ReseaRch aRticle

ISSN 1743-5889Nanomedicine (2010) 5(3), 397–40810.2217/NNM.10.15 © 2010 Future Medicine Ltd 397

Liver and brain imaging through dimercaptosuccinic acid-coated iron oxide nanoparticles

Magnetic nanoparticles have the potential to revolutionize current approaches to clinical diagnostic and therapeutic treatment [1–3]. The use of small superparamagnetic iron oxide nan-oparticles as contrast agents in MRI has gained wide acceptance for imaging vascular leakage, macrophages and for cell tracking [4–13]. A therapeutic drug linked to such nanoparticles would allow monitoring of targeted drug deliv-ery and controlled release by the application of an external magnetic field [14]. Currently availa-ble commercial contrast agents are poorly taken up by cells; although well-suited for magnetic imaging, they do not permit tissue and intra-cellular drug delivery [6]. A particularly promis-ing approach is the use of these nanoparticles for the detection and treatment of active lesions in neurodegenerative disease, since nanoparticles are able to cross the blood–brain barrier (BBB) in certain conditions, although with potential risk to health [15].

Intense interest is currently focused on improvement of magnetic nanoparticle design in terms of magnetic core, coating material and functional ligands [7,16]. Commercial products are usually synthesized by coprecipi-tation and are coated by diverse polymers or molecules. Hydrodynamic size and coating determine nanoparticle biodistribution and targeting of the organ or tissue to be imaged. Dextran- (Feridex®; Guerbet, Aulnay-sous-Bois, Parise, France) and carboxydextran-(Resovist®; Schering, Berlin, Germany) coated small paramagnetic iron oxide (SPIO) with hydrodynamic sizes of more than 100 nm are used to image the liver [6], whereas ultra small

paramagnetic iron oxide (USPIO) with hydro-dynamic sizes less than 50 nm are preferred for angiography and tumor permeability applica-tions [6,17]. Particle and aggregate size reduction nonetheless diminishes the magnetic properties and thus, image contrast. Moreover, magneti-cally controlled drug targeting and delivery require nanoparticles with high magnetization and the possibility of surface derivatization for drug attachment.

New synthetic methods have been developed in the last 10 years to improve crystallinity by nonhydrolytic thermal decomposition of organic precursors, which allows synthetic control of particle features such as size, magnetic dopants, magneto–crystalline phases and surface chem-istry [8,18]. These innovative nanoparticles show superior magnetic properties and MR contrast enhancement effects compared with those of conventional MR contrast agents [7].

Here, we synthesized magnetic nanoparticles by thermal decomposition of iron acetylaceto-nate in an organic medium [19], which assured formation of uniform, highly crystalline nano-particles with enhanced magnetic properties, including saturation magnetization values near those measured for the bulk material [20,21]. Water stability was attained by ligand exchange with dimercaptosuccinic acid (DMSA), which provides high stability in aqueous media and free ligand groups for further biomolecule con-jugation [22–24]. The attachment of cytokines to DMSA-coated magnetic nanoparticles pre-pared by this method shows promise as a sys-tem for controlled local drug release in cancer therapy [25].

Background & aim: Uptake, cytotoxicity and interaction of improved superparamagnetic iron oxide nanoparticles were studied in cells, tissues and organs after single and multiple exposures. Material & method: We prepared dimercaptosuccinic acid-coated iron oxide nanoparticles by thermal decomposition in organic medium, resulting in aqueous suspensions with a small hydrodynamic size (< 100 nm), high saturation magnetization and susceptibility, high nuclear magnetic resonance contrast and low cytotoxicity. Results: In vitro and in vivo behavior showed that these nanoparticles are efficient carriers for drug delivery to the liver and brain that can be combined with MRI detection.

KeywoRds: blood–brain barrier disruption n brain imaging n magnetic nanoparticles n NMR contrast agent n NMR imaging n UsPIo iron oxide

Raquel Mejías2, Sonia Pérez-Yagüe2, Alejandro G Roca1, Nicolás Pérez3, Ángeles Villanueva4, Magdalena Cañete4, Santos Mañes2, Jesús Ruiz-Cabello5, Marina Benito6, Amilcar Labarta3, Xavier Batlle3, Sabino Veintemillas-Verdaguer1, M Puerto Morales1†, Domingo F Barber2 & Carlos J Serna1

†Author for correspondence: 1Instituto de Ciencia de Materiales de Madrid/CSIC, Sor Juana Inés de la Cruz 3, Campus de Cantoblanco, Madrid 28049, Spain Tel.: +34 913 348 995 Fax: +34 913 720 623 puerto@icmm.csic.es 2Centro Nacional de Biotecnología/CSIC, Spain 3Universitat de Barcelona, Spain4Universidad Autónoma de Madrid, Madrid, Spain 5Universidad Complutense de Madrid & CIBER de Enfermedades Respiratorias, Madrid, Spain 6Hospital Gregorio Marañón, Madrid, Spain

ReseaRch aRticle Mejías, Pérez-Yagüe, Roca et al. Dimercaptosuccinic acid-coated iron oxide nanoparticles imaging ReseaRch aRticle

Nanomedicine (2010) 5(3)398 future science group

We prepared aqueous suspensions of uniform magnetic iron oxide nanoparticles of distinct sizes (SPIOs and USPIOs) and described their structural, colloidal, magnetic and relaxo metric characterization. These DMSA-coated iron oxide nanoparticles were used in a systematic study of cytotoxicity and particle internalization patterns in a human cervical carcinoma cell line (HeLa). In addition, we measured the extent of postinjection nanoparticle clearance and bio-accumulation in several organs, as well as the effect of repeated nanoparticle exposure in mice.

Finally, we tested delivery of these contrast agents into the healthy brain by transient, reversible hyperosmotic disruption of the BBB in rats. The BBB has a major role in main-taining the stable environment necessary for normal brain function. It is a semipermeable dynamic capillary membrane system, con-sisting of endothelial cells that contribute to its impermeability through the presence of tight junctions [26]. We disrupted the BBB by injecting hyperosmotic mannitol prior to nano-particle injection. In a normal brain injected intravenously with iron oxide nanoparticles, there is no iron oxide-induced MRI signal change or iron staining in the cerebral paren-chyma or CSF compartments [27]. However, in certain pathological situations, iron oxide nano particles can reach the brain, although the precise mechanism of uptake and brain distri-bution remains unknown [28]. SPIO or USPIO MRI enhancement can be observed in the brain only following situations such as BBB leakage, intravascular trapping or peripheral phagocyte infiltration [27].

Materials & methods�n Synthesis of aqueous colloidal

suspensions of magnetite nanoparticlesMagnetite nanoparticles of different sizes were synthesized by thermal decomposition of Fe(acac)

3 in the presence of oleic acid and

oleylamine as surfactants and organic solvents with different boiling points (bps). The small-est particles (4 nm) were synthesized using phe-nyl ether as solvent (sample APh) [29]. A mix-ture of 5.32 g of Fe(acac)3 (15 mmol), 16.93 g of 1,2-dodecanediol (75 mmol), 14.19 g of oleic acid (45 mmol), 17.27 g of oleylamine (45 mmol) and 150 ml of phenyl ether was introduced into a four-necked flask and heated up to 200°C for 120 min with mechanical stirring and under a nitrogen f low. Then, the solution was heated to reflux (bp 254°C) for 30 min in a N

2 atmosphere. To remove

impurities, the solution was mixed at room temperature with ethanol and centrifuged at 5650 g for 10 min, and the supernatant was discarded. Finally, nanoparticles were mixed with 40 ml of hexane and 0.1 ml of oleic acid, and centrifuged twice at 5650 g for 10 min and pure and stable hydrophobic magnetite suspen-sions were obtained. Following a similar pro-cedure, nanoparticles with 9 nm of size were obtained by using trioctylamine (sample AOn, bp 365°C) as solvent. Powders from these sus-pensions were obtained by precipitation with ethanol and drying at room temperature under a N

2 flow. It should be emphasized that this

method can be scaled-up to large production.Ligand exchange reaction of oleic acid for

DMSA was used to transform hydrophobic magnetite nanoparticles into hydrophilic ones, following an established procedure [30]. First, particles were coagulated from the hydrophobic suspension (50 mg/5 ml) by adding ethanol, cen-trifuged (2825 g, 10 min), and the solution was eliminated. Then, a mixture of 25 ml of toluene and a solution of 90 mg of DMSA in 5 ml of dimethyl sulfoxide was added to the coagulated particles, sonicated for 5 min, and mechanically stirred for 24 h. After that, toluene was added to the mixture reaction and centrifuged again, and the supernatant containing the oleic acid-coated particles was discarded. Finally, the pre-cipitated nanoparticles were successively mixed and centrifuged with ethanol and acetone several times to remove free oleic acid molecules. A new final step was introduced in the surface modi-fication process that consisted of the dispersion of the nanoparticles in alkaline water before its redispersion at pH 7 [23]. After that, the disper-sion was dialyzed, its pH was adjusted to 7, and finally, it was filtered through a 0.2 µm pore size syringe. Hydrophilic samples APhS and AOnS were named after the corresponding hydropho-bic samples APh and AOn. Both hydrophilic powders obtained by filtration and hydrophilic suspensions in water and agar were analyzed in this work.

�n Structural, colloidal, magnetic & relaxometric characterizationParticle size and size distribution were studied by transmission electron microscopy (TEM) using a 200-keV JEOL-2000 FXII microscope (Tokyo, Japan). TEM samples were prepared by placing one drop of a dilute magnetite nano-particle suspension in water on a carbon-coated copper grid and allowing the solvent to evapo-rate at room temperature. Average particle size

ReseaRch aRticle Mejías, Pérez-Yagüe, Roca et al. Dimercaptosuccinic acid-coated iron oxide nanoparticles imaging ReseaRch aRticle

www.futuremedicine.com 399future science group

(DTEM) and distribution were evaluated by measuring the largest internal dimension of at least 300 particles.

Magnetic characterization was carried out in a vibrating sample magnetometer (MLVSM9 MagLab 9 T, Oxford Instruments, Oxford, UK) at room temperature and 5 K. Magnetization curves were recorded by saturating the sample in a 5 T field and sweeping the field range between 5 and -5 T at 0.3 T/min.

Hydrophilic suspensions obtained after lig-and exchange were evaluated by the hydro-dynamic size and z potential evolution versus pH and time in a Zetasizer Nano ZS device (Malvern Instruments, Worcestershire, UK). z potential was measured at 0.01 M KNO

3

potassium nitrate. Iron content in suspensions was determined by chemical elemental ana lysis (PerkinElmer 2400CHN, MA, USA).

Relaxation time was measured in a MiniSpec MQ60 (Bruker, Karlsruhe, Germany) at 37°C with a 1.5 T magnetic field to evaluate the effi-ciency of the hydrophilic suspensions as contrast agents. Samples were stabilized by adding a 5% hot agar solution, for a final agar concentration of 3%. The relaxation rates R

1,2 (1/T

1,2, s-1) were

obtained from the relaxation times (T1,2

, s), and converted to relaxivities (r

1,2, s-1mM-1) by sub-

tracting the agar contribution and dividing by the Fe concentration, according to the equation R

1,2 = R0

1,2 + r

1,2[Fe], where R0

1,2 (s-1) is the relax-

ation rate in the absence of contrast agent and [Fe] is the contrast agent concentration (mM). The relaxivity is related to the proton relaxation rate per unit of contrast medium concentration.

�n Cell interaction & cytotoxicityHuman cervical carcinoma cell line cells were cultured (5% CO

2, 37°C) as monolayers in

Dulbecco’s modified Eagle’s medium with 50 µ/ml penicillin, 50 µl/ml streptomycin and 10% fetal bovine serum (all from Gibco, Paisley, Scotland). Cells were seeded into 24 multiwell plates at an initial density of 3.5 × 103 cells/well. Treatment was initiated 3 days after plating (approximately 70% confluence). To analyze nanoparticle inter-nalization, HeLa cells were seeded on 10-mm square glass coverslips and incubated (1–24 h) with different concentrations of nanoparticles (0.05, 0.1 and 0.5 mg Fe/ml). Culture medium was then removed, cells washed three times with phosphate-buffered saline (PBS) and analyzed immediately using bright field microscopy.

For iron detection, cells were fixed in ice-cold methanol (5 min), stained with an equal volume of 2% hydrochloric acid and 2% potassium

ferrocyanide trihydrate (Prussian blue; 15 min), and counterstained with 0.5% neutral red (3 min). Preparations were then washed with distilled water, air dried and mounted in DePeX (Serva, Germany). Analysis and photography were performed in an Olympus BX61 microscope with bright field illumination.

Human cervical carcinoma cell line cell via-bility was determined using the methyl thiazol tetrazolium bromide assay (MTT; Sigma, MO, USA). At 24 h after cell incubation with nano-particles, MTT was added to each well (50 µg/ml final concentration; 3 h, 37°C). The forma-zan formed in the cells was dissolved by addition of 0.5 ml dimethyl sulfoxide/dish; OD (optical density)

570 nm was measured (Tecan Spectra

Fluor spectrophotometer). Cell survival was expressed as the percent absorption of treated cells DMSA relative to that of controls (PBS). Results are expressed as the mean ± SD of six or more experiments.

�n In vivo biodistribution of nanoparticlesBlood clearance studies and MR imaging of rats before and after nanoparticle injection were performed to evaluate their utility as a contrast agent for MRI; for these experiments, we used an ultra-shielded Bruker Biospec 7/210 nuclear mag-netic resonance spectrometer operating at 7 T (1H frequency 300 MHz) with a maximum gradient strength of 750 mT/m. A region of interest of 4 mm diameter was selected for the signal inten-sity measurement in each organ and its decrease was relative to precontrast value (1). Male Wistar rats (6–8 weeks old from our own inbred stock colony; average weight 300 g; two per experi-ment) were anesthetized with a gas mixture of oxygen and 5–6% sevoflurane; this concentra-tion was reduced (to ~1%) to maintain anesthe-sia. Respiratory rate and body temperature were monitored and recorded constantly with an MRI compatible small animal monitoring system (SA Instruments). Body temperature was maintained at 36.5°C and respiratory rate at approximately 50 bmp by adjusting the gas mixture.

Nanoparticle half-life in blood was determined after intravenous injection (85 µmol Fe/kg body weight, dose currently used for nuclear magnetic resonance commercial products). Blood samples (0.3 ml) were drawn from the tail vein before injection, and once every 10 min for 90 min postinjection. To determine the nuclear mag-netic relaxation times T

1 and T

2, blood samples

were diluted in PBS to a total Fe concentration less than 1 mM. Blood half-life of nanoparticles

ReseaRch aRticle Mejías, Pérez-Yagüe, Roca et al. Dimercaptosuccinic acid-coated iron oxide nanoparticles imaging ReseaRch aRticle

Nanomedicine (2010) 5(3)400 future science group

was calculated by fitting mean blood 1/T1 and

1/T2 values as a function of time. Fe concentra-

tion was determined by inducted coupled plasma (ICP) after HCl/KMnO

4 digestion.

For abdominal and brain MR imaging, groups of rats were anesthetized and scanned. Coronal (abdomen) and axial (brain) images were recorded using a 7.2 cm3 quadrature (birdcage) coil. For the brain, nine slices were acquired with a fast spin echo sequence used with the following MR parameters: 3.5 × 3 cm2

field of view, two

excitations, 256 × 256 acquisition matrix, 1 mm slice thickness, 1500 ms recovery time (T

R), and

46 ms effective echo time (TE). For abdominal

imaging, a single slice was acquired with a very fast gradient-echo pulse sequence, fast imaging with steady-state precession (steady state free pre-cession) with the following parameters: 6 × 6 cm2 field of view, eight excitations, 256 × 256 matrix, 1 mm slice thickness, and 2/15 ms T

E/T

R. Rats

were imaged with the same parameters and spa-tial localization before and several times (90 min maximum) after contrast administration. For data comparison and quantification, images were reconstructed using the same gray scale.

Evolution of the contrast in liver and brain was followed as a function of time for 90 min in different rats. Only nanoparticles with the small-est hydrodynamic size (< 50 nm) were used in this experiment to help brain permeability. The BBB was overcome by first injecting 2 ml 25% mannitol solution in 0.9% NaCl (30 s infusion time); after 2 min, we injected 0.8 ml nano-particles (3 mg Fe/ml; 140 µmolFe/kg body weight; 30 s infusion time).

Finally, we studied the effect of repeated nanoparticle exposure to determine clearance and bioaccumulation using 12-week-old female C57BL/6 mice (Harlan Laboratories, IN, USA). Mice received subcutaneous or intravenous injec-tions of 100 µl PBS or a DMSA-coated nano-particle suspension (3 mg/ml Fe) three times per week for 3 weeks. Higher nanoparticle doses are used in this case, since partial degradation of the particles and transformation to ferritin is expected after 1 week. Mice were euthanized 30 min after the last injection; liver, spleen and kidneys were harvested and lyophilized, and ana-lyzed using a SQUID Magnetometer (CA, USA).

All animal procedures were handled following EU guidelines.

�n Results & discussionWe synthesized hydrophobic magnetite nano-particles by decomposition of iron acetylaceto-nate in phenyl ether and octadecene, solvents

with different boiling temperatures, to obtain particle sizes of 4 and 9 nm, respectively. Particles were uniform in size and shape, with a polydispersity degree (standard deviation/mean size) less than 0.20 (Figure 1A & B).

For biomedical applications, it is essential to stabilize these nanoparticles in an aqueous phase. Several studies have discussed a variety of methodological improvements, including ligand exchange with tetramethylammonium hydroxide [31], phosphine oxide polymers [32] or DMSA [23,33] to enhance nanoparticle stability in aqueous solution. Hydrophobic oleic acid-coated magnetite nanoparticles were surface modified with DMSA to render aqueous-stable suspensions at pH 7 with a high negative surface charge (Figure 1C, D & Figure 2A).

The exchange of oleic acid for DMSA mol-ecules results in some aggregation, due to a reduction in interparticle distance caused by the shorter carbon chain of DMSA (Figure 1C & D). Hydrodynamic sizes calculated from the inten-sity distributions for 4 and 9 nm particles were 65 and 70 nm, respectively, and the polydisper-sity degree (PDI) was less than 0.25 (Figure 2B), classifying these suspensions as SPIO suspen-sions. Hydrodynamic sizes of 30 nm can also be obtained for 4 nm nanoparticles by extending the sonication treatment, which yields a USPIO suspension (PDI < 0.2; Figure 2B). However, for 9 nm nanoparticles it was impossible to achieve stable high concentrated suspensions with low hydrodynamic size probably due to the presence of stronger magnetic interactions. Transmission electron microscopy images of 4 nm DMSA nan-oparticles in water showed individual particles as well as different-sized aggregates, always less than 50 nm (Figure 1C & D). Similar hydrodynamic sizes have been observed for dextran- or functionalized polyvinyl alcohol-coated iron oxide nanoparticles prepared by coprecipitation [34], but not for iron oxide particles prepared by thermal decomposition of organic precursors.

Magnetic behaviour at room temperature was reversible, showing no hysteresis for hydrophobic and hydrophilic nanoparticles, as predicted for particles less than 10 nm (Figure 2C) [35]. This con-firms that the particles are superparamagnetic at room temperature, another requirement for biomedical applications [36]. The high saturation magnetization values at room temperature for these particles (73 and 68 emu/g for hydropho-bic and hydrophilic particles, respectively) com-pared with magnetite nanoparticles prepared by other methods are explained by their highly crys-talline character and by the covalent bonding of

ReseaRch aRticle Mejías, Pérez-Yagüe, Roca et al. Dimercaptosuccinic acid-coated iron oxide nanoparticles imaging ReseaRch aRticle

www.futuremedicine.com 401future science group

oleic acid to the surface, which greatly reduces surface spin disorder [37]. Covalently bonded nanoparticles were recently reported to have bulk-like magnetic and electronic properties, whereas nanoparticles with adsorbed coatings demonstrate particle-like properties, with greatly reduced magnetization [21].

During the exchange process to aque-ous medium, surface oxidation of magnet-ite to maghemite takes place, which could be responsible for the 10% reduction in saturation magnetization for the DMSA nanoparticles (Figure 2C). In the case of both oleic acid and DMSA, carboxylic groups are bonded to the nanoparticle surface; the distinct bond strength between the coating molecule and the surface does not appear to affect the magnetic proper-ties. Slightly different bonding strengths would be predicted due to the mild temperature con-ditions for DMSA nanoparticle formation, in contrast to the high temperatures used for oleic acid nanoparticle synthesis.

Both longitudinal (r1) and transversal (r

2)

relaxivities indicate the potency of an MRI con-trast agent to generate contrast. Longitudinal relaxivity (r

1) was always small, independent of

particle and hydrodynamic size (7–26 s-1mM-1), as predicted for superparamagnetic contrast agents [38]. Transversal relaxivity increased from 90 to 317 s-1mM-1 as nanoparticle size increased from 4 to 9 nm, for samples with similar aggre-gate size (65–70 nm; Figure 2D). The highest trans-versal relaxivity value, r

2 = 317 s-1mM-1, was much

higher than those of commercial contrast agents of similar hydrodynamic size; this is explained by the higher crystallinity of particles prepared by thermal decomposition in organic media com-pared with particles prepared by coprecipitation. For the smallest particles (4 nm), the transversal relaxivity parameter was 90 s-1mM-1 for a 65 nm hydrodynamic diameter. Nonetheless, the r

2

value for 30 nm hydrodynamic size particles was not lower, but was even higher (126 s-1mM-1). USPIOs are predicted to provide less contrast enhancement per particle compared with SPIOs, due to the reduction in the magnetic field gra-dient created by the particle [38]. However, the high-quality USPIOs prepared in this study showed improved magnetic properties, assuring a high field gradient similar to SPIO samples. Their higher relaxivity values are most likely due to the larger water volume affected by the field gradient and the mobility of neighboring water molecules. These two factors gain importance with hydrodynamic size reduction, providing an additional contribution to relaxivity.

Commercial nanoparticles with hydro-dynamic sizes of approximately 150 nm have r

2 values of 100–120 s-1mM-1, which is reduced

to half (65 s-1mM-1) when hydrodynamic size is reduced to 30 nm, and to 33 s-1mM-1 for 7 nm hydrodynamic size [11]. In general, a reduction in particle and hydrodynamic size leads to a reduc-tion in the transversal relaxivity value, which is always larger for high crystalline particles such as those prepared here. The highest r

2 values

(> 200 s-1mM-1) are thus reported for magnetic nanoparticles such as manganese and cobalt fer-rites, although the contribution of the aggrega-tion state is unclear [33]. Saturation magnetiza-tion, aggregation state and spatial distribution determine the nuclear magnetic resonance con-trast produced by magnetic SPIOs. The relax-ivity values for high quality magnetic particles with very small hydrodynamic sizes (USPIOs) are also affected by the volume and the spatial distribution of neighboring water molecules.

As the question of toxicity must be addressed for DMSA nanoparticles intended for clinical use, we studied the biological effects of these iron oxide nanoparticles on a standard human tumor cell line (HeLa) to determine their internalization

25 nm

150 nm 30 nm

Figure 1. Transmission electron microscopy images of magnetite nanoparticles. (A) 4 nm and (B) 9 nm oleic acid-coated magnetite nanoparticles synthesized by thermal decomposition of iron acetylacetonate, and (C & d) aqueous suspension of 4 nm dimercaptosuccinic acid. nanoparticles with 30 nm of hydrodynamic size.

ReseaRch aRticle Mejías, Pérez-Yagüe, Roca et al. Dimercaptosuccinic acid-coated iron oxide nanoparticles imaging ReseaRch aRticle

Nanomedicine (2010) 5(3)402 future science group

efficiency at different doses and exposure times, as well as their intracellular localization and cytotox-icity. Cells cultured with DMSA samples showed internalized nanoparticles only after 24 h incu-bation with the maximum concentration tested (0.5 mg Fe ml-1) (Figure 3A). The intracellular dis-tribution pattern consisted of cytoplasmic spots of different sizes, always extranuclear. Similar results were independently obtained for hydro-dynamic sizes of less than 100 nm on the particle size. This is in agreement with similar subcellular nano particle distribution in HeLa and other cell types [39–41], and appears to indicate that nano-particles enter the cell through endocytosis.

The degree of cell survival was evaluated by the standard MTT (Figure 3B). Cytotoxicity ana-lysis after incubation of HeLa cells with increas-ing nanoparticle concentrations showed that cell culture viability was not notably affected or modified by nanoparticles at 24 h post-treatment (90–100% viability compared with controls). Nanoparticles must be internalized efficiently to overcome nonspecific targeting and short half-life

in blood, although internalization might pro-voke toxicity. The surface charge of iron oxide nanoparticles has an important role in cell inter-nalization. Neutral surfaces are well tolerated, although cell uptake is low [6]; cationic surfaces induce no reaction following uptake by HeLa [40] or brain-derived endothelial and microglial cells [34]. Anionic surfaces increase cell uptake, but can also cause increased cytotoxicity depending on the nature of the coating [40]. Following their internalization, anionic DMSA nanoparticles caused no cytotoxicity at the doses tested. Their small particle hydrodynamic size and anionic surface render this material ideal for cell labeling.

We performed blood clearance studies and MR imaging in male Wistar rats before and after nanoparticle injection to evaluate their utility as contrast agents for in vivo MRI. Mean blood 1/T

1

and 1/T2 values as a function of time were fitted

to a monoexponential clearance function, and the blood half-life of nanoparticles was calculated (data not shown). Blood half-life was 3 min for suspen-sions with hydrodynamic sizes in the 65–70 nm

0 2 4 6 8 10 12 14-60

-60

-80

-40

-40

-20

-20

0

0

20

20

40

40

60

60

80

ζ p

ote

nti

al (

mV

)

pH

DMSA coatedUncoated

-5 -4 -3 -2 -1 0 1 2 3 4 5

Mag

net

izat

ion

(em

u/g

)

Applied field (T)

Oleic acidDMSA

0

0 0

0100 200 300 400 500

5

10

1530 nm65 nm70 nm

Inte

nsi

ty

Hydrodynamic diameter (nm)

Hyd

rod

ynam

ic d

iam

eter

(n

m)

Particle size (nm)

r2 (s

-1mM

-1)

20

40

60

80

4 6 8 10

90

180

270

360

450

Figure 2. Physicochemical characteristics of dimercaptosuccinic acid nanoparticles. (A) Surface charge as a function of pH for uncoated and DMSA-coated nanoparticles. (B) Hydrodynamic size distribution of aqueous suspensions of DMSA nanoparticles calculated by dynamic light scattering at pH 7. (C) Magnetization curves for 4 nm oleic acid- and DMSA-coated nanoparticles at room temperature (298K). (d) Hydrodynamic diameter (filled symbols) and transversal relaxivity values (open symbols) as a function of magnetic nanoparticle size for small paramagnetic iron oxide (squares) and ultra small paramagnetic iron oxide (stars) aqueous suspensions of DMSA nanoparticles. DMSA: Dimercaptosuccinic acid.

ReseaRch aRticle Mejías, Pérez-Yagüe, Roca et al. Dimercaptosuccinic acid-coated iron oxide nanoparticles imaging ReseaRch aRticle

www.futuremedicine.com 403future science group

range (SPIO samples) and 15 min for suspensions with smaller hydrodynamic sizes (30 nm). The short blood circulation time of the SPIO nano-particles prepared in this study is related to their hydrodynamic size and the nature of the coating; a limited blood half-life is predicted for highly charged particles. Neutral particles, that is those coated with dextran or polyethylene glycol, have a blood half-life of hours, whereas citrate-coated maghemite particles have a 30-min blood half-life despite their small hydrodynamic size (7 nm) [42]. For in vivo applications such as drug delivery, cell engineering, tissue repair or diagnostics, DMSA nanoparticles must be further coated, for example with hydrophilic polymers such as polyethylene glycol or a drug, to reduce the surface charge and thereby increase their blood half-life.

For abdominal imaging, a single slice was acquired with a T

2-weighted very fast gradient-

echo pulse sequence, fast imaging with steady-state precession (steady state free precession), with the same parameters and spatial localiza-tion, before and once every 10 min (90 min maximum) after nanoparticle administration. All scanned rats showed a clear signal decrease 5 min after SPIO injection delineated mainly in liver and spleen; representative abdominal images are shown at 60 min postinjection of 9 nm DMSA nano particles showing the contrast change in liver (Figure 4A). DMSA nanoparticles accumu-lated rapidly in this organ through the sinusoi-dal or discontinuous capillaries, which have large endothelial openings (> 1000 nm diameter) that allow SPIO entry. The kidney has fenestrated capillaries with pores in the endothelial cells (60–80 nm diameter) that only allow diffusion of much smaller particles, whereas CNS continu-ous capillaries, with a sealed endothelium (2 nm), permit diffusion exclusively of small molecules such as water and ions.

We studied the effect of repeated SPIO nano-particle exposure to determine clearance and bioaccumulation using 12-week-old female C57BL/6 mice. The literature shows few post-administration studies of biodistribution and transformation of iron-containing drugs, due to difficulties in detecting trace metals in organic tissue or in identifying a few crystalline species in a much larger matrix [43–46]. Magnetic measure-ments are sensitive and offer substantial informa-tion, as they are specific for transition metals and allow characterization of a big portion of the sam-ple. Tested mice showed various degrees of nano-particle uptake, depending on the organ being observed. Magnetic data enabled quantitative estimation of the amount of magnetic material

in each organ by comparison of the normalized saturation magnetization of mouse organs to that of the nanoparticles alone (Figures 2C & 4B). Figure 4B shows the magnetization curves for lyophilized samples of liver, spleen and kidney, together with the mass fraction of the accumulated iron oxide nanoparticles with respect to total organ mass. The magnetization behaviour of an organ is the sum of the magnetization behaviour of the par-ticles (superpara magnetic at room temperature and ferromagnetic at 4.2 K), natural ferritin (paramagnetic) and the tissue (diamagnetic). Depending of the weight fraction of each com-ponent, the magnetic behaviour could be domi-nated by one of the components. In the case of the kidney, the amount of particles is so small that it is the diamagnetic component that determines the magnetic behaviour at high magnetic fields. A large amount of ferritin is naturally found in the spleen, so even the control samples show a predominant paramagnetic behavior for all field values up to 50 kOe. The signal of the kidney and liver controls starts as ferromagnetic but becomes predominantly diamagnetic at moder-ate fields, in accordance with the lower amounts of ferritin found in these organs. The intravenous route appeared to be the most efficient form of administration to liver, which demonstrated the greatest uptake; uptake was lower in spleen and much lower in kidney (Figure 4B). In the intra-venous repeated exposure protocol, with a total dose of 2.4 mg of iron oxide nanoparticles after nine injections, the percent of magnetic nanopar-ticle uptake per organ was an average of 35% for liver, 1.5% for spleen and 0.5% for the kidney.

Su

rviv

ing

fra

ctio

n (

%)

0

20

40

60

80

100

Control 0.05 0.1 0.5

Figure 3. Cell interaction and cytotoxicity of iron oxide nanoparticles. (A) HeLa cells cultured 24 h with 4 nm dimercaptosuccinic acid nanoparticles (65 nm hydrodynamic size) at the maximum concentration (0.5 mg Fe/ml) and stained with Prussian blue reaction for iron oxide detection. Scale bar: 5 µm; (B) methyl thiazol tetrazolium cytotoxicity ana lysis after HeLa cell incubation for 24 h with increasing concentrations of the dimercaptosuccinic acid nanoparticles (0.05, 0.1 and 0.5 mg Fe/mL). Phosphate buffer solution control sample is used as reference.

ReseaRch aRticle Mejías, Pérez-Yagüe, Roca et al. Dimercaptosuccinic acid-coated iron oxide nanoparticles imaging ReseaRch aRticle

Nanomedicine (2010) 5(3)404 future science group

It should be noted that the ana lysis of the mag-netic curves was carried out on the assumption that the initial particles remain unaltered dur-ing all the measurements. However, degradation of the particles is expected to start 1 week after administration as previously reported for other

iron compounds intravenously injected [47]. By contrast, subcutaneous injection of the same nanoparticle dose yielded no appreciable uptake in these organs compared with controls (Figure 4B). In this case, DMSA nanoparticles probably undergo a slower degradation for more than a

-0.01

-0.00

0.01

-50 -30 -10 10 30 50

Field (kOe)

-0.1

-0.0

0.1

Mag

net

izat

ion

(em

u/g

) Liver

-50 -30 -10 10 30 50

Field (kOe)

-0.01

-0.00

0.01

Mag

net

izat

ion

(em

u/g

) Kidney

Field (kOe)

1.2

0.8

0.4

0.0

-0.4

Spl

een

1

Spl

een

1

Spl

een

2

Spl

een

2

Live

r 2

Live

r 2

Live

r 1

Live

r 1

Kid

ney

1

Kid

ney

1

Kid

ney

2

Kid

ney

2

Mas

s fr

acti

on

(x

10-3)

-50 -30 -10 10 30 50

-0.1

-0.0

0.1

Mag

net

izat

ion

(em

u/g

)

Control 1Control 2iv. 1iv. 2sc. 1sc. 2

Spleen

Precontrast

Liver

Postcontrast

iv.sc.

0.10

0.08

0.06

Figure 4. In vivo biodistribution of small paramagnetic iron oxide nanoparticles after intravenous and subcutaneous injection. (A) Coronal fast spin echo MRI of rat abdomen at 0 (left) and 60 min (right) post-injection of SPIO 9 nm particles. (B) Magnetization curves at 5 K of lyophilized samples of mouse liver, spleen and kidney after nanoparticle treatment, compared with magnetization curves of control mice (no nanoparticles injected). Labels 1 and 2 indicate two different mice. The quantification of the mass fraction of accumulated iron oxide nanoparticles relative to total organ mass was obtained by comparing the normalized saturation magnetization of organs to those of the nanoparticles alone (Figure 2C). Insets show a detail of the positive field branches of samples labeled subcutaneous; horizontal and vertical scales are the same as those in the main graph. iv.: Intravenous; sc.: Subcutaneous; SPIO: Small paramagnetic iron oxide.

ReseaRch aRticle Mejías, Pérez-Yagüe, Roca et al. Dimercaptosuccinic acid-coated iron oxide nanoparticles imaging ReseaRch aRticle

www.futuremedicine.com 405future science group

month before they are eliminated, as observed for intramuscularly injected iron oxyhydroxide nanoparticles [43].

We studied the extent of biodistribution to, and clearance from, liver and brain using MRI for 30 nm hydrodynamic size suspensions (USPIO), after a single intravenous injection of DMSA nanoparticles into rats (Figure 5). MRI signal intensity decreased as a function of time, and the maximum contrast was observed in the liver after approximately 15 min. No change in brain sig-nal was observed following similar protocol. The trend was distinct when using osmotic disruption of the BBB, a technique to increase the delivery of suspensions to the brain that has been used clini-cally for more than 10 years [48]. Relative signal contrast in brain initially increased (in the first 5–10 min), but later decreased to reach normal intensity values after 1 h (Figure 5). Mannitol solu-tions disrupt the BBB transiently and reversibly by shrinking endothelial cells and opening the tight junctions, aiding nanoparticle entry from the interstitial fluid of brain parenchyma into the cerebrospinal fluid compartments [48,49].

Magnetic resonance imaging signal changes showed evolution of brain contrast for 1 h post-injection (Figure 6). Once nanoparticles crossed the BBB, they were rapidly distributed throughout the brain (5 min). The time-course of DMSA nano-particle residence in the brain was 1 h, and no residual material was observed thereafter (Figure 6). Nanoparticles accumulated transiently in lateral, third and fourth ventricles and most likely in some blood vessels of the brain. In addition to selectivity, retention time of these nanoparticles in the brain is long enough to allow efficient treatment, as it determines total drug exposure time to tissue.

ConclusionThe way in which the chemical and biophysical properties of nanoparticles define their interac-tion with cells, tissues and organs was addressed here, to ascertain the feasibility of magnetic nanoparticle use in biomedical applications. We prepared aqueous suspensions of uniform mag-netic iron oxide nanoparticles of distinct sizes (SPIO and USPIO) and studied their in vivo and in vitro applications. DMSA nanoparticles with an iron oxide core diameter of 4 nm and hydro-dynamic sizes less than 50 nm have outstanding magnetic properties and very high nuclear mag-netic resonance relaxivity values (126 s-1mM-1). These qualities improve MRI signal in low con-trast images, and thus the resolution limit of the nuclear magnetic resonance technique; contrast change is relative to nanoparticle concentration

in tissue, magnetic properties and water acces-sibility. DMSA nanoparticles were internalized efficiently and caused no cytotoxicity at the doses tested. Their small particle hydrodynamic size and anionic surface make this material ideal for cell labeling.

Our clearance and bioaccumulation studies reveal that DMSA nanoparticles with hydro-dynamic sizes of more than 50 nm are rapidly accumulated in liver and spleen and can be used as contrast agents or drug delivery systems to these organs if they are properly functional-ized. The intravenous route appears to be the most efficient form of administration to liver, which showed much greater uptake than spleen and kidney. DMSA nanoparticles with hydro-dynamic sizes of less than 50 nm were deliv-ered into a normal brain by transient, reversible hyperosmotic disruption of the BBB. Retention time of these nanoparticles in the brain was approximately 1 h, which would allow efficient treatment protocols. This time-frame allows accumulation in target tissues, which is one of the primary challenges for further use of mag-netic nanoparticles. The high negative charge of DMSA permits surface derivatization for drug attachment. In summary, the in vitro and in vivo behavior of DMSA iron oxide nanoparticles showed that they are efficient carriers for drug delivery to the liver and, combined with MRI

0 10 20 30 40 50 60 70 80

0.2

0.4

0.6

0.8

1.0 Brain

Liver

Time after injection (min)

Rel

ativ

e co

ntr

ast

in N

MR

imag

es

Figure 5. Time course of magnetic resonance imaging contrast negative enhancement in liver and brain after intravenous injection of ultra small paramagnetic iron oxide 4 nm nanoparticles in two different rats. Magnetic resonance imaging signal in brain was measured after blood–brain barrier disruption by intravenous injection of hyperosmotic mannitol.

ReseaRch aRticle Mejías, Pérez-Yagüe, Roca et al. Dimercaptosuccinic acid-coated iron oxide nanoparticles imaging ReseaRch aRticle

Nanomedicine (2010) 5(3)406 future science group

detection of active lesions in neurodegenerative diseases could also be used for diagnosis and treatment in the brain.

AcknowledgementsThe authors would like to thank F Wandosell for advice in the interpretation of rat brain images, the SQUID Magnetometry Facility of the University of Barcelona for magnetic measurements and C Mark for editorial assistance.

Financial & competing interests disclosureRaquel Mejías receives a predoctoral fellowship from the Spanish Ministry of Science and Innovation (MICINN) FPU program. Sonia Pérez-Yagüe holds a contract supported by the Spanish National Research Council (CSIC; Intramural Grant 200820I084 to Domingo F Barber). This work was supported by the Spanish Ministry of Science and Innovation (NAN2004–08805-C01–04 to Carlos J Serna, MAT2008–01489 to Sabino Veintemillas-Verdaguer, CSD2007–00010 to Mª Puerto Morales, SAF2008–05512

0 5 10

20

30 40

50

15 25

35

45 55

Figure 6. Rat brain nuclear magnetic resonance pre- (0 min) and post-contrast images (every 5 min) for 60 min postinjection with ultra small paramagnetic iron oxide 4 nm particles with hydrodynamic sizes <50 nm. The blood–brain barrier was overcome by first injecting a mannitol solution. Arrows indicate the areas of interest. Top: third ventricle; laterals: lateral ventricles; bottom: recess inferior colliculus. In vitro and in vivo behavior showed that dimercaptosuccinic acid-iron oxide nanoparticles are efficient carriers for drug delivery to liver and brain that can be combined with magnetic resonance imaging detection.

ReseaRch aRticle Mejías, Pérez-Yagüe, Roca et al. Dimercaptosuccinic acid-coated iron oxide nanoparticles imaging ReseaRch aRticle

www.futuremedicine.com 407future science group

Bibliography1 Cheon J, Lee JH: Synergistically integrated

nanoparticles as multimodal probes for nanobiotechnology. Acc. Chem. Res. 41, 1630–1640 (2008).

2 Cormode DP, Skajaa T, van Schooneveld MM et al.: Nanocrystal core high-density lipoproteins: a multimodality contrast agent platform. Nano Lett. 8, 3715–3723 (2008).

3 Li Z, Wei L, Gao M, Lei H: One-pot reaction to synthesize biocompatible magnetite nanoparticles. Adv. Mater. 17, 1001–1005 (2005).

4 Bjornerud A, Johansson L: The utility of superparamagnetic contrast agents in MRI: theoretical consideration and applications in the cardiovascular system. NMR Biomed. 17, 465–477 (2004).

5 Bulte JW, Kraitchman DL: Iron oxide MR contrast agents for molecular and cellular imaging. NMR Biomed. 17, 484–499 (2004).

6 Corot C, Petry KG, Trivedi R et al.: Macrophage imaging in central nervous system and in carotid atherosclerotic plaque using ultrasmall superparamagnetic iron oxide in magnetic resonance imaging. Invest. Radiol. 39, 619–625 (2004).

7 Jun YW, Lee JH, Cheon J: Chemical design of nanoparticle probes for high-performance magnetic resonance imaging. Angew. Chem. Int. Ed. Engl. 47, 5122–5135 (2008).

8 Kim J, Piao Y, Hyeon T: Multifunctional nanostructured materials for multimodal imaging, and simultaneous imaging and therapy. Chem. Soc. Rev. 38, 372–390 (2009).

9 Lanza GM, Winter PM, Caruthers SD et al.: Magnetic resonance molecular imaging with nanoparticles. J. Nucl. Cardiol. 11, 733–743 (2004).

10 Mulder WJ, Strijkers GJ, van Tilborg GA, Griffioen AW, Nicolay K: Lipid-based nanoparticles for contrast-enhanced MRI and molecular imaging. NMR Biomed. 19, 142–164 (2006).

11 Raynal I, Prigent P, Peyramaure S, Najid A, Rebuzzi C, Corot C: Macrophage endocytosis of superparamagnetic iron oxide nanoparticles: mechanisms and comparison of ferumoxides and ferumoxtran-10. Invest. Radiol. 39, 56–63 (2004).

12 Sun C, Lee JS, Zhang M: Magnetic nanoparticles in MR imaging and drug delivery. Adv. Drug. Deliv. Rev. 60, 1252–1265 (2008).

13 Wunderbaldinger P, Josephson L, Weissleder R: Crosslinked iron oxides (CLIO): a new platform for the development of targeted MR contrast agents. Acad. Radiol. 9(Suppl. 2), S304–S306 (2002).

to Jesús Ruiz-Cabello, MAT2006–03999, MAT2009–08667 and CSD2006–00012 to Xavier Batlle, and SAF2008–00471 to Domingo F Barber), the Madrid (S-0505/MAT/0194 to Mª Puerto Morales and CCG08-CSIC/SAL-3451 to Domingo F Barber) and Cataluña regional governments (2009SGR856 to Amilcar Labarta), the Instituto de Salud Carlos III (RETICS Program, RD08/0075 (RIER) to Domingo F Barber) and the European Union (LSHB-CT-2006–037639 to Sabino Veintemillas-Verdaguer). 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 insti-tutional review board approval or have followed the princi-ples outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investi gations involving human subjects, informed consent has been obtained from the participants involved.

executive summary

� Uptake, cytotoxicity and interaction of dimercaptosuccinic acid-coated iron oxide nanoparticles were studied in cells, tissues and organs after single and multiple exposures.

� Magnetic nanoparticles synthesized by thermal decomposition of iron acetylacetonate in an organic medium show uniform size and shape, high crystallinity and enhanced magnetic properties.

� Aqueous suspensions of those nanoparticles of distinct sizes (small paramagnetic iron oxide and ultra small paramagnetic iron oxide) can be attained by ligand exchange with dimercaptosuccinic acid (DMSA), which provides high stability in aqueous media and free ligand groups for further biomolecule conjugation.

� DMSA nanoparticles with an iron oxide core diameter of 4 nm and hydrodynamic sizes of less than 50 nm have outstanding magnetic properties and very high nuclear magnetic resonance (NMR) relaxivity values (126 s-1 mM-1). These qualities improve magnetic resonance imaging signal-to-background ratios in low contrast images, and thus the resolution limit of the NMR technique.

� Human cervical carcinoma cells (HeLa) cultured with DMSA samples show internalized nanoparticles after 24 h incubation in the form of cytoplasmic spots of different sizes, always extranuclear. Their small particle hydrodynamic size and anionic surface make this material ideal for cell labeling.

� Clearance and bioaccumulation studies show that DMSA nanoparticles with hydrodynamic sizes over 50 nm are rapidly accumulated in liver and spleen and can be used as contrast agents or drug delivery systems to these organs. Magnetic data enabled quantitative estimation of the amount of magnetic material in each organ (35 for liver, 1.5 for spleen and 0.5% for kidney after intravenous injection).

� DMSA nanoparticles with hydrodynamic sizes of less than 50 nm can be delivered into a normal brain by transient, reversible hyperosmotic disruption of the blood–brain barrier. Retention time of these nanoparticles in brain was approximately 1 h, which would allow efficient treatment protocols. This time-frame allows accumulation in target tissues, which is one of the primary challenges for further use of magnetic nanoparticles for diagnosis and treatment of active lesions in neurodegenerative diseases.

� In summary, in vitro and in vivo behavior of DMSA iron oxide nanoparticles showed that they could be efficient carriers for drug delivery to the liver and the brain, combined with MRI detection.

ReseaRch aRticle Mejías, Pérez-Yagüe, Roca et al.

Nanomedicine (2010) 5(3)408 future science group

14 Arruebo M, Fernández-Pacheco R, Ibarra M, Santamaría J: Magnetic nanoparticles for drug delivery. Nano Today 2, 22–32 (2007).

15 Lockman PR, Oyewumi MO, Koziara JM, Roder KE, Mumper RJ, Allen DD: Brain uptake of thiamine-coated nanoparticles. J. Control. Release 93, 271–282 (2003).

16 Thorek DL, Tsourkas A: Size, charge and concentration dependent uptake of iron oxide particles by non-phagocytic cells. Biomaterials 29, 3583–3590 (2008).

17 Wagner S, Schnorr J, Pilgrimm H, Hamm B, Taupitz M: Monomer-coated very small superparamagnetic iron oxide particles as contrast medium for magnetic resonance imaging: preclinical in vivo characterization. Invest. Radiol. 37, 167–177 (2002).

18 Jang JT, Nah H, Lee JH, Moon SH, Kim MG, Cheon J: Critical enhancements of MRI and hyperthermic effects by dopant-controlled magnetic nanoparticles. Angew. Chem. Int. Ed. Engl. 48, 1234–1238 (2009).

19 Roca AG, Morales MP, O’Grady K, Serna CJ: Structural and magnetic properties of uniform magnetite nanoparticles prepared by high temperature decomposition of organic precursors. Nanotechnology 17, 2783–2788 (2006).

20 Guardia P, Batlle-Brugal B, Roca A et al.: Surfactant effects in magnetite nanoparticles of controlled size. J. Magn. Magn. Mater. 316, 756–759 (2007).

21 Pérez N, Bartolomé F, García L et al.: Nanostructural origin of the spin and orbital contribution of the magnetic moment in Fe

3-xO

4 magnetite nanoparticles. Appl. Phys.

Lett. 94, 093108 (2009).

22 Na HB, Lee JH, An K et al.: Development of a T

1 contrast agent for magnetic

resonance imaging using MnO nanoparticles. Angew. Chem. Int. Ed. Engl. 46, 5397–5401 (2007).

23 Roca AG, Veintemillas-Verdaguer S, Port M, Robic C, Serna CJ, Morales MP: Effect of nanoparticle and aggregate size on the relaxometric properties of MR contrast agents based on high quality magnetite nanoparticles. J. Phys. Chem. B. 113, 7033–7039 (2009).

24 Wan J, Cai W, Meng X, Liu E: Monodisperse water-soluble magnetite nanoparticles prepared by polyol process for high-performance magnetic resonance imaging. Chem. Commun. (Camb). 47, 5004–5006 (2007).

25 Mejias R, Costo R, Roca AG et al.: Cytokine adsorption/release on uniform magnetic nanoparticles for localized drug delivery. J. Control. Release 130, 168–174 (2008).

26 Schaller B, Graf R: Different compartments of intracranial pressure and its relationship to cerebral blood flow. J. Trauma. 59, 1521–1531 (2005).

27 Desestret V, Brisset JC, Moucharrafie S et al.: Early-stage investigations of ultrasmall superparamagnetic iron oxide-induced signal change after permanent middle cerebral artery occlusion in mice. Stroke 40, 1834–1841 (2009).

28 Chertok B, Moffat BA, David AE et al.: Iron oxide nanoparticles as a drug delivery vehicle for MRI monitored magnetic targeting of brain tumors. Biomaterials 29, 487–496 (2008).

29 Sun S, Zeng H, Robinson DB et al.: Monodisperse MFe

2O

4 (M= Fe, Co, Mn)

nanoparticles. J. Am. Chem. Soc. 126, 273–279 (2004).

30 Jun YW, Huh YM, Choi JS et al.: Nanoscale size effect of magnetic nanocrystals and their utilization for cancer diagnosis via magnetic resonance imaging. J. Am. Chem. Soc. 127, 5732–5733 (2005).

31 Salgueirino-Maceira V, Liz-Marzan LM, Farle M: Water-based ferrofluids from Fe

xPt

1-x

nanoparticles synthesized in organic media. Langmuir 20, 6946–6950 (2004).

32 Robinson DB, Persson HH, Zeng H et al.: DNA-functionalized MFe

2O

4 (M = Fe, Co or

Mn) nanoparticles and their hybridization to DNA-functionalized surfaces. Langmuir 21, 3096–3103 (2005).

33 Huh YM, Jun YW, Song HT et al.: In vivo magnetic resonance detection of cancer by using multifunctional magnetic nanocrystals. J. Am. Chem. Soc. 127, 12387–12391 (2005).

34 Cengelli F, Maysinger D, Tschudi-Monnet F et al.: Interaction of functionalized superparamagnetic iron oxide nanoparticles with brain structures. J. Pharmacol. Exp. Ther. 318, 108–116 (2006).

35 Morales MP, Veintemillas-Verdaguer S, Serna CJ et al.: Surface and internal spin canting in g-Fe

2O

3 nanoparticles. Chem.

Mater. 11, 3058–3064 (1999).

36 Pankhurst QA, Connolly J, Jones S, Dobson J: Applications of magnetic nanoparticles in biomedicine. J. Phys. D. Appl. Phys. 36, 167–181 (2003).

37 Roca A, Niznansky D, Poltierova-Vejpravova J et al.: Magnetite nanoparticles with no surface spin canting. J. Appl. Phys. 105, 114309 (2009).

38 Merbach A, Toth E: The Chemistry of Contrast Agents in Medical Magnetic Resonance Imaging. Wiley, New York, USA (2001).

39 Riviere C, Wilhelm C, Cousin F, Dupuis V, Gazeau F, Perzynski R: Internal structure of magnetic endosomes. Eur. Phys. J. E. Soft. Matter. 22, 1–10 (2007).

40 Villanueva A, Canete M, Roca AG et al.: The influence of surface functionalization on the enhanced internalization of magnetic nanoparticles in cancer cells. Nanotechnology 20(11), 115103 (2009).

41 Wilhelm C, Billotey C, Roger J, Pons JN, Bacri JC, Gazeau F: Intracellular uptake of anionic superparamagnetic nanoparticles as a function of their surface coating. Biomaterials 24, 1001–1011 (2003).

42 Taupitz M, Schnorr J, Abramjuk C et al.: New generation of monomer-stabilized very small superparamagnetic iron oxide particles (VSOP) as contrast medium for MR angiography: preclinical results in rats and rabbits. J. Magn. Reson. Imaging 12, 905–911 (2000).

43 Lazaro FJ, Abadia AR, Romero MS, Gutierrez L, Lazaro J, Morales MP: Magnetic characterisation of rat muscle tissues after subcutaneous iron dextran injection. Biochim. Biophys. Acta 1740, 434–445 (2005).

44 Ricketts CR, Cox JS, Fitzmaurice C, Moss GF: The iron-dextran complex. Nature 208, 237–239 (1965).

45 Skotland T, Sontum PC, Oulie I: In vitro stability analysis as a model for metabolism of ferromagnetic particles (Clariscan), a contrast agent for magnetic resonance imaging. J. Pharm. Biomed. Anal. 28, 323–329 (2002).

46 Rapoport SI: Osmotic opening of the blood–brain barrier: principles, mechanism, and therapeutic applications. Cell. Mol. Neurobiol. 20, 217–230 (1998).

47 Gutiérrez L, Lázaro FJ, Abadía AR et al.: Bioinorganic transformation of liver iron deposits observed by tissue magnetic characterisation in a rat model. J. Inorg. Biochem. 100, 1790–1799 (2006).

48 Cosolo WC, Martinello P, Louis WJ, Christophidis N: Blood–brain barrier disruption using mannitol: time course and electron microscopy studies. Am. J. Physiol. 256(2 Pt 2), R443–R447 (1989).

49 Gumerlock MK, Belshe BD, Madsen R, Watts C: Osmotic blood–brain barrier disruption and chemotherapy in the treatment of high grade malignant glioma: patient series and literature review. J. Neurooncol. 12, 33–46 (1992).