then, monodisperse dimercaptosuccinic acid coated magnetic nanoparticles (diameter of ) were administered intravenously to the animals. Lung and liver tissues were collected after the particle administration and the amount of particles in each tissue was determined by alternating current magnetic susceptibility measurements. Whereas the amount of particles that reaches the liver seems not to be affected by the anesthesia used, the amount of particles that reaches the lungs for inhaled isoflurane is three times less than for the intraperitoneally injected anesthetic. Index Terms—AC susceptibility, anesthesia, biodistribution, magnetic nanoparticles.
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I. INTRODUCTION
AGNETIC nanoparticles are currently being improved to be used in several biomedical applications such as magnetic carriers for drug delivery, contrast agents for medical imaging, or magnetic hyperthermia for cancer treatment [1]. In the last years, new synthesis techniques have been developed to produce particles with the most appropriated physicochemical properties for each biomedical application. However, the interactions between the particles and the organisms are still not completely controlled. In particular, the fate of the administered nanoparticles, a potential source of toxicity, is largely unknown and hence it deserves to be explored. One of the present challenges in nanomedicine is to determine the nanoparticle biodistribution after systemic administration [2]. Apart from developing better imaging methods for particle tracking [3] or quantitative methods for determining the amount of particles in each organ [4], it is fundamental to be able to understand all the factors that influence the particle distribution because that will allow us to be able to target specific tissues. The usually studied factors that influence the nanoparticle biodistribution are particle size, coating, composition and surface charge [5], [6]. Nevertheless, in addition to the physicochemical properties of the particles, it is also important to study other factors that may alter their biodistribution, such as particle administration route, doses or animal species. The aim of this work was to determine the influence of the type of anesthesia, used during nanoparticle administration, on their subsequent biodistribution. In the study, two types of anesthesia were tested and their magnetic nanoparticle biodistribution was monitored by alternating current (AC) magnetic susceptibility measurements. Based on this type of measurements, often used to characterize the magnetic properties of just Manuscript received July 08, 2012; revised September 12, 2012; accepted September 24, 2012. Date of current version December 19, 2012. Corresponding author: L. Gutiérrez (e-mail:
[email protected]). Color versions of one or more of the figures in this paper are available online at http://ieeexplore.ieee.org. Digital Object Identifier 10.1109/TMAG.2012.2221162
synthetized nanoparticles [7], [8], we developed a protocol that allows us to get quantitative particle concentrations in tissues [9], [10]. The particles selected for this study are dimercaptosuccinic acid coated magnetic nanoparticles (DMSA-MNP) whose efficiency has been previously proved for in vivo drug delivery applications [11]. II. MATERIALS AND METHODS A. Magnetic Nanoparticles Synthesis DMSA-MNP were prepared as reported previously by Roca et al. [12] with slight modifications. Briefly, a mixture of Fe(acac) (7.1 g, 20 mmol), 1, 2-dodecanediol (20.2 g, 100 mmol), oleic acid (16.9 g, 60 mmol) and oleylamine (16.0 g, 60 mmol) in 1-octadecene (200 ml) was stirred mechanically under atmosphere and heated at 200 C during 2 h and at 315 C (refluxed) for 30 min. The resulting solution was allowed to cool down to room temperature and washed several times with ethanol, using a permanent magnet to precipitate the particles and remove the supernatant. Then, the rests of ethanol were evaporated and the magnetite nanoparticles were dispersed in toluene. The particle suspension in toluene (80 ml, 0.9 mmol) was added to a solution of DMSA (360 mg, 2.0 mmol) in dimethyl sulfoxide (20 ml) and stirred mechanically for 48 h. After this treatment, DMSA coated nanoparticles precipitate as a black powder that was washed with ethanol several times and redispersed in water. Finally, pH was increased to 10 by adding NaOH and the resulting homogeneous dispersion was dialyzed for 3 days and filtered through a 0.2 mm-pore filter before bringing pH back to 7. B. Magnetic Nanoparticles Characterization Iron concentration of the DMSA-MNP dispersion was measured by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES). Particle core distribution was measured using a 200-KeV JEOL-2000 FXII transmission electron microscope (TEM). Samples were prepared by placing a drop of a dilute
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DMSA-MNP suspension on a carbon-coated copper grid and allowing the solvent to evaporate at room temperature. Hydrodynamic size, values from the intensity distributions, was measured by dynamic light scattering (DLS) using a Nano Sizer ZS (Malvern). C. Mouse Model Twelve-week-old female C57BL/6 mice (Harlan Laboratories) were used for the analysis . Animals were maintained in the Centro Nacional de Biotecnología (CNB) animal facility during the whole study. All mouse breeding and animal experiments were approved by the Ethics Committee for Animal Experimentation at the CNB in compliance with European Union legislation. In order to study the influence of the type of anesthesia on the particle biodistribution, mice were split into two groups and treated with two kinds of anesthetics: isoflurane (inhaled, 0.5% in oxygen) and a mixture of ketamine and xylazine (intraperitoneally injected). Animals received either a single dose of an intravenous injection of DMSA-MNP (300 g Fe/injection) or multiple doses (same dose, twice a week for two weeks) while under the effects of the anesthesia. Animals were sacrificed between 30 minutes and 24 hours after the particle administration. Lung and liver tissues were immediately collected, frozen and freeze-dried over night. D. Tissue Magnetic Characterization In the case of lung tissues, the whole organ was magnetically characterized, while in the case of livers, due to their greater size, the freeze-dried tissue was ground to powder in order to get a homogeneous sample and only part of it was used for the measurements. Tissue samples were transferred into gelatine capsule sample holders for their magnetic characterization in a Quantum Design MPMS-XL SQUID magnetometer with an AC option. AC Magnetic susceptibility measurements were performed in the temperature range 5–200 K, with an AC frequency of 1 Hz and amplitude of 0.25 mT.
Fig. 1. DMSA-MNP characterization: (a) Transmission electron micrograph and (b) DLS determined hydrodynamic size.
E. Magnetic Nanoparticle Quantification in Tissues To determine the concentration of DMSA-MNP in tissues the several steps described below were done following previous proposed guidelines [9], [10]. First, the temperature dependence of the out-of-phase magnetic susceptibility, , profile per mass of iron for several standards, that consisted of gel solutions of the DMSA-MNP with different particle concentrations, hence with different degrees of dipolar interactions, was measured. Then, the profile per mass of tissue for the different organs was measured and compared with the profile of the standards to select the standard that best represents the tissue profile both in shape and in temperature. Eventually, the iron concentration in the tissue in the form of DMSA-MNP is obtained from the number that multiplied by the profile of the standard yields the best fit with the tissue profile. The use of is especially adequate for quantification purposes because either paramagnetic or diamagnetic species do not contribute to the out-of-phase susceptibility. In addition, it is possible to distinguish the DMSA-MNP from other superparamagnetic biogenic species such as ferritin, because they differ in the temperature position of their maxima [13].
Fig. 2. Temperature dependence of the out-of-phase component of the AC magnetic susceptibility scaled to their maxima of the agar dilutions of the DMSA-MNP (D1–D4) and the lung and liver freeze-dried tissues from animals that were treated with two different anesthetics (isoflurane or ketamine/xylazine) during the magnetic nanoparticles administration and a single dose of DMSA-MNP.
III. RESULTS AND DISCUSSION Uniform nanoparticles with TEM core diameter of and hydrodynamic diameter of 110 nm were synthetised (Fig. 1). The DLS results suggest a small degree of particle aggregation in the suspension. AC magnetic susceptibility measurements of the DMSAMNP, dispersed in agar and prepared at different particle concentrations, have been previously carried out [10]. Briefly, the magnetic susceptibility of the DMSA-MNP presents a single peak in both the in-phase and the out-of-phase components,
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Fig. 3. Temperature dependence of the out-of-phase component of the magnetic susceptibility per mass of lung and liver freeze-dried tissues from animals that were treated with two different anesthetics (isoflurane and ketamine/xylazine) during the single dose of DMSA-MNP administration.
indicative of the blocking of the particle magnetic moments. The location in temperature of the AC susceptibility peak of the DMSA-MNP samples with different dipolar interactions goes from about 70 K, for the most concentrated samples, to 40 K, for the most diluted ones, as a result of different particle aggregation [10] (Fig. 2). Magnetic measurements have been performed in lung and liver tissues of animals that were anesthetized in two different ways before the magnetic nanoparticle administration in order to study the influence of the anesthetic on the MNP biodistribution. These two organs, liver and lungs, were chosen because DMSA-MNP were detected in them after intravenous injection [14], [15]. It is known that the liver is usually the main organ that accumulates iron-containing MNP [16]. Our interest in the lungs is based in the fact that a preferential targeting of the DMSA coated particles to this tissue has been previously reported [14]. The other organ that usually accumulates MNP is the spleen although the percentage of particles that goes to this organ is usually much lower than the other two [10], [16]. The presence of DMSA-MNP in lung and liver tissues was confirmed by AC magnetic susceptibility measurements (Fig. 2). The temperature location of the out-of-phase susceptibility maxima for the lung and liver tissues, around 65–75 K, is quite similar to that of the most concentrated agar solution of DMSA-MNP (D1) (Fig. 2). Slight differences in the out-of-phase susceptibility profile can be observed between the tissues and the agar dilutions of DMSA-MNP. In particular, the out-of-phase susceptibility maxima for the lung tissues seem to be located at slightly higher temperatures than in the case of the liver tissues and the agar solutions of DMSA-MNP. This fact may indicate stronger dipolar interactions between particles in the lungs, as a result of a slightly higher aggregation of the particles. The height of the out-of-phase susceptibility maxima per mass of sample can be used as a surrogate measure of the amount of particles in the tissue. In Fig. 3, results from the AC magnetic susceptibility characterization of tissues from animals that received a single dose of DMSA-MNP are presented. In this figure, it can be observed that the height of the out-of-phase susceptibility maxima of the liver tissues from animals treated with the two kinds of anesthesia does not differ much, whereas striking differences can be observed when comparing the amount of par-
Fig. 4. Concentration of iron in the form of magnetic nanoparticles in lung and liver tissues as determined from the magnetic data. The horizontal bars indicate mean values into each animal group. (A) Lungs. (B) Liver.
ticles in the lung tissues of animals that received different anesthetic treatments. In particular, the height of the maxima of the lung tissues from the mouse treated with the mixture of ketamine/ xylazine is three times higher than that of the lung tissues from the mouse treated with isoflurane. It is known that isoflurane produces systemic vasodilation [17] and lowers the cardiac output, although it is still not clear why it changes the particle accumulation in the lungs and not in the liver tissues. The concentration of iron in the form of MNP in the tissues has been calculated following the method previously described [9], [10]. For the animals that received multiple doses of DMSA-MNP, these calculations resulted in the following iron concentrations: and mg/g dry tissue respectively for the liver and lungs of the mice anesthetized with ketamine/xylazine and and mg/g dry tissue respectively for the liver and the lungs of the mice anesthetized with isoflurane (Fig. 4). Differences in the iron concentration between the two treatments are not significantly different for the liver tissues while they significantly differ when comparing the lung tissues . These results indicate that the anesthesia used during the particle administration influences the particle distribution, in particular, changing the amount of particles that reaches the lungs, which could be a source of toxicity. Therefore, this parameter should be taken into account for future studies on the biodistribution of nanoparticles or when comparing results from different studies. The amount of particles that reach the lungs follow the same anesthesia dependent pattern for animals treated with a single
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MSPS) (PI060549 to FJL; Cooperative Research Thematic Network program (RETICS) and ResearchNetwork in Inflammation and Rheumatic Diseases (RIER) RD08/0075/0015 to DFB) and EU-FP7 MULTIFUN project (n 262943). LG holds a Sara Borrell postdoctoral contract (CD09/00030) from the ISCIIIMSPS. REFERENCES
Fig. 5. Percentage of the administered magnetic nanoparticles found in each organ from animals treated with a single dose of DMSA-MNP and different anesthetics during the particle administration. Values have been calculated from the total amount of iron found in each whole tissue in the form of MNP from the magnetic quantification with respect to the total amount of injected iron. Whereas the amount of MNP that reaches the liver is similar when using both anesthetics, the amount of MNP that accumulates in the lungs is three times lower when using isoflurane in comparison with ketamine/xylazine. (A) Ketamine/xylazine. (B) Isoflurane.
dose of DMSA-MNP (Fig. 4). However, while the different number of DMSA-MNP doses seems not to affect the concentration of iron in the form of particles in the lung tissues, multiple doses give rise to an increased concentration of iron in the form of MNP in the liver tissues with respect to animals treated with just a single dose. For the animals that just received a single injection of 300 g of MNP, the percentage of particles that were found in each organ was calculated (results shown in Fig. 5). The rest of the particles that have not been found in the liver and lungs (53–56% of the injected particles) may probably be still either in the blood stream or in the spleen, which is the other organ where these particles have been found to accumulate [10]. IV. CONCLUSION There is a clear influence of the anaesthesia used during magnetic nanoparticle administration on particle biodistribution within the different tissues. In particular, we have found that inhaled isoflurane (0.5% in oxygen) reduces the amount of magnetic nanoparticles that that reaches the lungs when compared with the use of a mixture of ketamine and xylazine intraperitoneally injected. Therefore, it will be fundamental to take this parameter into account when comparing different studies on magnetic nanoparticle biodistributions. ACKNOWLEDGMENT This work was partially supported by grants rom the Spanish Ministry of Economy and Competitiveness (MAT2011-23641 and CSD2007-00010 to MPM, SAF-2011-23639 to DFB), the Madrid regional government CM (S009/MAT-1726 to MPM), the ISCIII- Spanish Ministry for Health & Social Policy (ISCIII-
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