Materials Science and Engineering C 33 (2013) 746–751
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Nitric oxide donor superparamagnetic iron oxide nanoparticles Miguel M. Molina a, Amedea B. Seabra b, Marcelo G. de Oliveira c, Rosangela Itri a, Paula S. Haddad b,⁎ a b c
Instituto de Física, Universidade de São Paulo, São Paulo, São Paulo, 05508–090, Brazil Departamento de Ciências Exatas e da Terra, Universidade Federal de São Paulo, Diadema, SP, 09972–270, Brazil Instituto de Química, Universidade Estadual de Campinas, Campinas, São Paulo, 13083–970, Brazil
a r t i c l e
i n f o
Article history: Received 16 August 2012 Received in revised form 14 October 2012 Accepted 30 October 2012 Available online 12 November 2012 Keywords: Magnetite nanoparticles Nitric oxide S-nitrosothiol Iron oxide nanoparticles Nanomaterials Nanotechnology
a b s t r a c t This work reports a new strategy for delivering nitric oxide (NO), based on magnetic nanoparticles (MNPs), with great potential for biomedical applications. Water-soluble magnetic nanoparticles were prepared through a co-precipitation method by using ferrous and ferric chlorides in acidic solution, followed by a mercaptosuccinic acid (MSA) coating. The thiolated nanoparticles (SH-NPs) were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), and vibrating sample magnetometry (VSM). The results showed that the SH-NPs have a mean diameter of 10 nm and display superparamagnetic behavior at room temperature. Free thiol groups on the magnetite surface were nitrosated through the addition of an acidified nitrite solution, yielding nitrosated magnetic nanoparticles (SNO-NPs). The amount of NO covalently bound to the nanoparticles surface was evaluated by chemiluminescense. The SNO-NPs spontaneously released NO in aqueous solution at levels required for biomedical applications. This new magnetic NO-delivery vehicle has a great potential to generate desired amounts of NO directed to the target location. © 2012 Elsevier B.V. All rights reserved.
1. Introduction It is well known that the endogenous molecule nitric oxide (NO) is involved in several physiological processes, such as the control of vascular tone, the inhibition of platelet aggregation, smooth muscle cell replication, immune response, neuronal communication, and wound healing [1]. On the other hand, several pathologies are associated to dysfunctions in endogenous NO production. NO is considered a unique biomolecule due to its chemical nature: small size, lack of charge and high lipophilicity, which makes NO a highly diffusible molecule capable of diffusing through cell membranes to interact with intracellular targets, without the action of membrane channels or receptors. [2]. As a free radical in the biological medium, NO can readily react with biomolecules, leading to its inactivation. Therefore, there is great interest in the development of NO-releasing drugs and matrices which are capable of stabilizing and releasing NO locally, directly into different tissues and organs [3]. In recent decades, many different systems have been proposed for drug delivery, such as micelles [4–6], nanoparticles [7,8] and polymers [9]. In these systems, the drug may be captured, attached, adsorbed, or encapsulated in or on nanomatrices [10]. Among the nanostructured materials, magnetic nanoparticles (MNPs) appear as good candidates
⁎ Corresponding author at: Departamento de Ciências e da Terra, Universidade Federal de São Paulo, Rua Artur Riedel, 275- Diadema, SP, CEP: 09972–270, Brazil. Tel.: + 55 11 3319 3300; fax: + 55 11 4043 6428. E-mail address: [email protected] (P.S. Haddad). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.10.027
for drug delivery [11–17], in part due to their low toxicity, and mainly because they can be guided in vivo to the specific target sites by an external magnetic field [18]. In this context, iron oxide particles, such as magnetite (Fe3O4) or maghemite (γ-Fe2O3), are the most common MNPs used for biomedical applications. These NPs are of comparable size to important biological systems (at diameters smaller than 100 nm), presenting superparamagnetic behavior at room temperature [19–22], high effective surface areas, low sedimentation rates, and improved diffusion through tissues [1,23]. In general, in vitro studies of iron oxide MNPs demonstrated little or no toxicity of this material, indicating the biocompatibility of this nanovector [24,25]. Moreover, coated nanoparticles are found to be less toxic compared to uncoated nanoparticles, due to the presence of the biocompatible coating, which also lower protein adsorption on nanoparticle surface [26]. Similarly, in vivo studies based on intravenous/intraperitoneal administrations of iron oxide MNPs coated with different biocompatible ligands showed no long-term implications of their use when administered at clinically relevant concentrations [27]. In vivo, iron oxide nanoparticles are reported to be metabolized to iron ions, which are incorporated to the biological iron storage pool, such as erythrocytes, indicating the safe use of this nanomaterial [27]. Moreover, these nanoparticles have already been tested in phase I trial in patients with prostate cancer and no systemic toxicity was reported after several months post application [28]. Taken together, these results indicate that iron oxide nanoparticles are safe for several biomedical applications. Specific interactions with biomolecules usually require chemical modifications on the MNP surface. To our best knowledge, this is the
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first work that describes the synthesis and characterization of surfacemodified MNPs as vehicles to carry and deliver NO molecules. 2. Materials and methods 2.1. Materials Mercaptosuccinic acid (MSA), sodium nitrite (NaNO2), iron (III) chloride hexahydrate (FeCl3.6H2O), iron(II) chloride tetrahydrate, ammonium hydroxide (Sigma Aldrich Ch. Co., Inc., USA) and hydrochloric acid (12 mol/L, Synth, USA) were used as received. Aqueous solutions were prepared using analytical grade water from a Millipore Milli-Q Gradient filtration system. 2.2. Methods 2.2.1. Synthesis of magnetic nanoparticles The NPs were synthesized by using a co-precipitation method, as previously reported [29]. In brief, 4.0 mL of FeCl3·6H2O and 1.0 mL of FeCl2·4H2O (molar ratio 2:1), prepared in 1.0 mol/L HCl, were mixed and stirred, while a volume of 50 mL of NH4OH (0.7 mol/L) was added as precipitator. At this stage, the solution was centrifuged and the precipitate was decanted, followed by the addition of 6.0 mL of oleic acid. This mixture was then stirred for 20 min. The solution was centrifuged several times and the new precipitate was washed several times with ethanol and acetone, leading to a NP covered with oleic acid. 2.2.2. Adsorption of mercaptosuccinic acid Oleic acid coated-NPs (~ 10.0 mg) were suspended in 1.0 mL of toluene while MSA was dissolved in dimethyl sulfoxide (DMSO). The two solutions were mixed and vigorously stirred for 14 h producing a black powder that was isolated by centrifugation. This procedure led to ligand exchange and, hence, to the formation of water stable thiol-containing NPs (SH-NPs). 2.2.3. Nitrosation of MSA-coated MNPs An aqueous suspension of MSA-coated NPs was filtered in a Microcon centrifugal filter device containing ultrafiltration membranes (MWCO 10-kDa molar mass cut-off filter, Millipore, Billerica, MA, USA). The SH-NPs were washed with water and centrifuged 5 times for 10 min at 13,400 g in a Micromax RF centrifuge (Thermo IEC, Milford, MA, USA). The thiol groups present on the surface of MNPs were nitrosated through the addition of an acidified sodium nitrite solution. In this step, an amount of 4.6 mg of filtered SH-NPs was suspended in 1.0 mL of deionised water containing 5 μL of 6.0 mol/L aqueous HCl. A volume of 200 μL of aqueous sodium nitrite (60 mmol/L) was added to the SH-MNPs. After 15 min of incubation at room temperature, the nanoparticles suspension was filtered by centrifugal ultrafiltration and washed with deionised water, as described above. 2.2.4. Fourier transform infrared (FTIR) spectroscopy Dry Fe3O4-MSA (1:40) nanoparticles were mixed with pure potassium bromide (KBr) powder using a w/w sample:KBr ratio of 1:100. These mixtures were ground into fine powders, pressed in a mechanical press to form translucent pellets and analyzed using a Bomen B-100 spectrometer (Hartmann & Braun, Baptiste, Quebec, Canada). A pure KBr pellet was used as background. The FTIR spectra were registered from 700 to 4000 cm −1 at a resolution of 2 cm −1. 2.2.5. X-ray diffraction (XRD) The diffractograms were obtained with approximately 200 mg of powdered Fe3O4-MSA onto a glass substrate of 2 × 2 cm. The measurements were performed in reflection set-up, with a conventional X-ray generator (CuKα radiation of 1.5418 Å and a graphite monochromator) coupled to a scintillation detector. The angular
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scanning performed on all samples ranged from 20 to 70º with 0.05º step-width at 5 s per angle. The average size of the nanoparticles was calculated from the full width at half maximum of the (311) reflection (spinel structure) using the Scherrer's equation [30]. 2.2.6. Transmission electron microscopy (TEM) Photomicrographies of the nanoparticles were obtained using a Philips CM200 transmission electron microscope (TEM) with an energy dispersive spectrometer, operating at 160 kV. 2.2.7. Vibrating sample magnetometry (VSM) A VSM was used to obtain the magnetization versus magnetic field loop (M versus H) at room temperature up to H = 20 kOe. The apparatus was calibrated with a Ni pattern. The magnetization measurements were carried out on a known quantity of powdered sample, slightly pressed and conditioned in cylindrical Lucite holders. 2.2.8. Detection of total free NO released from nitrosated ultra filtered MNPs The total amount of NO released from nitrosated MNPs (SNO-NPs) was measured by chemiluminescence using a Sievers chemiluminescence NO analyzer® (NOA 280i, GE Analytical, Boulder, CO, USA). Aliquots of 10 and 100 μL of aqueous suspensions of SNO-NPs (3.8 mg of nanoparticles/mL), and controls (aqueous suspension of SH-MNPs, and aqueous solution of sodium nitrite) were injected into the sampling compartment which contained 5.0 mL of an aqueous solution of ascorbic acid (160 mmol/L at pH 11). This condition allowed the detection of free NO released from S-NO groups present on nanoparticles surface, without the influence of nitrite. Calibration curves were obtained with aqueous solutions of S-nitrosoglutathione, which were freshly prepared and immediately analyzed (data not shown). 2.2.9. Kinetics of NO release from SNO-NPs in aqueous solution The NO release profile from nitrosated nanoparticles was obtained in real time by chemiluminescence. SNO-NPs were freshly prepared by adding 2.20 mg of SH-MNPs to 200 μL of deionized water and homogenized. After homogenization, aliquots of 10 μL of this suspension were added to 3.0 mL of deionized water that contained 10 μL of aqueous HCl (0.6 mol/L). A volume of 30 μL of aqueous NaNO2 (50 mmol/L) was added to the SH-NPs suspension. The final suspension was homogenized, protected from light with an aluminum foil, and kept at room temperature (25 °C) for kinetic measurements. The stability of S-NO groups present on the surface of the MNPs was kinetically monitored at 25 °C, in the dark, by injecting 5.0 μL of the sample into the NO analyzer, at different time intervals over 6.7 h. Before each injection, the samples were vigorously homogenized in a vortex. The experiments were performed in duplicate. 3. Results and discussion The transfer of NPs from the organic to the aqueous phase provides biostability to the particles at physiological pH. Synthetic MNPs are generally surface-modified with hydrophobic ligands, becoming unstable in aqueous suspension. In this work, the nanoparticles were initially coated with oleic acid and were therefore insoluble in water. Replacement of oleic acid by MSA led to MNPs containing sulphydryl (SH) groups on the surface, as identified by FTIR spectroscopy. This technique is useful to identify the most important stretching vibrations of the MSA ligand attached on the particle surface [31]. Fig. 1 shows the IR spectrum of the synthesized NP coated with MSA at the molar ratio 1:40. The sharp band at 580 cm −1 is a fingerprint of Fe–O–Fe bond [32,33]. On the other hand, the peaks at 3437 cm −1 (νOH), 1707 cm −1 (νCO), 1611 cm −1 (νassymCOO) and 1408 cm −1 (νsymCOO) are assigned to the carboxylic group. It is possible to observe that there are two small peaks at 2845 and 2924 cm −1 that can be are attributed to the νSH of the free thiol group of the MSA
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M.M. Molina et al. / Materials Science and Engineering C 33 (2013) 746–751 Table 1 Particle size (diameter) and values of saturation magnetization of sample 1: NPs coated with oleic acid and of sample 2: MSA-coated NPs obtained by XRD, TEM and VSM, respectively. The uncertainties are displayed in parenthesis.
Fig. 1. FTIR spectrum of Fe3O4-MSA (molar ratio 1:40) nanoparticles in the 400– 4000 cm−1 range. The bands at 3437 (2924 and 2845), 1707, 1611, 1408 and 580 cm−1 are associated with the νOH, νSH, ν(CO), νasym(COO), νsym(COO) and Fe–O–Fe vibrations, respectively [33].
ligand [34]. Furthermore, the absence of bands between 400 and 500 cm −1 indicates that there was no dimerization of thiol groups (S-S) during the preparation procedures. Fig. 2 shows the X-ray diffractograms from two synthesized samples: MNPs coated with oleic acid (sample 1) followed by MSA exchange at molar ratio 1:40 (sample 2), respectively, along with their characterization as the inverted spinel structure [35]. The diffraction measurements demonstrated that the crystallographic structure of MNPs corresponds to magnetite (Fe3O4) (JCPDS 20–596). Crystallite sizes from 10 to 12 nm (Table 1) were obtained from Scherrer's equation [30] by taking into account the (311) Bragg's reflection. The morphologies and size distribution of the NPs were analyzed by transmission electron microscopy. Fig. 3A shows a representative image from sample 1. As can be observed, NPs are well defined with spherical contours, although they clearly present aggregation. The analysis of the size histogram displayed in Fig. 3B reveals a broad distribution with a mean dimension of 12 nm and polydispersity of 20%. On the other hand, the replacement of oleic acid by MSA favors MNP dispersion (Fig. 3C, sample 2). The size histogram (Fig. 3D) shows that the NP average size increases from 12 to 14 nm; however, the polydispersity decreases to 8%. Analysis of the size distribution histograms (Fig. 3B and D) indicates that ligand exchange favors the stabilization of larger particles. A careful observation of the contrast of the MNPs in the TEM images
Fig. 2. X-ray powder diffraction from samples coated with oleic acid (sample 1) and with MSA (sample 2) displaying the Bragg peak reflections of magnetite. The diffractograms are displaced for better visualization.
Sample
DRX (nm)
TEM (nm)
VSM σ (emu/g)
1 2
12.4 (0.6) 12.7 (0.5)
12.3 14.4
72.6 (0.5) 71.6 (0.5)
(Fig. 3A and C) reveals in some particles a dark-core light-shell type of contrast. Such a kind of contrast may be related to differences in the mean atomic number of the material in these two regions, suggesting that the nanoparticles are formed by a Fe-oxide shell with an inner oxygen-poor core. This core–shell morphology is in agreement with the formation of Fe3O4 during the synthesis [36]. The magnetization analysis, displayed in Fig. 4, was carried out through isothermal magnetic measurements in an applied field at room temperature. The hysteresis loops show MNP superparamagnetic behavior. Values of saturation magnetization around 72 emu/g were found (Table 1), compatible with that found for Fe3O4 magnetite [37], in spite of the ligand exchange from sample 1 to sample 2. Therefore, the combined results obtained by VSM, FTIR, DRX and TEM give support to conclude that the ligand exchange produced crystalline SH-NPs with narrow size distribution and superparamagnetic behavior at room temperature. In the present study, thiol groups present on the nanoparticle surface (SH-NPs) were nitrosated by the addition of sodium nitrite in acidified solution, leading to the formation of SNO-NPs (Scheme 1). In acidified aqueous solution, nitrite (NO2−) is in equilibrium with nitrous acid (HNO2), according to Eq. (1):
þ
NO2 ðaqÞ þ H ðaqÞ⇄HNO2 ðaqÞ
ð1Þ
Thus, nitrous acid is considered the nitrosating agent of SH groups leading to the formation of SNO groups [38]. The yield of the S-nitrosation reaction can be controlled by adjusting the molar ratio of available –SH groups to HONO (Scheme 1). Both –SNO and the remaining free –SH groups exposed on the surface of the MNPs will confer them hydrophilicity, assuring their stability in aqueous suspension, as desired for future applications. Fig. 5 shows representative chemiluminescence data for injections of (i) 100 μL of aqueous non-nitrosated MSA-coated magnetic nanoparticles (SH-NPs) and (ii) 100 μL of aqueous sodium nitrite, as control samples. The arrows in Fig. 5 indicate the injections. As expected, no signals of NO were detected for the control samples. Peaks (iii) and (iv) correspond to injections of 100 and 10 μL, respectively, of aqueous SNO-NPs. Peaks (iii) and (iv) correspond to the total NO released from SNO-MNPs. The presence of intense peaks can be observed for the SNO-NPs samples and the intensity of NO detection was found to be higher for the injection of 100 μL of SNO-NPs samples (peak (iii)), compared to the injection of only 10 μL of the same sample (peak (iv)). These results show that SH-NPs were readily nitrosated by the addition of sodium nitrite at low pH (Scheme 1). It was found that the amount of NO released from the particle surface of aqueous suspension of 3.8 mg of SNO-NPs was 160 μmol L −1 as assayed by chemiluminescence. This result showed that NO was successfully bound to the MSA-coated MNPs, leading to a new platform for NO delivery system, with great potential to be used in several NO-based biomedical applications. Particularly, the amount of NO released (μmol L −1) might be used to promote wound healing in vivo, as previously reported [39]. Concerning the NO release profile over time, Fig. 6 shows the total NO released from SNO-NPs after 1.0, 2.3, 4.2 and 6.7 h at 25 °C in the dark, measured by chemiluminescence. One can observe that
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Fig. 3. (A) TEM image of sample 1: NPs covered with oleic acid. (B) Corresponding size histogram of sample 1. Size: 12 nm; polydispersity: 20%. (C) TEM image of sample 2: MSA-coated NPs. (D) Corresponding size histogram of sample 2. Size: 14 nm; polydispersity: 8%. The scale bar corresponds to 100 nm for all images.
this new material is able to spontaneously release NO in aqueous suspension, in the dark, and at room temperature, for several hours. As previously reported for low molar mass S-nitrosothiols (RSNOs), such as S-nitrosoglutathione (GSNO), the S-NO groups undergo homolytic bond cleavage with free NO release [40].
The decomposition products after the NO release from the NP-SNO are dimmers of the mercaptosuccinic acid bound by a sulfur bridge, according to the reactions: •
•
NP−MSA−SNO→NP−MSA−S þ NO •
•
NP−MSA−S þ NP−MSA−S →NP−MSA−S−S−MSA−NP
ð2Þ ð3Þ
where NP-MSA-SNO represent the S-nitroso mercaptosuccinic acid molecules adsorbed on the NPs surface, NP-MSA-S • represents the
Fig. 4. Magnetization curves at room temperature for sample 1: NPs coated with oleic acid and sample 2: MSA-coated NPs.
Scheme 1. (Left panel) S-nitrosation of MSA-coated magnetic nanoparticles (SH-NPs) by nitrous acid (HNO2), generated from acidified aqueous sodium nitrite, leading to the formation of (right panel) S-nitrosated nanoparticles (SNO-NPs).
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nanoparticles. This tool can be used to increase local NO delivery from the nanoparticle directly to the target site of application. 4. Conclusion
Fig. 5. Representative chemiluminescence data for NO release from S-NO groups present on the surface of NPs. The arrows indicate the following injections: (i) 100 μL of aqueous solution of non-nitrosated MSA-coated magnetic nanoparticles (SH-NPs); (ii) 100 μL of aqueous solution of sodium nitrite; (iii) and (iv) 100 and 10 μL, respectively, of aqueous suspension of SNO-NPs.
thiyl radical formed after the homolitic S-N bond cleavage, and NP-MSA-S-S-MSA-NP the resulting dimmers which are expected to remain adsorbed on the NPs surface. Although desorption of the dimmers from the NPs surface cannot be excluded, it is unlikely that this will happen in significant extent, considering that the strong adsorption established between the carboxyl groups of the dimmer and the hydrated iron oxide surface remain unchanged. This NO release profile might find biomedical applications, which require a NO flux released locally over a range of hours. For example, by adjusting the dose of NO release, this material might be used to accelerate/promote wound healing or to coat blood-contact medical devices to inhibit platelet adhesion and thrombus formation [41]. In both cases, a sustained NO release directly to the site of application is desired, for a time frame of hours. In addition, due to the versatile chemistry of S-NO groups, it is possible to modulate the rates of NO release from SNO-NPs in order to adjust it to a particular biomedical application. For instance, the rates of NO release can be greatly accelerated by stimulation of the materials with visible light [40]. Therefore, this new material acts as a new vehicle to carry NO, it may load high amounts of NO, and deliver it in a sustained manner for several hours. Although this is the first work based on the preparation of NO-releasing iron oxide magnetic nanoparticles, the NO release profile from this new material is in accordance with other NO-releasing nanomaterials based on –S-NO modified dendrimers, and NO-releasing silica nanoparticles, already reported in the literature [42–46]. One major advantage of this material is the superparamagnetic behavior of
Fig. 6. NO release profile from SNO-NPs in aqueous suspension, at 25 °C, in the dark for up to 6.7 h.
This work describes the preparation and characterization of MNPs as new NO-delivery platform. Firstly, NPs were prepared through the co-precipitation method, and coated with (MSA), leading to the formation of a stable aqueous dispersion of thiolated nanoparticles (SH-NPs). The free thiol (SH) groups of MSA ligand were used as sites to covalently bind NO. SH-NPs were S-nitrosated leading to the formation of SNO-NPs, which acted as NO donors in aqueous solution. This new NO-releasing nanomaterial might find important biomedical applications, since due to the importance of NO in vivo, vehicles that act as NO carriers and donors are extremely attractive for diverse pharmacological applications [45,46]. Toxicological evaluations of NO-releasing MNPs are currently in progress. Acknowledgements The authors acknowledge the financial support of FAPESP (proc. nr. 2001/10125-0) and CNPq (proc. nr. 310209/2010 and 309390/ 2011-7). Thanks are also due to Elisa Ferreira for her assistance with the NOA, Dr. Mauricio S. Baptista (IQUSP) for the use of laboratory facilities, Dr. Antonio Domingues (IFUSP) for the VSM measurements Simone Ferreira and Pedro K. Kiyohara for technical assistance with TEM measurements and Prof. Carol Hollingworth Collins for assistance in revising the manuscript. R. Itri, A.B. Seabra and M.G. Oliveira acknowledge CNPq research fellowships. P.S. Haddad acknowledges FAPESP research fellowship. M.M. Molina is a recipient of a Capes fellowship. References [1] L.J. Ignarro, Nitric Oxide, Biology and Pathobiology, Academic Press, San Diego, CA, 2000. [2] F.S. Dioguardi, J. Nutrigenet. Nutrigenomics 4 (2011) 90–98. [3] A.B. Seabra, N. Duran, J. Math. Chem. 20 (2010) 1624–1637. [4] Y. Matsumura, Jpn. J. Clin. Oncol. 38 (2008) 793–802. [5] H.M. Aliabadi, M. Shahin, D.R. Brocks, A. Lavasanifiar, Clin. Pharmacokinet. 47 (2008) 619–634. [6] M. Yokoyama, J. Artif. Organs 8 (2011) 238–244. [7] K.E. Uhrich, S.M. Cannizzaro, R.S. Langer, K.M. Shakesheff, Chem. Rev. 99 (1999) 3181–3198. [8] E. Soussan, S. Cassel, M. Blanzat, I. Rico-Lattes, Angew. Chem. Int. Ed. 48 (2009) 274–288. [9] E. Gullotti, Y. Yeo, Mol. Pharm. 6 (2009) 1041–1051. [10] R. Singh, J.W. Lillard, Exp. Mol. Pathol. 86 (2009) 215–223. [11] P. Tartaj, M.P. Morales, S. Veintemillas-Verdaguer, T.G. Carreno, C.J. Serna, J. Phys. D: Appl. Phys. 36 (2003) R182–R197. [12] D.L. Huber, Small 1 (2005) 482–501. [13] B. Fang, S. Gon, M. Park, K.N. Kumar, V.M. Rotello, K. Nusslein, M.M. Santore, Colloids Surf. B 87 (2011) 109–115. [14] J.E. Rosen, F.X. Gu, Langmuir 27 (2011) 10507–10513. [15] L.A. Thomas, L. Dekker, M. Kallumadil, P. Southern, M. Wilson, S.P. Nair, Q.A. Pankhurst, I.P. Parkin, J. Mater. Chem. 19 (2009) 6529–6535. [16] M. Jeun, S. Bae, A. Tomitaka, Y. Takemura, K.H. Park, S.H. Paek, K.W. Chung, Appl. Phys. Lett. 95 (2009) 2501–2503. [17] T.K. Nguyen, L. Thanha, A.W. Greena, Nano Today 5 (2010) 213–230. [18] P.S. Haddad, A.B. Seabra, in: A.I. Martinez (Ed.), Iron Oxides: Structure, Properties and Applications, Nova Science Publishers, 2012. [19] C.P. Bean, I.S. Jacobis, J. Appl. Phys. 30 (1956) 1448–1452. [20] B.D. Cullity, C.D. Graham, Introduction to magnetic materials, 1st ed., Addison-Wesley Publishing Company Inc., 1972. [21] M.P. Morales, S. Veintemillas-Verdaguer, M.I. Montero, C.J. Serna, A. Roig, L. Casas, B. Martinez, F. Sandiumenge, Chem. Mater. 11 (1999) 3058–3064. [22] X. Batlle, A. Labarta, J. Phys. D: Appl. Phys. 35 (2002) R15–R42. [23] D. Portet, B. Denizot, E. Rump, J.J. Lejeune, P. Jallet, J. Colloid Interface Sci. 238 (2001) 37–42. [24] A. Petri-Fink, B. Steitz, A. Finka, J. Salaklang, H. Hofmann, Eur. J. Pharm. Biopharm. 68 (2008) 129–137. [25] M. Mahmoudi, M.A. Shokrgozar, A. Simchi, M. Imani, A.S. Milani, P. Stroeve, H. Vali, U.O. Hafeli, S. Bonakdar, J. Phys. Chem. C 113 (2009) 2322–2331. [26] M. Mahmoudi, H. Hofmann, B. Rothen-Rutishauser, A. Petri-Fink, Chem. Rev. 112 (2012) 2323–2338. [27] Y.-F. Li, C. Chen, Small 7 (2011) 2965–2980.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Nitric oxide donor superparamagnetic iron oxide nanoparticles Miguel M. Molina a, Amedea B. Seabra b, Marcelo G. de Oliveira c, Rosangela Itri a, Paula S. Haddad b,⁎ a b c
Instituto de Física, Universidade de São Paulo, São Paulo, São Paulo, 05508–090, Brazil Departamento de Ciências Exatas e da Terra, Universidade Federal de São Paulo, Diadema, SP, 09972–270, Brazil Instituto de Química, Universidade Estadual de Campinas, Campinas, São Paulo, 13083–970, Brazil
a r t i c l e
i n f o
Article history: Received 16 August 2012 Received in revised form 14 October 2012 Accepted 30 October 2012 Available online 12 November 2012 Keywords: Magnetite nanoparticles Nitric oxide S-nitrosothiol Iron oxide nanoparticles Nanomaterials Nanotechnology
a b s t r a c t This work reports a new strategy for delivering nitric oxide (NO), based on magnetic nanoparticles (MNPs), with great potential for biomedical applications. Water-soluble magnetic nanoparticles were prepared through a co-precipitation method by using ferrous and ferric chlorides in acidic solution, followed by a mercaptosuccinic acid (MSA) coating. The thiolated nanoparticles (SH-NPs) were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), and vibrating sample magnetometry (VSM). The results showed that the SH-NPs have a mean diameter of 10 nm and display superparamagnetic behavior at room temperature. Free thiol groups on the magnetite surface were nitrosated through the addition of an acidified nitrite solution, yielding nitrosated magnetic nanoparticles (SNO-NPs). The amount of NO covalently bound to the nanoparticles surface was evaluated by chemiluminescense. The SNO-NPs spontaneously released NO in aqueous solution at levels required for biomedical applications. This new magnetic NO-delivery vehicle has a great potential to generate desired amounts of NO directed to the target location. © 2012 Elsevier B.V. All rights reserved.
1. Introduction It is well known that the endogenous molecule nitric oxide (NO) is involved in several physiological processes, such as the control of vascular tone, the inhibition of platelet aggregation, smooth muscle cell replication, immune response, neuronal communication, and wound healing [1]. On the other hand, several pathologies are associated to dysfunctions in endogenous NO production. NO is considered a unique biomolecule due to its chemical nature: small size, lack of charge and high lipophilicity, which makes NO a highly diffusible molecule capable of diffusing through cell membranes to interact with intracellular targets, without the action of membrane channels or receptors. [2]. As a free radical in the biological medium, NO can readily react with biomolecules, leading to its inactivation. Therefore, there is great interest in the development of NO-releasing drugs and matrices which are capable of stabilizing and releasing NO locally, directly into different tissues and organs [3]. In recent decades, many different systems have been proposed for drug delivery, such as micelles [4–6], nanoparticles [7,8] and polymers [9]. In these systems, the drug may be captured, attached, adsorbed, or encapsulated in or on nanomatrices [10]. Among the nanostructured materials, magnetic nanoparticles (MNPs) appear as good candidates
⁎ Corresponding author at: Departamento de Ciências e da Terra, Universidade Federal de São Paulo, Rua Artur Riedel, 275- Diadema, SP, CEP: 09972–270, Brazil. Tel.: + 55 11 3319 3300; fax: + 55 11 4043 6428. E-mail address: [email protected] (P.S. Haddad). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.10.027
for drug delivery [11–17], in part due to their low toxicity, and mainly because they can be guided in vivo to the specific target sites by an external magnetic field [18]. In this context, iron oxide particles, such as magnetite (Fe3O4) or maghemite (γ-Fe2O3), are the most common MNPs used for biomedical applications. These NPs are of comparable size to important biological systems (at diameters smaller than 100 nm), presenting superparamagnetic behavior at room temperature [19–22], high effective surface areas, low sedimentation rates, and improved diffusion through tissues [1,23]. In general, in vitro studies of iron oxide MNPs demonstrated little or no toxicity of this material, indicating the biocompatibility of this nanovector [24,25]. Moreover, coated nanoparticles are found to be less toxic compared to uncoated nanoparticles, due to the presence of the biocompatible coating, which also lower protein adsorption on nanoparticle surface [26]. Similarly, in vivo studies based on intravenous/intraperitoneal administrations of iron oxide MNPs coated with different biocompatible ligands showed no long-term implications of their use when administered at clinically relevant concentrations [27]. In vivo, iron oxide nanoparticles are reported to be metabolized to iron ions, which are incorporated to the biological iron storage pool, such as erythrocytes, indicating the safe use of this nanomaterial [27]. Moreover, these nanoparticles have already been tested in phase I trial in patients with prostate cancer and no systemic toxicity was reported after several months post application [28]. Taken together, these results indicate that iron oxide nanoparticles are safe for several biomedical applications. Specific interactions with biomolecules usually require chemical modifications on the MNP surface. To our best knowledge, this is the
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first work that describes the synthesis and characterization of surfacemodified MNPs as vehicles to carry and deliver NO molecules. 2. Materials and methods 2.1. Materials Mercaptosuccinic acid (MSA), sodium nitrite (NaNO2), iron (III) chloride hexahydrate (FeCl3.6H2O), iron(II) chloride tetrahydrate, ammonium hydroxide (Sigma Aldrich Ch. Co., Inc., USA) and hydrochloric acid (12 mol/L, Synth, USA) were used as received. Aqueous solutions were prepared using analytical grade water from a Millipore Milli-Q Gradient filtration system. 2.2. Methods 2.2.1. Synthesis of magnetic nanoparticles The NPs were synthesized by using a co-precipitation method, as previously reported [29]. In brief, 4.0 mL of FeCl3·6H2O and 1.0 mL of FeCl2·4H2O (molar ratio 2:1), prepared in 1.0 mol/L HCl, were mixed and stirred, while a volume of 50 mL of NH4OH (0.7 mol/L) was added as precipitator. At this stage, the solution was centrifuged and the precipitate was decanted, followed by the addition of 6.0 mL of oleic acid. This mixture was then stirred for 20 min. The solution was centrifuged several times and the new precipitate was washed several times with ethanol and acetone, leading to a NP covered with oleic acid. 2.2.2. Adsorption of mercaptosuccinic acid Oleic acid coated-NPs (~ 10.0 mg) were suspended in 1.0 mL of toluene while MSA was dissolved in dimethyl sulfoxide (DMSO). The two solutions were mixed and vigorously stirred for 14 h producing a black powder that was isolated by centrifugation. This procedure led to ligand exchange and, hence, to the formation of water stable thiol-containing NPs (SH-NPs). 2.2.3. Nitrosation of MSA-coated MNPs An aqueous suspension of MSA-coated NPs was filtered in a Microcon centrifugal filter device containing ultrafiltration membranes (MWCO 10-kDa molar mass cut-off filter, Millipore, Billerica, MA, USA). The SH-NPs were washed with water and centrifuged 5 times for 10 min at 13,400 g in a Micromax RF centrifuge (Thermo IEC, Milford, MA, USA). The thiol groups present on the surface of MNPs were nitrosated through the addition of an acidified sodium nitrite solution. In this step, an amount of 4.6 mg of filtered SH-NPs was suspended in 1.0 mL of deionised water containing 5 μL of 6.0 mol/L aqueous HCl. A volume of 200 μL of aqueous sodium nitrite (60 mmol/L) was added to the SH-MNPs. After 15 min of incubation at room temperature, the nanoparticles suspension was filtered by centrifugal ultrafiltration and washed with deionised water, as described above. 2.2.4. Fourier transform infrared (FTIR) spectroscopy Dry Fe3O4-MSA (1:40) nanoparticles were mixed with pure potassium bromide (KBr) powder using a w/w sample:KBr ratio of 1:100. These mixtures were ground into fine powders, pressed in a mechanical press to form translucent pellets and analyzed using a Bomen B-100 spectrometer (Hartmann & Braun, Baptiste, Quebec, Canada). A pure KBr pellet was used as background. The FTIR spectra were registered from 700 to 4000 cm −1 at a resolution of 2 cm −1. 2.2.5. X-ray diffraction (XRD) The diffractograms were obtained with approximately 200 mg of powdered Fe3O4-MSA onto a glass substrate of 2 × 2 cm. The measurements were performed in reflection set-up, with a conventional X-ray generator (CuKα radiation of 1.5418 Å and a graphite monochromator) coupled to a scintillation detector. The angular
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scanning performed on all samples ranged from 20 to 70º with 0.05º step-width at 5 s per angle. The average size of the nanoparticles was calculated from the full width at half maximum of the (311) reflection (spinel structure) using the Scherrer's equation [30]. 2.2.6. Transmission electron microscopy (TEM) Photomicrographies of the nanoparticles were obtained using a Philips CM200 transmission electron microscope (TEM) with an energy dispersive spectrometer, operating at 160 kV. 2.2.7. Vibrating sample magnetometry (VSM) A VSM was used to obtain the magnetization versus magnetic field loop (M versus H) at room temperature up to H = 20 kOe. The apparatus was calibrated with a Ni pattern. The magnetization measurements were carried out on a known quantity of powdered sample, slightly pressed and conditioned in cylindrical Lucite holders. 2.2.8. Detection of total free NO released from nitrosated ultra filtered MNPs The total amount of NO released from nitrosated MNPs (SNO-NPs) was measured by chemiluminescence using a Sievers chemiluminescence NO analyzer® (NOA 280i, GE Analytical, Boulder, CO, USA). Aliquots of 10 and 100 μL of aqueous suspensions of SNO-NPs (3.8 mg of nanoparticles/mL), and controls (aqueous suspension of SH-MNPs, and aqueous solution of sodium nitrite) were injected into the sampling compartment which contained 5.0 mL of an aqueous solution of ascorbic acid (160 mmol/L at pH 11). This condition allowed the detection of free NO released from S-NO groups present on nanoparticles surface, without the influence of nitrite. Calibration curves were obtained with aqueous solutions of S-nitrosoglutathione, which were freshly prepared and immediately analyzed (data not shown). 2.2.9. Kinetics of NO release from SNO-NPs in aqueous solution The NO release profile from nitrosated nanoparticles was obtained in real time by chemiluminescence. SNO-NPs were freshly prepared by adding 2.20 mg of SH-MNPs to 200 μL of deionized water and homogenized. After homogenization, aliquots of 10 μL of this suspension were added to 3.0 mL of deionized water that contained 10 μL of aqueous HCl (0.6 mol/L). A volume of 30 μL of aqueous NaNO2 (50 mmol/L) was added to the SH-NPs suspension. The final suspension was homogenized, protected from light with an aluminum foil, and kept at room temperature (25 °C) for kinetic measurements. The stability of S-NO groups present on the surface of the MNPs was kinetically monitored at 25 °C, in the dark, by injecting 5.0 μL of the sample into the NO analyzer, at different time intervals over 6.7 h. Before each injection, the samples were vigorously homogenized in a vortex. The experiments were performed in duplicate. 3. Results and discussion The transfer of NPs from the organic to the aqueous phase provides biostability to the particles at physiological pH. Synthetic MNPs are generally surface-modified with hydrophobic ligands, becoming unstable in aqueous suspension. In this work, the nanoparticles were initially coated with oleic acid and were therefore insoluble in water. Replacement of oleic acid by MSA led to MNPs containing sulphydryl (SH) groups on the surface, as identified by FTIR spectroscopy. This technique is useful to identify the most important stretching vibrations of the MSA ligand attached on the particle surface [31]. Fig. 1 shows the IR spectrum of the synthesized NP coated with MSA at the molar ratio 1:40. The sharp band at 580 cm −1 is a fingerprint of Fe–O–Fe bond [32,33]. On the other hand, the peaks at 3437 cm −1 (νOH), 1707 cm −1 (νCO), 1611 cm −1 (νassymCOO) and 1408 cm −1 (νsymCOO) are assigned to the carboxylic group. It is possible to observe that there are two small peaks at 2845 and 2924 cm −1 that can be are attributed to the νSH of the free thiol group of the MSA
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M.M. Molina et al. / Materials Science and Engineering C 33 (2013) 746–751 Table 1 Particle size (diameter) and values of saturation magnetization of sample 1: NPs coated with oleic acid and of sample 2: MSA-coated NPs obtained by XRD, TEM and VSM, respectively. The uncertainties are displayed in parenthesis.
Fig. 1. FTIR spectrum of Fe3O4-MSA (molar ratio 1:40) nanoparticles in the 400– 4000 cm−1 range. The bands at 3437 (2924 and 2845), 1707, 1611, 1408 and 580 cm−1 are associated with the νOH, νSH, ν(CO), νasym(COO), νsym(COO) and Fe–O–Fe vibrations, respectively [33].
ligand [34]. Furthermore, the absence of bands between 400 and 500 cm −1 indicates that there was no dimerization of thiol groups (S-S) during the preparation procedures. Fig. 2 shows the X-ray diffractograms from two synthesized samples: MNPs coated with oleic acid (sample 1) followed by MSA exchange at molar ratio 1:40 (sample 2), respectively, along with their characterization as the inverted spinel structure [35]. The diffraction measurements demonstrated that the crystallographic structure of MNPs corresponds to magnetite (Fe3O4) (JCPDS 20–596). Crystallite sizes from 10 to 12 nm (Table 1) were obtained from Scherrer's equation [30] by taking into account the (311) Bragg's reflection. The morphologies and size distribution of the NPs were analyzed by transmission electron microscopy. Fig. 3A shows a representative image from sample 1. As can be observed, NPs are well defined with spherical contours, although they clearly present aggregation. The analysis of the size histogram displayed in Fig. 3B reveals a broad distribution with a mean dimension of 12 nm and polydispersity of 20%. On the other hand, the replacement of oleic acid by MSA favors MNP dispersion (Fig. 3C, sample 2). The size histogram (Fig. 3D) shows that the NP average size increases from 12 to 14 nm; however, the polydispersity decreases to 8%. Analysis of the size distribution histograms (Fig. 3B and D) indicates that ligand exchange favors the stabilization of larger particles. A careful observation of the contrast of the MNPs in the TEM images
Fig. 2. X-ray powder diffraction from samples coated with oleic acid (sample 1) and with MSA (sample 2) displaying the Bragg peak reflections of magnetite. The diffractograms are displaced for better visualization.
Sample
DRX (nm)
TEM (nm)
VSM σ (emu/g)
1 2
12.4 (0.6) 12.7 (0.5)
12.3 14.4
72.6 (0.5) 71.6 (0.5)
(Fig. 3A and C) reveals in some particles a dark-core light-shell type of contrast. Such a kind of contrast may be related to differences in the mean atomic number of the material in these two regions, suggesting that the nanoparticles are formed by a Fe-oxide shell with an inner oxygen-poor core. This core–shell morphology is in agreement with the formation of Fe3O4 during the synthesis [36]. The magnetization analysis, displayed in Fig. 4, was carried out through isothermal magnetic measurements in an applied field at room temperature. The hysteresis loops show MNP superparamagnetic behavior. Values of saturation magnetization around 72 emu/g were found (Table 1), compatible with that found for Fe3O4 magnetite [37], in spite of the ligand exchange from sample 1 to sample 2. Therefore, the combined results obtained by VSM, FTIR, DRX and TEM give support to conclude that the ligand exchange produced crystalline SH-NPs with narrow size distribution and superparamagnetic behavior at room temperature. In the present study, thiol groups present on the nanoparticle surface (SH-NPs) were nitrosated by the addition of sodium nitrite in acidified solution, leading to the formation of SNO-NPs (Scheme 1). In acidified aqueous solution, nitrite (NO2−) is in equilibrium with nitrous acid (HNO2), according to Eq. (1):
þ
NO2 ðaqÞ þ H ðaqÞ⇄HNO2 ðaqÞ
ð1Þ
Thus, nitrous acid is considered the nitrosating agent of SH groups leading to the formation of SNO groups [38]. The yield of the S-nitrosation reaction can be controlled by adjusting the molar ratio of available –SH groups to HONO (Scheme 1). Both –SNO and the remaining free –SH groups exposed on the surface of the MNPs will confer them hydrophilicity, assuring their stability in aqueous suspension, as desired for future applications. Fig. 5 shows representative chemiluminescence data for injections of (i) 100 μL of aqueous non-nitrosated MSA-coated magnetic nanoparticles (SH-NPs) and (ii) 100 μL of aqueous sodium nitrite, as control samples. The arrows in Fig. 5 indicate the injections. As expected, no signals of NO were detected for the control samples. Peaks (iii) and (iv) correspond to injections of 100 and 10 μL, respectively, of aqueous SNO-NPs. Peaks (iii) and (iv) correspond to the total NO released from SNO-MNPs. The presence of intense peaks can be observed for the SNO-NPs samples and the intensity of NO detection was found to be higher for the injection of 100 μL of SNO-NPs samples (peak (iii)), compared to the injection of only 10 μL of the same sample (peak (iv)). These results show that SH-NPs were readily nitrosated by the addition of sodium nitrite at low pH (Scheme 1). It was found that the amount of NO released from the particle surface of aqueous suspension of 3.8 mg of SNO-NPs was 160 μmol L −1 as assayed by chemiluminescence. This result showed that NO was successfully bound to the MSA-coated MNPs, leading to a new platform for NO delivery system, with great potential to be used in several NO-based biomedical applications. Particularly, the amount of NO released (μmol L −1) might be used to promote wound healing in vivo, as previously reported [39]. Concerning the NO release profile over time, Fig. 6 shows the total NO released from SNO-NPs after 1.0, 2.3, 4.2 and 6.7 h at 25 °C in the dark, measured by chemiluminescence. One can observe that
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Fig. 3. (A) TEM image of sample 1: NPs covered with oleic acid. (B) Corresponding size histogram of sample 1. Size: 12 nm; polydispersity: 20%. (C) TEM image of sample 2: MSA-coated NPs. (D) Corresponding size histogram of sample 2. Size: 14 nm; polydispersity: 8%. The scale bar corresponds to 100 nm for all images.
this new material is able to spontaneously release NO in aqueous suspension, in the dark, and at room temperature, for several hours. As previously reported for low molar mass S-nitrosothiols (RSNOs), such as S-nitrosoglutathione (GSNO), the S-NO groups undergo homolytic bond cleavage with free NO release [40].
The decomposition products after the NO release from the NP-SNO are dimmers of the mercaptosuccinic acid bound by a sulfur bridge, according to the reactions: •
•
NP−MSA−SNO→NP−MSA−S þ NO •
•
NP−MSA−S þ NP−MSA−S →NP−MSA−S−S−MSA−NP
ð2Þ ð3Þ
where NP-MSA-SNO represent the S-nitroso mercaptosuccinic acid molecules adsorbed on the NPs surface, NP-MSA-S • represents the
Fig. 4. Magnetization curves at room temperature for sample 1: NPs coated with oleic acid and sample 2: MSA-coated NPs.
Scheme 1. (Left panel) S-nitrosation of MSA-coated magnetic nanoparticles (SH-NPs) by nitrous acid (HNO2), generated from acidified aqueous sodium nitrite, leading to the formation of (right panel) S-nitrosated nanoparticles (SNO-NPs).
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nanoparticles. This tool can be used to increase local NO delivery from the nanoparticle directly to the target site of application. 4. Conclusion
Fig. 5. Representative chemiluminescence data for NO release from S-NO groups present on the surface of NPs. The arrows indicate the following injections: (i) 100 μL of aqueous solution of non-nitrosated MSA-coated magnetic nanoparticles (SH-NPs); (ii) 100 μL of aqueous solution of sodium nitrite; (iii) and (iv) 100 and 10 μL, respectively, of aqueous suspension of SNO-NPs.
thiyl radical formed after the homolitic S-N bond cleavage, and NP-MSA-S-S-MSA-NP the resulting dimmers which are expected to remain adsorbed on the NPs surface. Although desorption of the dimmers from the NPs surface cannot be excluded, it is unlikely that this will happen in significant extent, considering that the strong adsorption established between the carboxyl groups of the dimmer and the hydrated iron oxide surface remain unchanged. This NO release profile might find biomedical applications, which require a NO flux released locally over a range of hours. For example, by adjusting the dose of NO release, this material might be used to accelerate/promote wound healing or to coat blood-contact medical devices to inhibit platelet adhesion and thrombus formation [41]. In both cases, a sustained NO release directly to the site of application is desired, for a time frame of hours. In addition, due to the versatile chemistry of S-NO groups, it is possible to modulate the rates of NO release from SNO-NPs in order to adjust it to a particular biomedical application. For instance, the rates of NO release can be greatly accelerated by stimulation of the materials with visible light [40]. Therefore, this new material acts as a new vehicle to carry NO, it may load high amounts of NO, and deliver it in a sustained manner for several hours. Although this is the first work based on the preparation of NO-releasing iron oxide magnetic nanoparticles, the NO release profile from this new material is in accordance with other NO-releasing nanomaterials based on –S-NO modified dendrimers, and NO-releasing silica nanoparticles, already reported in the literature [42–46]. One major advantage of this material is the superparamagnetic behavior of
Fig. 6. NO release profile from SNO-NPs in aqueous suspension, at 25 °C, in the dark for up to 6.7 h.
This work describes the preparation and characterization of MNPs as new NO-delivery platform. Firstly, NPs were prepared through the co-precipitation method, and coated with (MSA), leading to the formation of a stable aqueous dispersion of thiolated nanoparticles (SH-NPs). The free thiol (SH) groups of MSA ligand were used as sites to covalently bind NO. SH-NPs were S-nitrosated leading to the formation of SNO-NPs, which acted as NO donors in aqueous solution. This new NO-releasing nanomaterial might find important biomedical applications, since due to the importance of NO in vivo, vehicles that act as NO carriers and donors are extremely attractive for diverse pharmacological applications [45,46]. Toxicological evaluations of NO-releasing MNPs are currently in progress. Acknowledgements The authors acknowledge the financial support of FAPESP (proc. nr. 2001/10125-0) and CNPq (proc. nr. 310209/2010 and 309390/ 2011-7). Thanks are also due to Elisa Ferreira for her assistance with the NOA, Dr. Mauricio S. Baptista (IQUSP) for the use of laboratory facilities, Dr. Antonio Domingues (IFUSP) for the VSM measurements Simone Ferreira and Pedro K. Kiyohara for technical assistance with TEM measurements and Prof. Carol Hollingworth Collins for assistance in revising the manuscript. R. Itri, A.B. Seabra and M.G. Oliveira acknowledge CNPq research fellowships. P.S. Haddad acknowledges FAPESP research fellowship. M.M. Molina is a recipient of a Capes fellowship. References [1] L.J. Ignarro, Nitric Oxide, Biology and Pathobiology, Academic Press, San Diego, CA, 2000. [2] F.S. Dioguardi, J. Nutrigenet. Nutrigenomics 4 (2011) 90–98. [3] A.B. Seabra, N. Duran, J. Math. Chem. 20 (2010) 1624–1637. [4] Y. Matsumura, Jpn. J. Clin. Oncol. 38 (2008) 793–802. [5] H.M. Aliabadi, M. Shahin, D.R. Brocks, A. Lavasanifiar, Clin. Pharmacokinet. 47 (2008) 619–634. [6] M. Yokoyama, J. Artif. Organs 8 (2011) 238–244. [7] K.E. Uhrich, S.M. Cannizzaro, R.S. Langer, K.M. Shakesheff, Chem. Rev. 99 (1999) 3181–3198. [8] E. Soussan, S. Cassel, M. Blanzat, I. Rico-Lattes, Angew. Chem. Int. Ed. 48 (2009) 274–288. [9] E. Gullotti, Y. Yeo, Mol. Pharm. 6 (2009) 1041–1051. [10] R. Singh, J.W. Lillard, Exp. Mol. Pathol. 86 (2009) 215–223. [11] P. Tartaj, M.P. Morales, S. Veintemillas-Verdaguer, T.G. Carreno, C.J. Serna, J. Phys. D: Appl. Phys. 36 (2003) R182–R197. [12] D.L. Huber, Small 1 (2005) 482–501. [13] B. Fang, S. Gon, M. Park, K.N. Kumar, V.M. Rotello, K. Nusslein, M.M. Santore, Colloids Surf. B 87 (2011) 109–115. [14] J.E. Rosen, F.X. Gu, Langmuir 27 (2011) 10507–10513. [15] L.A. Thomas, L. Dekker, M. Kallumadil, P. Southern, M. Wilson, S.P. Nair, Q.A. Pankhurst, I.P. Parkin, J. Mater. Chem. 19 (2009) 6529–6535. [16] M. Jeun, S. Bae, A. Tomitaka, Y. Takemura, K.H. Park, S.H. Paek, K.W. Chung, Appl. Phys. Lett. 95 (2009) 2501–2503. [17] T.K. Nguyen, L. Thanha, A.W. Greena, Nano Today 5 (2010) 213–230. [18] P.S. Haddad, A.B. Seabra, in: A.I. Martinez (Ed.), Iron Oxides: Structure, Properties and Applications, Nova Science Publishers, 2012. [19] C.P. Bean, I.S. Jacobis, J. Appl. Phys. 30 (1956) 1448–1452. [20] B.D. Cullity, C.D. Graham, Introduction to magnetic materials, 1st ed., Addison-Wesley Publishing Company Inc., 1972. [21] M.P. Morales, S. Veintemillas-Verdaguer, M.I. Montero, C.J. Serna, A. Roig, L. Casas, B. Martinez, F. Sandiumenge, Chem. Mater. 11 (1999) 3058–3064. [22] X. Batlle, A. Labarta, J. Phys. D: Appl. Phys. 35 (2002) R15–R42. [23] D. Portet, B. Denizot, E. Rump, J.J. Lejeune, P. Jallet, J. Colloid Interface Sci. 238 (2001) 37–42. [24] A. Petri-Fink, B. Steitz, A. Finka, J. Salaklang, H. Hofmann, Eur. J. Pharm. Biopharm. 68 (2008) 129–137. [25] M. Mahmoudi, M.A. Shokrgozar, A. Simchi, M. Imani, A.S. Milani, P. Stroeve, H. Vali, U.O. Hafeli, S. Bonakdar, J. Phys. Chem. C 113 (2009) 2322–2331. [26] M. Mahmoudi, H. Hofmann, B. Rothen-Rutishauser, A. Petri-Fink, Chem. Rev. 112 (2012) 2323–2338. [27] Y.-F. Li, C. Chen, Small 7 (2011) 2965–2980.
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