International Journal of Nanomedicine Dovepress

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Open Access Full Text Article O riginal R esearch

Folate-targeted polymeric micelles loaded

with ultrasmall superparamagnetic iron oxide:
combined small size and high MRI sensitivity
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
9 June 2012
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Guo-bin Hong 1,2 Abstract: Targeted delivery of contrast agents is a highly desirable strategy for enhancing
Jing-xing Zhou 2 diagnostic efficiency and reducing side effects and toxicity. Water-soluble and tumor-targeting
Ren-xu Yuan 3 superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized by loading hydrophobic
SPIONs into micelles assembled from an amphiphilic block copolymer poly(ethylene glycol)-
1
Department of Radiology, Fifth
Affiliated Hospital, Zhuhai, poly(ε-caprolactone) (PEG-PCL) bearing folate in the distal ends of PEG chains. Compared to
2
Department of Radiology, Sun Yat- the water-soluble SPIONs obtained by small molecular surfactant coating, ultrasmall SPION
sen Memorial Hospital, Guangzhou,
encapsulation with PEG-PCL micelles (PEG-PCL-SPIONs) simultaneously increases transverse
3
School of Chemistry and Chemical
Engineering, Sun Yat-sen University, (r2) and decreases longitudinal (r1) magnetic resonance (MR) relaxivities of water proton in
Guangzhou, China micelle solution, leading to a notably high r2/r1 ratio up to 78, which makes the PEG-PCL-SPIONs
a highly sensitive MR imaging (MRI) T2 contrast agent. The mean size of folate-attached SPION
micelles (Fa-PEG-PCL-SPIONs) is 44 ± 3 nm on average, ideal for in vivo MRI applications
in which long circulation is greatly determined by small particle size and is highly desirable.
Prussian blue staining of BEL-7402 cells over-expressing folate receptors, after incubation
with micelle-containing medium, demonstrated that folate functionalization of the magnetic
particles significantly enhanced their cell uptake. The potential of Fa-PEG-PCL-SPIONs as
a potent MRI probe for in vivo tumor detection was assessed. At 3 hours after intravenous
injection of the Fa-PEG-PCL-SPION solution into mice bearing subcutaneous xenografts of
human BEL-7402 hepatoma, a 41.2% signal intensity decrease was detected in the T2-weighted
MR images of the tumor, indicating the efficient accumulation of Fa-PEG-PCL-SPIONs in the
tumor tissue.
Keywords: tumor targeting, magnetic resonance imaging, polymeric micelles,
superparamagnetic iron oxide

Introduction
As one of the most powerful diagnostic techniques in clinical medicine, magnetic
resonance imaging (MRI) has attracted much attention, and considerable effort has
been made to improve its resolution and contrast quality in recent years.1 Targeted
delivery of contrast agents is a highly desirable strategy for simultaneously enhancing
contrast effect and reducing side effects and toxicity. This technique is extremely useful
for detecting early signs of diseases. Superparamagnetic nanocrystals are well-known
Correspondence: Jing-xing Zhou excellent MRI probes which can noninvasively monitor in vivo events even at molecu-
Department of Radiology, Sun Yat-sen
Memorial Hospital, Sun Yat-sen lar and cellular level. To date, most interest has been focused on superparamagnetic
University, Guangzhou 510120, China iron oxide nanoparticles (SPIONs), magnetite (Fe3O4) and maghemite (γ-Fe2O3).2
Tel +86 20 8133 2243
Fax +86 20 8133 2702
Magnetite nanoparticles are usually prepared through a conventional copre-
Email [email protected] cipitation method in the aqueous phase. Although this method is suitable for mass

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Dovepress © 2012 Hong et al, publisher and licensee Dove Medical Press Ltd. This is an Open Access article
http://dx.doi.org/10.2147/IJN.S25739 which permits unrestricted noncommercial use, provided the original work is properly cited.
Hong et al Dovepress

production of magnetic nanoparticles,3 it requires careful magnetic nanoparticles could be encapsulated into the
adjustment of the pH value of the solution for particle for- hydrophobic core of polymeric micelle, resulting in an
mation and stabilization, and it is difficult to control size ultrasensitive MRI T2 contrast agent.12 These approaches
and size distribution, particularly when the desired particle have been demonstrated successfully for preparing mag-
size is smaller than 20 nm. By contrast, high-temperature netic nanoparticles with high monodispersity and stability
decomposition strategies have been developed recently in aqueous solution. It is noted that water-soluble SPIO
to produce monodisperse and highly crystalline iron nanoparticles reported in such work mostly lack in specificity
oxide nanoparticles.3,4 Magnetic nanoparticles produced towards a pathological site, which potentially limits their in
by high-temperature decomposition are potentially more vivo applications. Indeed, most of the convincing successes
advantageous for biomedical applications such as magnetic in in vivo applications of SPIO-based nanoparticles as
resonance imaging and magnetic cell separation, in which MRI probes have been achieved with nanoparticles smaller
well-defined particle size and uniform size distribution are than 50 nm.13,14 Further research towards a combination of
often required to achieve long circulation or efficient cell reducing particle size for long circulation, enhancing MRI
uptake. However, nanoparticles thus prepared are coated sensitivity, and introducing molecular targeting to guide
with a layer of surfactant molecules to prevent them from site-specific delivery is much needed.
aggregation. The long hydrocarbon chains of the surfac- In this study, we developed a type of tumor-targeted and
tants covering the SPIONs lead to high hydrophobicity of water-dispersible SPIO nanoparticle smaller than 50 nm in
particle surface, hence the biological applications of these diameter. By loading hydrophobic SPIO nanocrystals into
nanoparticles are greatly restricted because of their poor polymeric micelles assembled from a diblock copolymer
dispersibility in aqueous solution.5 Furthermore, particles of polyethylene glycol (PEG) and biodegradable poly-ε-
that have a highly hydrophobic surface can be efficiently caprolactone (PCL), we were able to load ultrasmall SPIO
coated with plasma components and thus rapidly removed nanocrystal into a micelle core, thus decreasing the particle
from the circulation. Therefore, surface modification of these size significantly. Because folic acid receptor is overexpressed
magnetic iron oxide nanocrystals is essential for most of in a wide range of cancer cells,15 and folate-functionalized
their bio-related applications.6 An ideal surface modification PEG-PCL micelles have been used for targeting delivery of
should (a) stabilize the nanoparticles in biological surround- a multidrug-resistance modulator, FG020326,16 we attached
ings with a pH of about 7.4 and particularly at physiological folate onto the surface layer of the polymeric micelles to
salt concentration, (b) provide functional groups at the par- achieve functional tumor-targeting micelles. We further exam-
ticle surface for further derivatization (eg, attaching receptor ined and evaluated the MRI T2 sensitivity of the ultrasmall
ligands for cell-specific uptake), and (c) suppress the uptake SPIO-loaded targeting micelles (Fa-PEG-PCL-SPION), and
of nanoparticles by the reticuloendothelial system.7 So far, tested their potential use as a potent MRI T2 probe for in vivo
several methods have been explored for converting hydro- tumor detection on a 1.5T clinical MRI scanner.
phobic SPIO nanocrystals into hydrophilic ones, typically
including ligand exchange,8,9 bipolar molecule,5 and polymer Materials and methods
stabilization.12 These modifications have the advantages of Materials
avoiding potential exposure of hydrophobic SPION surface ε-Caprolactone (ε-CL; Sigma-Aldrich, St Louis, MO)
and adsorption of blood proteins, and may allow a prolonged and allyl alcohol (Guangzhou Chemical Reagent Factory,
blood circulation. Nowadays, more interest is being focused Guangzhou, China) were both purified by vacuum distilla-
on amphiphilic polymers because of their advantage of tion over calcium hydride (CaH2). Tetrahydrofuran (THF;
high colloidal stability over small molecular surfactants. Sigma-Aldrich) was dried by refluxing the chemical over
For example, hydrophobic iron oxide nanoparticles were a sodium-potassium alloy and distilled under dry argon.
encapsulated within an amphiphilic polymer shell con- 18-Crown-6 (Sigma-Aldrich) was vacuum-dried overnight
sisting of poly-(maleic anhydride-alt-1-tetradecene) and at 46°C. 2-Aminoethanethiol hydrochloride, folic acid,
cross-linked by bis(6-aminohexyl)amine, and ABA-type N-hydroxysuccinimide (NHS), naphthalene, potassium
triblock copolymer consisting of poly(propylene oxide) ­persulfate (K2S2O8), and dicyclohexylcarbodiimide (DCC)
and poly(ethylene oxide) (Pluronic F127) was also used to were purchased from Sigma-Aldrich. Ethylene oxide (EO,
transfer the hydrophobic SPIONs into water-soluble ones.11 99% purity), stored inside a gas tank, was obtained from
In addition, Ai et al reported that clusters of hydrophobic Foshan Kedi Gas Chemical Industry (Foshan, China).

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Dovepress Targeting tumors with SPION-loaded micelles

Phenyl ether (99%), benzyl ether (99%), 1,2-hexadecanediol stirring, and then allowing evaporation of THF and formation
(97%), oleic acid (99%), oleylamine (.70%) and iron (III) of micelles. The micelle solution was first bubbled with nitro-
acetylacetonate were purchased from Sigma-Aldrich and gen for 1 hour to remove oxygen, and then K2S2O8 (0.8 molar
used without further purification. BEL-7402 cells, a human equivalent of allyl-PEG-PCL) and 2-aminoethanethiol hydro-
hepatic carcinoma cell line with overexpressed surface chloride (tenfold molar equivalent of allyl-PEG-PCL) were
receptors for folic acid, were obtained from the Experimental added to the above solution. Subsequently, the micelle solution
Animal Center of Sun Yat-sen University. was sealed in a nitrogen atmosphere and stirred for 5 hours
at 52°C. Unreacted 2-aminoethanethiol hydrochloride and
Methods K2S2O8 were removed by dialysis against water for 24 hours at
Synthesis of allyl-terminated diblock copolymer room temperature (MW cutoff: 8 kDa). The obtained micelle
of PCL and PEG (allyl-PEG-PCL) solution was immediately freeze-dried (yield $ 78%).
The copolymer was synthesized by sequential anionic ring- 1
H-NMR (CDCl3): δ = 1.40 (m, 2H, −COCH2CH2CH2
opening polymerization of EO and ε-CL in one pot using CH2CH2O−), 1.65 (m, 4H, −COCH2CH2CH2CH2CH2O−),
potassium alkoxide as an initiator.16 THF solution (4 mL) 2.31 (t, 2H, −COCH 2CH 2CH 2CH 2CH 2O−), 2.65∼2.70
of potassium naphthalide was allowed to mix with 0.5 mL (m, 4H, −CH2SCH2−), 2.94 (t, 3H, H2NCH2CH2S−), 3.65
allyl alcohol, and then the mixture was stirred for 30 minutes (s, 4H,−CH2CH2O−) 4.05 (t, 2H, −COCH2CH2CH2CH2
into a flame-dried reaction flask equipped with a magnetic CH2O−), 4.2 (s, 2H, −CH2CH2OCO−).
stirring bar and two capillary gas inlets for EO and argon,
respectively. Subsequently, 20 mL anhydrous THF and 1.5 g Preparation of folate-conjugated copolymer
18-crown-6 predissolved in 5 mL anhydrous THF in another (folate-PEG-PCL)
flamed flask were then transferred into the first reaction Briefly, folic acid (1 g) dissolved in anhydrous dimethyl
flask under argon. After stirring for another 30 minutes, the sulfoxide (DMSO; 30 mL) was reacted overnight with NHS
mixture was cooled with a salted ice-water bath of −5°C. (0.9 g) in the presence of DCC (0.5 g) under argon at room
A precalculated amount of dry EO was slowly blown and temperature, and 1,3-dicyclohexylurea (DCU) was removed
condensed into the reaction mixture. Afterwards, the EO by filtration. Subsequently, the above activated folate solution
polymerization was conducted at 0°C for 24 hours and then (3 mL) was added to a DMSO solution (5 mL) containing
at room temperature for 3 days to ensure a thorough conver- NH2-PEG-PCL (0.4 g) and triethylamine (0.05 mL). The
sion of EO. In the second step, a predesigned amount of ε-CL reaction was performed at room temperature for 10 hours
was injected into the reaction flask under argon protection under argon. The resulting solution was centrifuged and fil-
and then polymerized at room temperature for 48 hours. tered. The filtrate thus obtained was dialyzed against water for
The polymerization was finally quenched by adding a small 24 hours (MW cutoff: 1 kDa). The aqueous solution inside the
amount of acetic acid. The crude copolymer collected by dialysis bag was then freeze-dried. The powdery sample was
precipitation in hexane was redissolved in dichloromethane redissolved in THF (3 mL), and the filtrate was added drop-
and added to tenfold diethyl ether under vigorous stirring. wise to distilled water under stirring. After overnight evapo-
A white powder was sequentially isolated by filtration and ration of THF, the resultant micelle solution was dialyzed
washed with hexane and diethyl ether. against water for 5 days to completely remove unreacted
1
H-NMR (CDCl3): δ = 1.40 (m, 2H, −COCH2CH2CH2 folic acid and any residual THF. The micelle solution was
CH2CH2O−), 1.65 (m, 4H, −COCH2CH2CH2CH2CH2O−), finally freeze-dried to yield a solid powder (yield $ 82%).
2.31 (t, 2H, − COCH 2 CH 2 CH 2 CH 2 CH 2 O− ), 3.65 To evaluate the conversion rate of NH2-PEG-PCL into folate-
(s, 4H, −CH2CH2O−), 4.05 (t, 2H, −COCH2CH2CH2CH2 PEG-PCL, copolymer was dissolved in DMSO and folate
CH2O−), 4.2 (s, 2H, −CH2CH2OCO−), 5.15∼5.30 (q, 1H, absorbance at 363 nm was measured by a Unico (Shanghai,
CH2 = CH−), 5.80 (m, 2H, CH2 = CH−). China) UV-2000 UV-Vis spectrophotometer to quantify the
folate mass content in the sample. Absorbance of folate at
Conversion of allyl-PEG-PCL 363 nm in DMSO with various concentrations was measured
into NH2-PEG-PCL to generate a calibration curve.
The reaction was conducted in an aqueous micelle solution, 1
H-NMR (DMSO-d6): δ = 1.40 (m, 2H, −COCH2CH2CH2
which was prepared by slowly adding a THF solution (2 mL) CH2CH2O−), 1.65 (m, 4H, −COCH2CH2CH2CH2CH2O−),
of allyl-PEG-PCL (0.5 g) into distilled water (20 mL) under 2.31 (t, 2H, −COCH 2CH 2CH 2CH 2CH 2O−), 2.65∼2.70

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(m, 4H, −CH2SCH2−), 3.2 (t, 3H, H2NCH2CH2S−), 3.65 by sonication and centrifuged at 3000 rpm for 10 minutes at
(s, 4H, −CH2CH2O−), 4.05 (t, 2H, −COCH2CH2CH2CH2- room temperature to remove any large aggregates. The PEG-
CH2O−), 4.2 (s, 2H, −CH2CH2OCO−), 4.45 (d, 2H, C9−H2 PCL-SPIONs formulations are shown in Table 1.
of folic acid), 6.61 (d, 2H, aromatic protons of folic acid),
7.60 (d, 2H, aromatic protons of folic acid), 8.62 (s, 1H, Dynamic light scattering (DLS) measurements
C7-H of folic acid). The diameter of PEG-PCL-SPIONs was examined at 25°C
using a Brookhaven (Holtsville, NY) laser light-scattering
Synthesis of hydrophobic Fe3O4 nanoparticles system consisting of a BI-200SM goniometer and a
(as synthesized SPIONs) BI-9000AT digital correlator. A 532-nm vertically polarized
Fe3O4 nanoparticles (SPIO) were synthesized according to a argon ion laser was used as the light source.
previously reported method.3 Briefly, iron (III) acetylacetonate
(2 mmol), 1,2-hexadecanediol (10 mmol), oleic acid (6 mmol), TEM measurements
oleylamine (6 mmol), and benzyl ether (20 mL) were mixed Transmission electron microscopy (TEM) images were
and magnetically stirred under a flow of nitrogen. The mixture obtained at room temperature in a JEOL (Tokyo, Japan)
was heated to 200°C for 2 hours and then, under a blanket of JEM-2010HR operated at 160 kV. Samples for TEM analysis
nitrogen, heated to reflux (300°C) for 1 hour. The black mixture were prepared by depositing a drop of the solution onto
was cooled to room temperature by removing the heat source. carbon-coated copper grids and dried at room temperature for
The product, 6-nm Fe3O4 nanoparticles, was then precipitated 24 hours. To avoid highly concentrated regions, we scanned
with ethanol, centrifuged (6000 rpm, 10 minutes) to remove the the samples near the border of the drop.
solvent, and redispersed into hexane. A black-brown hexane
dispersion of 6-nm Fe3O4 nanoparticles was then produced. Determination of SPIO-loading contents
The SPIO loading density of PEG-PCL-SPIONs was
determined using a polarized Zeeman Atomic Absorption
Synthesis of hydrophilic Fe3O4 nanoparticles
Spectrophotometer (Z-2000; Hitachi High Technologies,
(WSPIOs) by small molecular surfactant coating
Tokyo, Japan). Briefly, PEG-PCL-SPIONs were f irst
These hydrophilic Fe3O4 nanoparticles were synthesized
weighed before being suspended in 1 M HCl solution to
according to a previous report.3 Under ambient conditions, a
allow for polymer degradation and complete dissolution
hexane dispersion of hydrophobic Fe3O4 nanoparticles (about
of PEG-PCL-SPIONs. Iron concentration was determined
20 mg in 0.2 mL) was added to a suspension of tetrameth-
at the specific Fe-absorption wavelength (248.3 nm) based
ylammonium 11-aminoundecanoate (about 20 mg in 2 mL)
on a previously established calibration curve. SPIO loading
in dichloromethane. The mixture was shaken for about
density was calculated as the ratio of iron oxide over the total
20 minutes, during which time the particles precipitated and
weight of PEG-PCL-SPIONs.
were separated using a magnet. The solvent and nonmagnetic
suspensions were decanted, and the precipitate was washed
Magnetization measurements
once with dichloromethane and separated again using a mag-
The magnetization data of PEG-PCL-SPIONs were deter-
net to remove excess surfactants before drying under N2. The
mined using a MPMS XL-7 Quantum Design (San Diego, CA)
product was then dispersed in deionized water.

Preparation of PEG-PCL coated Fe3O4 nanoparticles Table 1 Properties of SPIONs with different formulations
PEG-PCL coated Fe3O4 nanoparticles (PEG-PCL-SPIONs) Micelle formulation Micelle size Fe3O4 content r1 r2 r2/r1
(nm) (wt%)
were prepared by solvent evaporation method. Briefly, 20 mg
Fa-PEG-PCL-SPIONsa 44 ± 3 33.5% 1.7 113 66
of allyl-PEG-PCL or Fa-PEG-PCL (a mixture of allyl-PEG-
PEG-PCL-SPIONs 38 ± 3 37.0% 1.4 110 78
PCL and folate-PEG-PCL containing 20% folate-PEG-PCL) Hydrophobic SPIO 6 77.0%b nd nd nd
and 2 mg SPIO were dissolved in THF (1.0 mL). The above WSPIOsc 6 3.2 40 12.5
solution was slowly added into 10 mL of deionized water Resovit 65 7.2 82 11.3

under sonication and stirred for 48 hours at room temperature Notes: r1 and r2 values are expressed as Fe mM-1 s-1. aSurface layer was a mixture of
Fa-PEG-PCL and PEG-PCL containing 20% Fa-PEG-PCL; bTested by TGA; cHydrophilic
to remove THF. These particles were separated by magnetic SPIO obtained by small molecular surfactant coating.
Abbreviations: Fa-PEG-PCL-SPIONs, folate-attached poly(ethylene glycol)-poly(ε-
field-guided accumulation and washed with water to remove
caprolactone) superparamagnetic iron oxide nanoparticles; WSPIOs, water-soluble
excessive polymer. Nanoparticles were resuspended in water superparamagnetic iron oxide nanoparticles; nd, not determined.

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Dovepress Targeting tumors with SPION-loaded micelles

SQUID magnetometer at 10 K and 300 K. The applied mag- was a significant difference among the MRI signal intensity
netic field was varied from 2 × 104 Oe to −2 × 104 Oe in order or the rate of signal intensity variety at four time points
to generate hysteresis loops. (0 h, 3 h, 6 h, 24 h), post hoc multiple comparison (Student
Newman–Keuls test) would be used to determine which
In vivo MR imaging time point differed significantly. P-values less than 0.05
Animals (nu/nu CD-1 male nude mice each weighing were considered to be statistically significant. All statistical
25 ± 3 g) were maintained on a folate-free diet for 1 week tests were two-sided and performed using the Statistical
before MR imaging. Subcutaneous tumor xenografts were Package for the Social Sciences (IBM, Armonk, NY) version
generated in anesthetized (10% chloralhydrate) mice by 13.0 software for Windows.
subcutaneous injection of 1 × 107 BEL-7402 cells in 200 µL
of serum-free cell culture medium. Animals were studied by Results and discussion
MR imaging when the subcutaneous xenografts reached a Preparation and characterization of
diameter of about 1 cm. Nanoparticle aqueous solution in
folate-targeted and SPIO-loaded micelles
0.9% soldium chloride was injected into a nude mouse bear-
Amphiphilic block copolymers, folate-PEG-PCL (Mn = 5.1 kD,
ing the BEL-7402 tumor through the tail vein. The applied
Mn(PEG) = 2.9 kD, Mn(PCL) = 0.87 kD) and allyl-PEG-PCL
dose was 10 mg Fe/kg of body weight, which is similar to
(Mn = 3.8 kD, Mn (PEG) = 2.9 kD, Mn (PCL) = 0.87 kD), were used
that recommended for commercial contrast agents such as
for micelle fabrication. They were synthesized by multistep
Resovist. The animals were scanned under an Intera 1.5 T
chemical reactions, as reported in a recent publication,16
MR scanner (Philips, Amsterdam, Netherlands) at room
(Figure 1). Magnetic nanocrystals for MR signal enhance-
temperature. A home-built 4.5 × 9-cm linearly polarized
ment must be well defined in structure and size because the
birdcage radio frequency coil was used for all studies. The
size, crystalline phase, and stoichiometry of these nano-
T2-weighted echo images were acquired before and after
crystals can affect the MR signals. In this research, Fe3O4
nanoparticle administration using the following parameters:
repetition time (TR)/echo time (TE), 5000/100 ms; field of
view, 150 mm; matrix, 256 × 256; slice thickness, 1.5 mm. THF _
K+ K+
At each experimental time point, six mice were scanned for
Potassium naphthalide
both targeting and nontargeting groups. The MRI T2 signal
Potassium + CH THF
intensities within the region of interest were measured. After naphthalide 2 CH — CH2OH CH 2 CH — CH2O−K +
MR imaging, two mice of each group were killed and the Ethylene oxide
tumors were further processed for Prussian blue staining.
CH 2 CH — CH2 OCH2CH2 O−K +
n
Prussian blue staining ε-caprolactone
Tumors were fixed in 10% formalin and cryoprotected with
O
18% sucrose solution. Tissues were then snap frozen in OCT CH 2 CHCH2 OCH2CH2 O C — (CH2)5 O H
n m
medium and sectioned at 8 µm. Sections were rinsed in dis- allyl-PEG-PCL
tilled water and then incubated for 30 minutes in a solution 1. HSCH2CH 2NH 2 ·HCl
2. LiOH
of 2% potassium ferrocyanide and 2% hydrochloric acid in
a 1:1 ratio. After Prussian blue staining, the sections were O
NH2CH2CH2SCH2CH2CH2 OCH2CH2 O CCH2CH2CH2CH2CH2O H
rinsed again and counterstained with 1% neutral red. n m
NH 2 PEG − PCL

Statistical analysis Folic acid

MRI signal intensity and rate of signal intensity variety

Folate-PEG-PCL
were expressed as mean ± standard deviation and median,
OH
O
respectively. One-way analysis of variance was performed Folic acid = N
N
N COOH
H HN
to determine significant changes in MRI signal intensity, H2N N N
COOH
and the Kruskal–Wallis test was used to determine signifi-
Figure 1 Synthetic approach of folate-PEG-PCL for SPION coating.
cant changes in rate of signal intensity variety. If a one-way Abbreviations: PEG-PCL, poly(ethylene glycol)-poly(ε-caprolactone); SPION,
analysis of variance or Kruskal–Wallis test showed that there superparamagnetic iron oxide nanoparticle.

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nanoparticles (SPIO) measuring 6 nm were prepared by apparently can be attributed to the hydrodynamic radius
the high-temperature decomposition method.3 As shown in of the polymeric coating on the iron oxide nanoparticles.
Figure 3A the SPIO nanoparticles are uniform in shape and This result is in agreement with that of Pluronic-coated iron
size. A comparison between the lattice spacing based on the oxide nanoparticles.18
selected area electron diffraction pattern (Figure 3C) with The hydrodynamic size of nanoparticles in physiological
that for bulk Fe3O4 indicates that the particle composition fluids is known to significantly affect their plasma half-life
is Fe3O4. time, biodistribution, and pharmacokinetic properties. Noting
The Fe3O4 nanoparticles prepared by the high-tempera- that the capillary diameter of the reticuloendothelial system
ture decomposition method are hydrophobic. A simple but is about 50 nm,19 the in vivo application of nanoparticles
effective method was adopted to make them water soluble would preferentially require sizes smaller than 50 nm to
and biocompatible. We hypothesized that the hydrophobic achieve long circulation. As to the in vivo MRI application,
segments of PEG-PCL could anchor at the interface of the SPIONs matching this size requirement are very useful for
hydrophobic surfactant shell around iron oxide nanoparticles imaging of the vascular compartment (magnetic resonance
and the hydrophilic PEG segments extend into the aque- angiography), and for perfusion imaging, neurofunctional
ous phase. Figure 2 depicts an amphiphilic copolymer of imaging, imaging of lymph nodes, receptor imaging, and
PEG-PCL being introduced to encapsulate the ultrasmall target-specific imaging.20 Although a lot of SPION-related
SPIO nanocrystal, which forms stable micellar dispersion MRI contrast agents have been reported thus far, develop-
in aqueous media. Figure 3 shows the TEM images of ment of SPIONs combining small size (for example, smaller
PEG-PCL-coated and-uncoated Fe3O4 nanocrystals. The than 50 nm) and high MRI sensitivities is still a hot topic in
PEG-PCL-coated Fe3O4 nanoparticles retain their original this field. Based on the previous work that multiple SPIONs
size of 6 nm without evidence of aggregation (Figure 3B). loading into polymeric micelle cores resulted in high MRI
The diameters of blank PEG-PCL micelle and PEG-PCL- T2 relaxivity and also significant size increase,12 the current
SPIONs were determined by dynamic light scattering, and work successfully reduced particle size by loading ultrasmall
results are shown in Figure 4 and Table 1. The diameter of SPIONs into polymeric micelle core.
blank PEG-PCL micelles is about 28 nm, which is smaller
than that of PEG-PCL-SPIONs (38 nm). The size of PEG- Magnetization and MRI sensitivity of
PCL-SPIONs is highly uniform in water and larger than that folate-targeted and SPIO-loaded micelles
of SPIO nanoparticles (6 nm) unencapsulated with polymeric The magnetization loops of the Fe3O4 nanoparticles and PEG-
micelle. Since dynamic light-scattering measurement pro- PCL-SPIONs measured at both 10 K and room temperature
vides information on the hydrodynamic size of particles, are shown in Figure 5. Both the Fe3O4 nanoparticles and
including the magnetite core and polymer coating layers, the PEG-PCL-SPIONs are ferromagnetic at 10 K with a coerciv-
increase of the particle diameter upon micellar encapsulation ity of 240 Oe and 146 Oe, respectively. At room temperature,

Fe3O4
Fe3O4

PEG PCL
Targeting ligand

Figure 2 Synthesis of monodisperse Fe3O4 nanoparticles coated with biodegradable diblock copolymer.
Abbreviation: PEG-PCL, poly(ethylene glycol)-poly(ε-caprolactone).

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Dovepress Targeting tumors with SPION-loaded micelles

A B A 100
10 K
a b 80
Hexane
Hexane 60
Water Water
40 300 K

M (emu g−1)
20
0
80 10 K
−20

M (emu g−1)
50 nm 50 nm
40 300 K
−40 0
C 1 – 440
1 −60 −40

2 2 – 511 −80
−80

34 −4 −2
H (kOe)
0 2 4
3 – 422 −100
5
6 4 – 400 −20 −15 −10 −5 0 5 10 15 20
H (kOe)
5 – 311

6 – 220 B
100
80 10 K
Figure 3 Transmission electron microscopy (TEM) images of synthesized 6 nm
SPIONs (A) and PEG-PCL-SPIONs (B). The insert (a) shows the synthesized 60
SPIONs were soluble in hexane, and PEG-PCL-SPIONs were easily dispersed in
water (b). The selected area electron diffraction (SAED) pattern of 6-nm Fe3O4 40 300 K

M (emu g−1)
nanoparticles is shown in (C).
Abbreviation: PEG-PCL-SPIONs, poly(ethylene glycol)-poly(ε-caprolactone) 20
superparamagnetic iron oxide nanoparticles. 0
80 10 K
−20

M (emu g−1)
40
300 K
the samples show typical superparamagnetic behavior, with −40 0

zero coercivity and remanence. Under a large external field, −60 −40

the magnetization of the particles aligns with the field direction −80
−80
−4 −2 0 2 4
and reaches its saturation value (saturation magnetization, σs). −100
H (kOe)

A saturation magnetization of 64.7 Fe emu/g was determined −20 −15 −10 −5 0 5 10 15 20

for the Fe3O4 nanoparticles. The value was 61.3 Fe emu/g for H (kOe)
PEG-PCL-SPIONs, indicating no obvious loss in magnetiza- Figure 5 Hysteresis loops of 6-nm Fe3O4 nanoparticles (A) and PEG-PCL-SPIONs
(B) measured at 300 K and 10 K.
tion, which is different from several reports that entrapment
Abbreviation: PEG-PCL-SPIONs, poly(ethylene glycol)-poly(ε-caprolactone)
of magnetic nanoparticles into hydrophobic polymers such superparamagnetic iron oxide nanoparticles.

100
as poly-DL-lactide-coglycolide and polylactides leads to a
loss of saturation magnetization value.21,22 The superparamag-
80 PEG-PCL-SPIONs netic character of the PEG-PCL coated Fe3O4 nanoparticles
PEG-PCL micelles
is important for biomedical applications where remanent
Intensity (%)

60 magnetization is undesirable. As MRI contrast agents, the

particles must rapidly relax their magnetic moment vectors
40 to random directions when the applied magnetic field is
removed.17
20
In addition to the particle size, MRI sensitivity is another
key property determining the application of SPIONs as
0
efficient contrast agents. Based on the MRI mechanism,
0 20 40 60 80 100 120
the image contrast comes from local differences in spin
Diameter (nm)
relaxation kinetics of hydrogen nuclei of water along the
Figure 4 DLS profiles of PEG-PCL-SPIONs and blank PEG-PCL micelle.
Abbreviations: DLS, dynamic light scattering; PEG-PCL-SPIONs, poly(ethylene
longitudinal and transverse planes of the main magnetic field
glycol)-poly(ε-caprolactone) superparamagnetic iron oxide nanoparticles. applied to the specimen. A contrast agent can alter the signal

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Hong et al Dovepress

intensity by selectively shortening hydrogen longitudinal increased by clustering r2 agents within nanocontainers such
(spin–lattice) relaxation time T1 and transverse (spin–spin) as polymeric micelle24 and liposome.10 In this case, the local Fe
relaxation time T2. The effect of an MRI contrast agent is concentration is high and maintainable, whatever the dilution
assessed based on its longitudinal and transverse relaxivities factor applied to the solution. This is apparently not a reason
r1 and r2, which reflect the ability of the contrast agent to alter leading to r2 increase upon coating ultrasmall SPIONs with
T1 and T2, respectively. A T2 agent provides enhancement polymer micelle, because such micellar encapsulation does
to negative MRI contrast and thus causes darkening of not change the local Fe concentration, ie, the stable dispersion
the interfered regions. The higher the r2/r1 ratio, the better of nonclustered 6-nm SPIONs in water, as shown in the TEM
the effectiveness of a T2 agent. Following the size and image Figure 3B. The phenomenon may be explained from
magnetization characterizations, we measured longitudinal the large magnetic field heterogeneity around the nanoparticle
and transverse relaxation times of the water protons (T1 and T2) through which water molecules diffuse. Diffusion induces
for PEG-PCL-SPION aqueous solutions. For comparison, dephasing of the proton magnetic moments and results in
water-soluble Fe3O 4 nanoparticles (WSPIOs) prepared T2 shortening. There exist three types of water molecules in
by mixing hydrophobic Fe3O4 nanoparticles with bipolar colloidal systems: “bound” water, strongly associated with
molecules (tetramethylammonium 11-aminoundecanoate) the polymeric chains by means of hydrogen bonds or polar
were also measured. As shown in Table 1, SPION interactions; “interfacial” water, characterised by hydropho-
coating with polymeric micelle concomitantly increased bic interaction with the macromolecule; and “bulk” water,
r2 relaxivities (110 Fe mM−1 s−1 vs 40 Fe mM−1 s−1) and whose properties are not affected by the presence of the
decreased r1 (1.4 Fe mM−1 s−1 vs 3.2 Fe mM−1 s−1), leading to polymeric matrix.25 T2 relaxivity of bound water is very high
significantly elevated r2/r1 values (78 vs 13). In comparison compared to that of bulk water. For the PEG-PCL-SPION
with the small molecular surfactant coating, SPION coating aqueous dispersions, the mobility of water molecules in the
with polymeric micelle leads to a remarkable r2/r1 increase diffusing layer is restricted because of hydrogen bonding
by a factor of six, strongly indicating greatly enhanced MRI between the water molecules and the hydrophilic chains of
T2 sensitivity. the PEG blocks, resulting in a large amount of bound water,
It is well known that the process of T1 shortening requires which should be one of the reasons for the high T2 relaxivities
direct interaction between protons and the magnetic parts of of PEG-PCL-SPIONs. Note that the r1 and r2 values of the
the contrast agent, and proton T1 relaxation in the regions of commercially available T2 agent Resovit were 7.2 and 82 Fe
interest is expedited by easy access of the water molecules to
the contrast agent. The r1 values of both PEG-PCL-SPION and
A B
WSPIO are significantly smaller than 20–30 Fe mM−1 s−1, typi-
cal for hydrophilic SPION in a dextran matrix (eg, Clariscan,
monocrystalline iron oxide nanoparticles [MION]-46).23
Unlike MION-46 and other T2 contrast agents whose coat-
ing layer is entirely hydrophilic, PEG-PCL-SPIONs and
0h 3h
WSPIOs in the present research comprise two hydrophobic
interlayers, ie, coatings of oleic acid and hydrophobic moiety C D
of small molecular surfactant or polymer, which can impede
the water molecules from contacting with the magnetic core
and consequently leads to the lower r1 value. Furthermore,
the formation of a thick coating layer of hydrophobic PCL
on the original Fe3O4 nanoparticles is hypothesized to be the
underlying cause for an even lower r1 of PEG-PCL-SPIONs 0h 3h

compared to WSPIOs, whose hydrophobic coating layer is

Figure 6 T2-weighted MRI images (TR/TE, 5000/100 ms) taken at 0 and 3 hours after
apparently thinner. T2 shortening in the presence of an MRI injecting 5 mg of Fe/kg of PEG-PCL-SPIONs (A and B) and Fa-PEG-PCL-SPIONs (C and D)
contrast agent is closely related to the local Fe concentration, via tail vein into a nude mouse bearing BEL-7402 tumor (about 1 cm in diameter).
Note: The white arrow indicates the xenograft tumor for determining T2-weighted
particle size, particle surface charge, particle surface func- signal intensity change.
Abbreviations: MRI, magnetic resonance imaging; TR, repetition time; TE, echo time;
tional groups, and the surface area that is accessible to the bulk Fa-PEG-PCL-SPIONs, folate-attached poly(ethylene glycol)-poly(ε-caprolactone)
water. Recent reports have demonstrated that r2 values can be superparamagnetic iron oxide nanoparticles.

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Dovepress Targeting tumors with SPION-loaded micelles

Table 2 Dependence of MRI T2-weighted signal intensity and signal intensity changes of tumor on the postinjection time
Hour(s) Fa-PEG-PCL-SPIO PEG-PCL-SPIO
MRI signal intensity MRI signal intensity MRI signal intensity MRI signal intensity
(x ± s) changes, ΔSI (x ± s) changes, ΔSI
(%, M) (%, M)
0 1691 ± 35 0 1822 ± 84 0
3 998 ± 30* –41.2* 1541 ± 81* –16.4*
6 1145 ± 71* –32.4* 1576 ± 108* –12.9*
24 1503 ± 81* –11.5* 1700 ± 77 –6.43
2 2
Statistical value F = 177.52 χ = 21.69 F = 12.56 χ = 19.98
Notes: *P , 0.05. At each experimental time point, six mice were scanned for both targeting and nontargeting groups. ΔSI was calculated according to the following equation:
ΔSI = (SIpost - SIpre)/SIpre × 100%, where SIpre and SIpost are signal intensities of pre- and postinjection, respectively.
Abbreviation: Fa-PEG-PCL-SPIO, folate-attached poly(ethylene glycol)-poly(ε-caprolactone) superparamagnetic iron oxide.

mM−1 s−1, respectively, corresponding to an r2/r1 value of 11.3 (T2-weighted) of 41.2% at 3 hours after injecting Fa-PEG-
at 1.5T in the manufacturer’s product summary. Therefore the PCL-SPIONs, while only 16.4% decrease at this time point
high r2/r1 value (herein 78) makes PEG-PCL-SPIONs a was observed when the same amount of nontargeting PEG-
very promising T2 contrast agent. To further evaluate their PCL-SPIONs were applied (n = 6). Moreover, at 6 hours after
potential as an effective MRI probe for tumor, we attached a injection, the T2 signal drop for Fa-PEG-PCL-SPIONs was
tumor-targeting ligand (folic acid) onto the micelle surface still as high as 32.4%, suggesting an ideal postinjection time
layer (Figure 2) and conducted the in vitro and in vivo MRI window for MRI scanning. By contrast, the T2 signal drop for
experiments. It is noteworthy that recently Qin et al reported PEG-PCL-SPIONs was only 12.9% at this time point. The
a similar T2 contrast agent prepared by coating SPION surface MRI T2 signal intensity was slowly recovered, along with
with Pluronic F127 copolymers.11 By comparison, SPIONs further extending postinjection time for MRI measurements. It
coated with the biodegradable PCL-PEG micelles should is noteworthy that a similar type of time-dependent intratumor
possess advantages in biosafety. In addition, the Pluronic- accumulation of an antibiofouling polymer-coated SPION
coated SPIONs do not have active targeting function for has been reported.26 As shown in Figure 7, tumor tissues
tumor, ie, no targeting ligand was introduced to the particle from Fa-PEG-PCL-SPION-administered mice showed a high
surface, and their in vivo performance was not tested yet in content of ferric ion accumulation (B), while this was not the
the mentioned literature. case in those tumor tissues from PEG-PCL-SPION-treated
mice (A). The results indicate that the targeting of Fa-PEG-
In vivo experiments PCL-SPIONs is very specific and in good agreement with the
So far, a wide variety of ligands such as antibodies and folic data obtained from MRI scanning.
acid have been used to target cell-surface biomarkers in In conclusion, our study demonstrate that Fa-PEG-PCL-
nanodelivery systems. This strategy enables nanoparticles to SPIONs not only have small particle size, but also high MRI
specifically enter malignant tumor cells, allowing an accurate sensitivity. Combining these characteristics with their specific
diagnosis at a stage when the disease is still treatable. In the tumor-targeting property, Fa-PEG-PCL-SPIONs can be used
current study, we tested the potential of Fa-PEG-PCL-SPIONs as a potential MRI contrast agent for in vivo tumor imaging.
for in vivo imaging applications. Magnetic micelle disper-
sions were injected into a nude mouse bearing the BEL-7402
tumor through the tail vein. Figure 6 shows the T2-weighted
images acquired before and after intravenous administration
of Fa-PEG-PCL-SPIONs into a mouse, which demonstrated
a great signal intensity decrease at a time point 3 hours after
Fa-PEG-PCL-SPION administration. The effect of negative
enhancement on the MRI T2-weighted signal intensity of
Figure 7 Prussian blue staining images of tumor tissues taken from mice at a time
tumor upon injecting magnetic micelle solution into mice point 3 hours after injection of PEG-PCL-SPIONs (A) and Fa-PEG-PCL-SPIONs (B).
Note: Blue stain density reflects the level of SPIO accumulation within tumor.
is summarized in Table 2. Regions of interest covering the
Abbreviation: Fa-PEG-PCL-SPIONs, folate-attached poly(ethylene glycol)-poly(ε-
entire tumor showed an average MRI signal intensity decrease caprolactone) superparamagnetic iron oxide nanoparticles.

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Summary 7. Thünemann AF, Schütt D, Kaufner L, et al. Maghemite nanoparticles

protectively coated with poly(ethylene imine) and poly(ethylene oxide)-
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synthesized by loading iron oxide ultrasmall nanoparticles 8. Nikolic MS, Krack M, Aleksandrovic V, et al. Tailor-made ligands for bio-
compatible nanoparticles. Angew Chem Int Ed. 2006;45:6577–6580.
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loaded micelles, Fa-PEG-PCL-SPIONs, are small (about the MRI relaxivity of manganese ferrite nanoparticle contrast agents.
40 nm) and favorable for long circulation and enhanced Nano Lett. 2007;7:2422–2427.
10. Yuan JJ, Armes SP, Takabayashi Y, et al. Synthesis of biocompatible
MRI T2. Folate functionalization increases the cell uptake poly[2-(methacryloyloxy)ethyl phosphorylcholine]-coated magnetite
of SPION-loaded micelle. Furthermore, the in vivo MRI nanoparticles. Langmuir. 2006;22:10989–10993.
11. Qin J, Laurent S, Jo YS, et al. A high-performance magnetic resonance
experiment and ex vivo histological study indicated that imaging T2 contrast agent. Adv Mater. 2007;19:1874–1878.
the Fa-PEG-PCL-SPIONs may build up in the tumor tissue, 12. Ai H, Flask C, Weinberg B, et al. Magnetite-loaded polymeric micelles
suggesting their potential in MRI diagnosis as a probe for as novel magnetic resonance probe. Adv Mater. 2005;17:1949–1952.
13. Lee JH, Huh YM, Jun YW, et al. Artificially engineered magnetic
folate receptor overexpressing tumor. nanoparticles for ultra-sensitive molecular imaging. Nat Med.
2007;13:95–99.
Acknowledgments 14. Huh YM, Jun YW, Song HT, et al. In vivo magnetic resonance detection
of cancer by using multifunctional magnetic nanocrystals. J Am Chem
This research were supported by the National Natural Science
Soc. 2005;127:12387–12391.
Foundation of China (30900357 to Guo-bin Hong), the 15. Leamon CP, Low PS. Folate-mediated targeting: from diagnostics to
Scientific and Technologic Projects of Guangdong Province, drug and gene delivery. Drug Discov Today. 2001;6:44–51.
16. Yang XQ, Deng WJ, Shuai XT, et al. Folate-functionalized polymeric
China (2007B031516012 to Jing-xing Zhou), the Project micelles for tumor targeted delivery of a potent multidrug-resistance
Supported by Guangdong Natural Science Foundation, China modulator FG020326. J Biomed Mater Res A. 2008;86:48–60.
17. Sahoo Y, Goodarzi A, Swihart MT, et al. Aqueous ferrofluid of
(9451008901001949 to Guo-bin Hong), the Fundamental
magnetite nanoparticles: fluorescence labeling and magnetophoretic
Research Funds for the Central Universities (10ykpy08 control. J Phys Chem B. 2005;109:3879–3885.
to Guo-bin Hong) and the Foundation for Distinguished 18. Jain TK, Morales MA, Sahoo SK, et al. Iron oxide nanoparticles for
sustained delivery of anticancer agents. Mol Pharm. 2005;2:194–205.
Young Talents in Higher Education of Guangdong, China 19. Kim DK, Mikhaylova M, Wang FH, et al. Starch-coated super-
(LYM09006 to Guo-bin Hong). paramagnetic nanoparticles as MR contrast agents. Chem Mater.
2003;15:4343–4351.
Disclosure 20. Weissleder R, Bogdanov A, Neuweltb EA, et al. Long-circulating iron
oxides for MR imaging. Adv Drug Deliv Rev. 1995;16:321–334.
The authors disclose no conflicts of interest. 21. Chattopadhyay P, Gupta RB. Supercritical CO2 based production of
magnetically responsive micro- and nanoparticles for drug targeting.
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