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IOP PUBLISHING NANOTECHNOLOGY
Nanotechnology 24 (2013) 015603 (12pp) doi:10.1088/0957-4484/24/1/015603

Preparation and in vitro evaluation of

folate-receptor-targeted SPION–polymer
micelle hybrids for MRI contrast
enhancement in cancer imaging
Shveta Mahajan1 , Veena Koul2 , Veena Choudhary1 , Gauri Shishodia3
and Alok C Bharti3
1
Centre for Polymer Science and Engineering, Indian Institute of Technology Delhi, New Delhi-110016,
India
2
Centre for Biomedical Engineering, Indian Institute of Technology Delhi, New Delhi-110016, India
3
Division of Molecular Oncology, Institute of Cytology and Preventive Oncology, Noida (UP)-201301,
India

E-mail: veenach@hotmail.com

Received 21 May 2012, in final form 7 November 2012

Published 7 December 2012
Online at stacks.iop.org/Nano/24/015603
Abstract
Polymer–SPION hybrids were investigated for receptor-mediated localization in tumour
tissue. Superparamagnetic iron oxide nanoparticles (SPIONs) prepared by high-temperature
decomposition of iron acetylacetonate were monodisperse (9.27 ± 3.37 nm), with high
saturation magnetization of 76.8 emu g−1 . Amphiphilic copolymers prepared from methyl
methacrylate and PEG methacrylate by atom transfer radical polymerization were conjugated
with folic acid (for folate-receptor specificity). The folate-conjugated polymer had a low
critical micellar concentration (0.4 mg l−1 ), indicating stability of the micellar formulation.
SPION–polymeric micelle clusters were prepared by desolvation of the SPION
dispersion/polymer solution in water. Magnetic resonance imaging of the formulation revealed
very good contrast enhancement, with transverse (T2 ) relaxivity of 260.4 mM−1 s−1 . The
biological evaluation of the SPION micelles included cellular viability assay (MTT) and
uptake in HeLa cells. These studies demonstrated the potential use of these nanoplatforms for
imaging and targeting.
S Online supplementary data available from stacks.iop.org/Nano/24/015603/mmedia
(Some figures may appear in colour only in the online journal)

1. Introduction field is removed [16, 17]. SPIONs have the advantage of

Superparamagnetic iron oxide nanoparticles (SPIONs) are
being extensively researched for biomedical applications
including targeted gene and drug delivery [1–3], magnetother-
mal therapy (e.g. cancer hyperthermia) [4–6] and contrast
enhancement for magnetic resonance imaging (MRI) [7–15].
Magnetite nanoparticles exhibit superparamagnetic behaviour
by virtue of their small sizes; i.e., typically in the 5–20 nm
range they magnetize strongly in an applied magnetic field
but lose the magnetization when the external magnetic
being chemically very stable and non-toxic. However, the
hydrophobicity of the SPION surface makes them prone to
phagocytic capture by macrophages: the specific uptake of
contrast agents may be enhanced by functionalization with
targeting ligands [18].
For prolonged circulation, nanoparticles have been
individually coated with dextran [19, 20], PEG [13, 21] and
other hydrophilic polymers [22–24]. Alternatively, SPIONs
could be encapsulated in amphiphilic block copolymers
to give spontaneously self-assembled nanostructures that

0957-4484/13/015603+12$33.00 1 c 2013 IOP Publishing Ltd Printed in the UK & the USA
Nanotechnology 24 (2013) 015603
combine the advantages of both magnetic particles and
polymeric core–shell systems. The hydrophilic block of the
polymer derivatized with a ligand endows it with targeting
ability while the hydrophobic core-forming block enables
delivery of hydrophobic drugs.
Controlled polymerization processes such as ATRP (atom
transfer radical polymerization) are used for the synthesis
of copolymers with controlled molecular weights, narrow
molecular weight distribution, and defined architectures [25].
ATRP is a robust polymerization technique with tolerance to a
variety of functional groups and monomers and is a facile and
efficient strategy to prepare architectures such as amphiphilic
block copolymers capable of micellization [26]. We used
ATRP for synthesis of PMMA–pPEGMA block copolymers.
Both PMMA and pPEGMA are biologically inert. PMMA
constitutes the hydrophobic block of the copolymer and
forms the core of the micelle in aqueous medium, capable
of encapsulating hydrophobic molecules such as therapeutic
agents or fluorescent labelling species. PEGMA endows
‘stealth’ characteristics and increases circulation half-life
after systemic administration. The polymers were derivatized
with folic acid for targeting the folate receptor, a glycosyl-
phosphatidylinositol-anchored receptor overexpressed by
several human cancers such as those of breast, lung, cervix,
prostate and lymphomas [27, 28], which has high binding
affinity with folic acid (Kd = 10−10 M). In literature,
liposomes, micelles and nanoparticles with folate conjugation
have been reported to exhibit enhanced cellular uptake by
receptor-mediated endocytosis [29].
In this work we prepared SPION–polymeric hybrids for
imaging of folate-receptor overexpressing cancers. Micelles
based on copolymers of methyl methacrylate (MMA) and
poly(ethylene glycol) methacrylate (PEGMA) bearing folic
acid on the termini of pendant PEG chains were combined
with SPIONs. The hydrophobic dye Nile red was encapsulated
in the core of micelles, as a fluorescent probe for biological
studies. Cell culture studies were performed to evaluate
folate-receptor-mediated uptake. The imaging potential of
polymer–SPION composites was investigated by loading
in agar gels and observation by MRI. The advantages
offered by this system over existing formulations are
manifold. At present, the SPION formulations available
commercially consist of an iron oxide core with a dextran
or carboxydextran coating. While they are biocompatible,
they are passively targeted and are localized in the organs of
the RES soon after administration. Encapsulation of SPIONs
within folate-modified polymer ensures concentration in the
tissue of interest. Furthermore, clustering within micellar
systems results in enhanced negative contrast at lower Fe
concentrations. Additionally, a hydrophobic agent, for therapy
or imaging, may be encapsulated in the hydrophobic micellar
core.
2. Methods
2.1. Materials
Iron (III) acetylacetonate (Fe(acac)3 ) and triethylene glycol
(TREG) were procured from Acros Organics (Fair Lawn,
2
S Mahajan et al
NJ, USA). Methyl methacrylate (MMA) was purchased from
Merck (Darmstadt, Germany). MMA was purified by washing
twice with 2% NaOH, then washing with distilled water until
neutral followed by drying over anhydrous sodium sulfate
overnight and vacuum distillation. Poly(ethylene glycol)
methacrylate (PEGMA), ethyl-2-bromo-isobutyrate (EBiB),
2, 20 -dimethyl dipyridine (dMdP), copper (I) bromide,
folic acid (FA), N, N 0 -dicyclohexylcarbodiimide (DCC),
4-dimethylaminopyridine (DMAP) and anhydrous toluene
were purchased from Sigma-Aldrich (St. Louis, MO, USA)
and used as received. Solvents were analytical grade or above
and used as received.
2.2. SPION synthesis and characterization
SPIONs were prepared by a high-temperature solution
decomposition method reported by Cai et al [30]. Briefly,
2 mmol (0.706 g) Fe(acac)3 was dissolved in 25 ml TREG
and heated to 180 ◦ C slowly for 30 min. This was followed
by rapid heating to reflux (∼280 ◦ C) and the solution was
kept at this temperature for another 60 min. The black
colloidal suspension of magnetite particles thus obtained was
flocculated with 50 ml ethyl acetate. The nanoparticles were
separated in a magnetic field, with decantation of supernatant,
redispersed in ethanol, reprecipitated with ethyl acetate and
separated again. The washing of SPIONs was repeated twice
to remove excess TREG.
The FTIR spectrum of SPIONs was recorded with a
Thermo Nicolet IR200 system using KBr pellets of the
sample. Particle size and morphological characterization were
done using HRTEM and selected area electron diffraction
(SAED) at an accelerating voltage of 200 kV (JEOL
2100F). Particle diameter was determined using ImageJ
software: an average diameter of 200 particles was reported.
Crystalline structure was studied by XRD (PANalytical, PW
3040/60 X’Pert PRO) under the following conditions—40 kV
tension and 30 mA current with Cu Kα radiation with
a monochromator (λ = 1.540 598 Å; 20◦ < 2θ < 80◦ ).
Magnetic properties were studied by vibration scanning
magnetometer (VSM, LakeShore Cryotronics) with field
varying between −5 kOe and 5 kOe at room temperature.
The iron oxide content in SPION was determined by TGA
in nitrogen atmosphere (Pyris 6 TGA, Perkin-Elmer). A
5 mg sample was heated from 50 to 800 ◦ C at the rate of
10 ◦ C min−1 .
2.3. Preparation of PMMA-b-pPEGMA copolymers
PMMA–pPEGMA copolymers were synthesized by ATRP.
First, the PMMA-Br macroinitiator was prepared by
polymerization of MMA using EBiB as initiator and a
CuBr/dMdP catalyst system. CuBr (301.3 mg, 2.1 mmol) and
dMdP (386.9 mg, 2.1 mmol) were taken in a dry Schlenk
flask equipped with a magnetic bead and sealed. This was
subjected to three cycles of vacuum degassing followed by
filling with nitrogen. Degassed dry toluene (10 ml) was added
followed by MMA (10.74 ml, 100.8 mmol) with a syringe
under a positive nitrogen pressure. The flask was lowered into
Nanotechnology 24 (2013) 015603
an oil bath at 80 ◦ C, initiator (0.309 ml, 2.1 mmol) was quickly
added under nitrogen flow and the flask was sealed. The
target molecular weight here was 5 kDa at 100% conversion
(monomer to initiator ratio [M]/[I] = 48). The polymerization
was carried out at this temperature for 2 h, after which
catalyst was removed by passing through a neutral alumina
column and the polymer precipitated with methanol as
non-solvent. PMMA-Br macroinitiator with target molecular
weight 10 kDa was prepared similarly.
To prepare the block copolymer, the macroinitiator,
i.e. PMMA-Br, was added at the beginning to the solvent to
dissolve followed by monomer (PEGMA), CuBr and dMdP.
The reaction was carried out at 80 ◦ C for 4 h and after
catalyst removal the copolymer was precipitated with hexane
as non-solvent.
The macroinitiator and copolymer were characterized by
GPC for molecular weight and molecular weight distribution.
GPC measurements were performed on a Waters system with
Styragel columns and THF as eluent. Polystyrene standards
(Shodex) were used for calibration. 1 H-NMR spectra were
recorded using a Bruker 300 MHz instrument with CDCl3 as
solvent.
2.4. Folate conjugation
Folate conjugation was carried out by carbodiimide coupling
between –OH groups of pendant PEG chains and carboxyl
groups from FA (Steglich esterification) [31]. FA, DCC
(1.1 equivalents of FA) and DMAP (5 mol% of DCC)
were dissolved in DMSO and allowed to stir for 6 h in
the dark at room temperature. The 1,3-dicyclohexylurea
precipitate formed was removed by filtration. A solution
of the PMMA–pPEGMA copolymer in DMF is added to
the activated FA solution and allowed to stir overnight at
50 ◦ C. The folate-conjugated product was dialysed against
DMSO for two days followed by water (MWCO 3.5 kDa).
A final dialysis was carried out with acetone as external
medium to facilitate easy recovery of sample by solvent
evaporation. The extent of folate conjugation was quantified
by UV–vis spectroscopy. A calibration curve was prepared
from folic acid solutions in DMSO with measurement
of folate absorbance at 363 nm (UV/VIS Spectrometer,
Perkin-Elmer Lambda 25). The copolymer was dissolved in
DMSO and absorbance at 363 nm was compared against the
calibration curve.
2.5. Preparation of polymeric micelles
A solution of the copolymer or folate-conjugated copolymer
in THF (1 mg ml−1 ) was taken in a flask fitted with a stirrer.
Water (10 ml) was added dropwise with stirring to induce
desolvation of the hydrophobic block in the polymer. Micelles
form with the hydrophilic pPEGMA constituting the shell and
PMMA in the core.
The morphology of the micelles was studied by AFM
(Nanoscope IIIa, Digital Instruments) and TEM (Philips CM
12). Dynamic light scattering (Zetasizer NanoZS, Malvern)
with 173◦ optics was used to determine size (sample
concentration—100 µg ml−1 ).
3
S Mahajan et al
2.6. Critical micellar concentration (CMC)
The pyrene fluorescence method was used to determine
the CMC of copolymers as well as the folate-conjugated
polymer [32, 33]. Aliquots of pyrene solution in THF (40 µl)
were added to glass test tubes and the solvent evaporated
at room temperature. 2 ml polymer solutions at different
concentrations were added to the test tubes containing the
pyrene residue such that the final pyrene concentration was
6 × 10−7 M. The solutions were allowed to equilibrate with
shaking for 24 h. The partitioning of pyrene in the micellar
phase was determined from the I3 /I1 ratio, i.e. the emission
peaks at λem = 383 and 373 nm respectively, with λex set at
339 nm. The CMC was determined from the inflection of the
curve of the I3 /I1 ratio plotted versus polymer concentrations.
Fluorescence spectra were recorded on a Perkin-Elmer LS-45
fluorescence spectrometer.
2.7. Preparation of SPION–micelle clusters
A THF solution of SPION (10% by weight of polymer)
was combined with the polymer solution and water
added dropwise with shaking. SPION–micelle clusters were
visualized by TEM and the content of iron oxide in the
formulation was determined by thermogravimetric analysis
(TGA). TGA of the SPION-loaded micelles was performed
using 5 mg samples. Heating was done in a nitrogen
atmosphere from 5 to 800 ◦ C at the rate of 10 ◦ C min−1 .
A schematic representation of the micellization and the
SPION–micelle cluster formed is given in figure 1.
2.8. Magnetic resonance imaging (MRI)
SPION–micelle suspensions were mixed in 2% w/v agar
solution in concentrations ranging between 0.01 and 0.50 mM
of iron. MRI images were obtained by a 7 T MRI scanner
(Bruker BioSpec 70/30 USR). The transverse relaxation
time (T2 ) for each sample concentration was determined by
acquisition of coronal images at various echo times (TE)
from 13 to 325 ms with a repetition time (TR) of 5000 ms.
Mono-exponential curve fitting of the signal intensity versus
time (TE) data (using Origin 6.1 software) yielded the
relaxation rates R2 (1/T2 ) for different iron concentrations
and the slope of the graph from plotting R2 versus iron
concentration gives the T2 relaxivity, which is a measure
of the sensitivity of a formulation for negative contrast
enhancement.
2.9. Biological evaluation—in vitro experiments
2.9.1. Cellular compatibility. MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide) assay was used to de-
termine biocompatibility of the SPION-loaded micelles [34].
HeLa cells, a cervical carcinoma cell line, were plated at a
concentration of 5000 cells/well and grown in 200 µl/well
of Dulbecco’s modified Eagle’s medium (Sigma) supple-
mented with 10% FCS and 1% streptomycin/penicillin until
sub-confluent. SPION-loaded micelles (unconjugated and
Nanotechnology 24 (2013) 015603
Figure 1. (a) Schematic representation of micellization of folate-conjugated copolymer pMMA–pPEGMA in water with entrapment of
SPIONs and Nile red. (b) A single SPION–micellar cluster with Nile red encapsulation in the core.
folic acid conjugated) were added to the wells at various
concentrations and incubated for 1, 6 and 24 h. The
supernatant was removed and MTT in cell medium was added
to the wells and incubated for a further 2 h. Purple formazan
crystals were solubilized with 0.1 N HCl in isopropanol.
Absorbance was measured at 540 nm with subtraction for
plate absorbance at 620 nm using a 96-well multiscanner
autoreader (BioTek PowerWave XS2, Vermont) with the lysis
buffer serving as blank. Cell viability was calculated as a
percentage with respect to the absorbance of untreated control
wells.
2.9.2. Particle uptake—in vitro iron quantification. For
demonstration of dose dependence of internalization ki-
netics, SPION-loaded micelles were incubated at different
concentrations with HeLa cells. The cells were seeded in
24-well plates at a density of 2 × 105 cells per well in
folate-free DMEM medium and grown for 24 h. After 24 h
the culture medium was replaced. SPION-loaded micelles
with and without folate conjugation were incubated with the
cells at concentrations of 2, 5, 10, 20, 30 and 50 µg ml−1
(Fe) for 6 h. Time kinetics of internalization were studied
by treating cells, grown in 12-well plates at a density of
3 × 105 , with the SPION micelles at a concentration of
20 µg ml−1 (Fe) for different time intervals. To demonstrate
folate-receptor-mediated uptake, cell cultures were treated
with folate-conjugated micelle SPIONs and unconjugated
micelle SPIONs in folate-free medium. A second set of cell
cultures was treated with micelle–SPION clusters in medium
supplemented with free folic acid, for inhibition control by
4
S Mahajan et al
receptor blocking. At the end of the incubation period, cells
were washed twice with PBS and the cell monolayers were
digested with 6 N HCl (100 µl) for 1 h. This was followed
by addition of a 5% solution of K4 [Fe(CN)6 ] (Merck) in
water (Milli-Q, 18.2 M cm resistivity). After 10 min the
absorbance was read at 690 nm in a multiwell plate reader
(BioTek PowerWave XS2). A standard curve was plotted from
a SPION dispersion of known concentration. Each experiment
was performed in triplicate with calculation of mean and
standard deviation.
2.9.3. Confocal laser scanning microscopy. HeLa cells were
grown on cover-slips until sub-confluent. Folate-conjugated
and unconjugated micelle–SPION clusters loaded with Nile
red were added to the cells at concentrations of 20 µg ml−1
(Fe) and incubated for 1, 6 and 24 h. After the incubation
period, the cells were washed with cold PBS and fixed with
3.7% paraformaldehyde. Following washing with PBS they
were counterstained with Hoechst for 15 min. The cells were
again washed with PBS. The cover-slips were mounted on
slides with mounting medium (Fluoromount, Sigma) and
observed under a confocal microscope (Olympus FluoView
FV1000).
2.10. Statistical analysis
Statistical analysis was done with GraphPad Prism, version
5.02, using the Student t-test and one-way ANOVA. P < 0.05
was considered statistically significant.
Nanotechnology 24 (2013) 015603 S Mahajan et al

Figure 2. SPION characterization: (a) TEM micrograph; (b) HRTEM of single crystallite showing lattice planes and the (440) lattice fringe
at 1.49 Å; (c) SAED pattern showing concentric rings with spots characteristic of crystallinity; (d) XRD pattern; (e) magnetization versus
magnetic field (M–H); (f) TGA.

3. Results and discussion 3.1. Preparation and characterization of SPIONs

Strategies to develop contrast agents for tumour-specific MR
imaging focus on the use of targeting ligands specific for
receptors that are overexpressed by tumours. The α-isoform
of the folate receptor is a highly specific tumour marker,
upregulated in several cancers of epithelial origin but with
limited expression in normal tissues. Additionally, the folate
receptor has a high binding affinity to FA (Kd ∼ 100 pM).
These factors render folate receptor targeting a good strategy
for tumour imaging. In this work SPIONs were prepared by
the thermal decomposition of an iron organic precursor and
combined with the amphiphilic copolymer PMMA–pPEGMA
(derivatized with FA) for MR imaging of folate-receptor
overexpressing cancers.
Magnetic nanoparticles were prepared by high-temperature
decomposition of Fe(acac)3 in solvent TREG. The method
confers the twin advantages of easy dispersibility of the
magnetic particles in water as they are coated with the
hydrophilic polyol ligand (TREG) in situ, and enhanced
magnetic properties as the high temperatures used increase
the particle crystallinity. The TEM micrograph reveals
spherical particles having the diameter 9.27 ± 3.37 nm
(figure 2(a)). A single iron oxide nanocrystal is shown
in the high-resolution TEM micrograph (figure 2(b)), in
which the individual lattice planes are clearly visible. The
measured d-spacing of 1.49 Å corresponds to that for the
(440) plane (1.484 Å) of magnetite. The SAED pattern

5
Nanotechnology 24 (2013) 015603
Figure 3. UV–vis spectra of M1 EG1 , FM1 EG1 and folic acid
(inset). Folic acid shows characteristic absorption at λmax = 363 nm.
(figure 2(c)) shows spots on rings, confirming the crystallinity
of the material. The diffraction pattern is consistent with
that of magnetite/maghemite (JCPDS file card 19-629) [35]
while the XRD pattern reveals peaks whose position
and intensity correspond to the cubic spinel structure of
bulk magnetite/maghemite (figure 2(d)). The d spacing as
(calculated from the 2θ values from XRD) with the respective
hkl indices is shown in table 1. The nanoparticles are
crystalline, and the peak broadening indicates the very small
size of the crystallites. Crystallite size as determined by the
Debye–Scherrer equation was found to be 8.74 nm, which
is close to size determined from TEM, indicating that the
nanoparticles are single crystals.
Magnetization was determined using VSM. The hystere-
sis loop for SPION (figure 2(e)) has the shape characteristic of
superparamagnetic behaviour: as the field increases from −5
to 5 kOe, the magnetization increases non-linearly; however,
when the field is decreased from 5 kOe and approaches
zero, the magnetization decreases to zero, depicting negligible
hysteresis (figure 2(e) inset). The saturation magnetization
(M) at room temperature (field H = 5 kOe) was found to
be 60.4 emu g−1 . The iron oxide content determined by
TGA was 78.7%; therefore, the corrected magnetization (Ms )
value is 76.8 emu g−1 iron oxide (IO), a value lower than
the 92 emu g−1 value of bulk magnetite. The difference is
accounted for by the very large surface-to-volume ratio of the
nanoparticles compared to their bulk counterparts, resulting in
a lowering in magnetization due to spin disorder in the surface
layer.
The magnetic nanoparticles were stabilized with triethy-
lene glycol (TREG) adsorbed on the nanoparticle surface.
TGA shows weight loss with an onset at 242 ◦ C and end
at 324 ◦ C corresponding to the loss of TREG (b.p. 280 ◦ C)
(figure 2(f)). TREG on the surface was further confirmed
by FTIR (supplementary information, figure S1 available at
stacks.iop.org/Nano/24/015603/mmedia) [24].
3.2. Polymerization
ATRP is a facile method for preparing block copolymers
from macroinitiators having halogen end groups. Here
6
S Mahajan et al
Table 1. d-spacing for sample SPION calculated from peak
positions in XRD pattern and compared with standard JCPDS
values for magnetite; corresponding hkl indices.
d-spacing of Fe
2θ d-spacing (Å) (Å, from JCPDS) hkl
30.67 2.960 2.967 220
36.01 2.492 2.532 311
43.11 2.097 2.099 400
51.94 1.759 1.714 422
57.47 1.602 1.615 511
63.33 1.467 1.484 440
PMMA-Br macroinitiators were prepared from MMA using
an EBiB initiator and Cu[I]/dMdP catalyst system, with
variation in monomer to initiator (EBiB) ratios. PMMA-
b-pPEGMA block copolymers were prepared by chain
extension of PMMA-Br macroinitiator, with varying PEGMA
to macroinitiator ratio. The compositions of the prepared
polymers as well as theoretical and observed molecular
weights are given in table 2.
The molecular weight of the PMMA-Br macroinitiator
was determined from 1 H-NMR taking the ratio of intensities
of the signal at 3.6 ppm (s) (–OCH3 , methacrylate) and 4.12
ppm (q) (–OCH2 CH3 , initiator). The following equation was
used to calculate the degree of polymerization:
I3.6 /3
DP = . (1)
I4.12 /2
The molecular weight from 1 H-NMR is in reasonable
agreement from the theoretical molecular weight as de-
termined from percentage conversion. However, Mn,GPC is
much higher; this is because polystyrene standards used for
calibration in GPC have a different hydrodynamic volume
in the solvent used for elution (THF). The polydispersity
decreases from 1.42 to 1.27 after copolymerization and
the absence of a shoulder on the lower molecular weight
side of the elution curve of the copolymer indicates
that all macroinitiator chain ends are active and initiate
polymerization of the second monomer.
FA conjugation was accomplished by carbodiimide
coupling between hydroxyl groups of pendant PEG chains in
the hydrophilic block and –COOH groups in FA molecules,
and confirmed by UV spectroscopy, where the FM1 EG1
polymer shows absorption at 363 nm, characteristic of
folic acid (figure 3). The amount of folate conjugated was
determined to be 0.31 ± 0.025 mmol of folic acid per gram
of polymer.
3.3. Micellization behaviour of folate-conjugated and
unconjugated PMMA–pPEGMA copolymers
Micelles were prepared from the copolymers M1 EG1 , M1 EG2
and M2 EG1 and the folate-conjugated polymer FM1 EG1 by
addition of water to the THF solutions of these polymers. The
addition of water induces thermodynamically driven micellar
self-assembly of the amphiphile, whereby the pPEGMA chain
ends constitute the micellar shell and the PMMA blocks are
driven to the core.
Nanotechnology 24 (2013) 015603 S Mahajan et al

Figure 4. (a) Pyrene emission spectra for M1 EG1 at concentrations 0.01 and 15 mg l−1 , λex = 349 nm. For determination of CMC
I383 /I373 is plotted as a function of concentration of the polymers: (b) M1 EG1 ; (c) M1 EG2 ; (d) M2 EG1 ; (e) FM1 EG1 .

Table 2. Polymer compositions, molecular weight and polydispersity.

Sample Ratio Monomer Polydispersity
designation [M]/[I]/[catalyst] conversion (%) MWTheo MWNMR Mn,GPC (GPC)
MMA: EBiB M1 48:1:1 73.3 3670 3184 10 501 1.42
M2 98:1:1 87.97 8800 7900 17 037 1.58
PEGMA: PMMA-Br M1 EG1 5:1:1 59.6 4743 — 13 861 1.27
M1 EG2 10:1:1 62.8 5931 — 19 780 1.31
M2 EG1 12.22:1:1 63.1 9676 — 19 686 1.50

7
Nanotechnology 24 (2013) 015603
Figure 5. TEM micrographs. (a) M1 EG1 . (b) A single micelle: core–shell morphology is visible. (c) FM1 EG1 . (d) FM1 EG1 –SPION cluster.
Pyrene, a hydrophobic fluorophore with high sensitivity
to polarity of the local medium, was used to determine the
CMC of the polymers. The relative intensities of the first
(I373 ) and third (I383 ) vibronic bands in the fluorescence
spectrum of pyrene show a strong dependence on medium
polarity [32]. In a polar environment there is enhancement of
the first band at the expense of the third band (figure 4(a)).
However, if the external medium has low polarity there
is lowering of the intensity of the band at 373 nm. In
micellization this is reflected in the sharp increase in the
curve of I383 /I373 ratio plotted versus concentration. The
concentration at the inflection point of the curve represents
the partitioning of pyrene into the hydrophobic interior of
micelles, and is defined as CMC.
CMC values for M1 EG1 , M1 EG2 and M2 EG1 were 4.2,
4.5 and 0.32 mg l−1 respectively (from figures 4(b)–(e)). It is
evident that as the size of the hydrophobic block increases
the CMC decreases, i.e. micellization occurs at a lower
polymer concentration. Furthermore, after folate conjugation
of M1 EG1 , the CMC drops to 0.4 mg l−1 . This CMC value
is quite low, which is advantageous in that the micelles will
remain stable even when considerable dilution occurs in in
vivo usage.
DLS measurements reveal that the micelles M1 EG1
possess a diameter of 95.82 nm with a narrow size
distribution (polydispersity index (PDI) 0.073). Micelles
formed from copolymer M1 EG2 have the diameter 110.3 nm
(PDI 0.11), slightly larger because the longer hydrophilic
8
S Mahajan et al
block contributes to the hydrodynamic radius of the micelle.
However, a considerable increase in size is observed in the
case of M2 EG1 micelles, 194.8 nm (PDI = 0.15), which
possess a large hydrophobic core.
Folate conjugation also results in an increase in size:
FM1 EG1 micelles have the diameter 118.7 nm (PDI
0.108). TEM micrographs of M1 EG1 and FM1 EG1 reveal
spherical micelles with a distinct core–shell morphology
(figures 5(a)–(c)).
SPIONs incorporated in the folate-conjugated copolymer
FM1 EG1 are shown in figure 5(d). SPIONs are clustered
in the hydrophilic part of the micelles, as their surface
has hydrophilic character due to adsorbed TREG (solvent)
molecules incorporated during nanoparticle synthesis. The
loading of SPIONs in the polymer was quantified by TGA.
It was found that the iron oxide content was 8.14 and
7.98 mg per 100 mg of FM1 EG1 –SPION and M1 EG1 –SPION
respectively. Inclusion of SPION does not affect micellar size.
3.4. Magnetic resonance imaging of SPION-loaded micelles
The transverse relaxation time T2 of SPION-loaded micelles
was determined using the following curve-fitting formula to
the plots of MRI-signal intensity versus echo time (TE) for
various Fe concentrations (figure 6):
  
TE
y = y0 + C exp − . (2)
T2
Nanotechnology 24 (2013) 015603
Figure 6. Magnetic resonance imaging of micellar-SPION clusters
(FM1 EG1 –SPION): (a) T2 -weighted images of clusters in phantom
agar gels; (b) T2 relaxation analysis curves; (c) T2 relaxation rate
(1/T2 ) as a function of Fe concentration (mM).
T2 values thus obtained were plotted against the different
Fe concentrations and the slope of this curve gave the T2
relaxivity (R2 ).
It is evident from figures 6(a) and (b) that with increasing
SPION concentration the signal darkening increases and the
relaxation curves become steeper. Signal enhancement is
given by the following relationship [36]:
Enhancement (%) = [(SISPION − SIblank )/SIblank ] × 100 (3)
where SISPION is the signal intensity from agar gel containing
polymeric micelles only and SIblank is the signal intensity
from SPION-loaded micelles. At a concentration of 0.05 mM
Fe (2.79 µg ml−1 Fe) the contrast enhancement is −65.6%,
while at the highest concentration used, 0.5 mM Fe
(27.9 µg ml−1 Fe), −92.2% contrast enhancement occurs
compared to the blank (TE = 13 ms).
T2 relaxivity (from the slope of the graph of
1/T2 versus Fe concentration, figure 6(c)) is found to
be 260.4 mM–1 s−1 , which is much higher than for
commercial formulations like the carboxydextran-coated
Resovist R (151.0 mM−1 s−1 ) [37]. Higher relaxivity means
that lower doses may be administered and still achieve
accurate differentiation of tissues in MRI.
3.5. Cytotoxicity of SPION–micelle clusters
Biocompatibility of the SPION-loaded micelles was evaluated
with HeLa cells, a cervical carcinoma cell line, by the
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S Mahajan et al
MTT assay for different concentrations and at various
time intervals (figures 7(a)–(c)). After 24 h of incubation
the cell viability at 20 µg ml−1 concentration was
90.3 and 92.8% for FM1 EG1 –SPION and M1 EG1 –SPION
formulations respectively (figure 7(a)), while at 50 µg ml−1
concentration 56.4 and 63.4% viable cells were observed with
FM1 EG1 –SPION and M1 EG1 –SPION (figure 7(b)). Based on
the cytotoxicity data, 20 µg ml−1 concentration was chosen
for cell uptake evaluations.
3.6. Cellular internalization by quantification of iron oxide
and confocal microscopy
The uptake of SPION-loaded folate-conjugated and uncon-
jugated micelles was evaluated in the HeLa cell line. These
cells overexpress the folate receptor. It is seen that the extent
of internalization of the folate-conjugated micelles increases
significantly as time of incubation increases from 1 to 24 h,
when the external medium is folic acid free (figure 8(a)). After
1 h incubation there is not a significant difference in uptake
of folate-conjugated (FM1 EG1 ) and unconjugated micelles
(M1 EG1 ), whereas after 6 h folate-conjugated micelles are
internalized to an extent nearly 11 times greater than the
unconjugated micelles. Similarly, after 24 h the folated
micelles are internalized by a factor 12 times higher than
unconjugated micelles. The difference in uptake is significant
even in the case when the external medium contains folic
acid as part of the cell-growth medium (figure 8(b)).
The internalization of folate-conjugated micelles when the
external medium contains folic acid is significantly lower than
when the medium is folate free, as folate receptors in the
cell membranes are blocked by competitive binding of folic
acid in the external medium. However, in this case too there
is an increase in intracellular concentration with time, as the
receptors lose the blocking folic acid moieties over time and
are recycled back to the cell surface.
The dependence of the extent of internalization on
concentration was studied by treating the cells with different
concentrations of the SPION micelles (figure 8(c)). It was
found that the uptake increases with the concentration of
formulation. A ‘levelling off’ of internalized iron oxide
was observed when the concentration was increased beyond
20–50 µg ml−1 , as the cells reach saturation.
Folate-specific cellular internalization was further studied
using confocal microscopy. HeLa cells were treated with
FM1 EG1 –SPION and M1 EG1 –SPION micelles loaded with
the hydrophobic fluorophore Nile red, which was used
as a tracker. Observations from confocal laser microscopy
corroborated the quantitative results from the cell uptake
experiments described above. Figures 9(a)–(e) show the
images of micelle–SPION clusters incubated with HeLa
cells for different time points in the presence and absence
of folic acid in the external medium. Figure 9(a) depicts
folate-conjugated micelles incubated with the cells for 24 h
when the culture medium was kept folate free. The red
micelles are seen internalized extensively in the cytoplasmic
region. Figure 9(b) shows unconjugated micelles in a similar
treatment, showing non-specific cellular internalization of
Nanotechnology 24 (2013) 015603 S Mahajan et al

Figure 7. Cytotoxicity evaluation of SPION-loaded micelles: HeLa cells were incubated with (a) 20 µg ml−1 Fe and (b) 50 µg ml−1 Fe of
folate-conjugated (FM1 EG1 ) and unconjugated (M1 EG1 ) micelle–SPION clusters and their viability was evaluated by MTT assay against
untreated control cells. (c) Dose response of FM1 EG1 –SPION and M1 EG1 –SPION clusters in terms of percentage cell viability of HeLa
cells after 24 h treatment. Error bars indicate mean ± SD (n = 3).

Figure 8. Intracellular localization of FM1 EG1 –SPION and M1 EG1 –SPION clusters in HeLa cells when (a) the external medium is folate
deficient or (b) when it contains folic acid. (c) Dose kinetics of internalization of folate-conjugated (FM1 EG1 –SPION) and unconjugated
(M1 EG1 –SPION) clusters. Error bars indicate mean ± SD (n = 3).

10
Nanotechnology 24 (2013) 015603
Figure 9. Confocal microscopy—(a) FM1 EG1 –SPION clusters
incubated with HeLa cells for 24 h, folate-deficient medium;
(b) M1 EG1 –SPION, 24 h incubation, folate-deficient medium;
(c) FM1 EG1 –SPION, 24 h incubation, folate-supplemented
medium; (d) FM1 EG1 –SPION, 6 h incubation, folate-deficient
medium; (e) FM1 EG1 –SPION, 1 h incubation, folate-deficient
medium.
micelles. The extent of internalization was distinctly less than
that of the folate-conjugated micelles, whose uptake is aided
by a folate-receptor-mediated endocytic pathway. The third
panel (figure 9(c)) also elegantly demonstrates the role of the
folate receptor in particle uptake. Here the cells were cultured
with folate-conjugated micelles in the presence of free folic
acid in the culture medium—the folate-mediated cellular
internalization of folate-conjugated micelles is observed to
be severely compromised. Figures 9(d) and (e) represent
folate-conjugated micelles incubated with the cells for 6 and
1 h respectively (folate-free medium). At 6 h of incubation
there was already a substantial concentration of micelles
within cells; however, none may be clearly seen after 1 h of
incubation time.
4. Conclusions
In conclusion, the nanohybrids were prepared with a high
T2 relaxivity of 260.4 mM−1 s−1 , which compares very
favourably against the values reported for commercially avail-
able MRI contrast agents (e.g. Resovist R , 151.0 mM−1 s−1 ;
Feridex IV, 98.3 mM−1 s−1 ). A high T2 relaxivity indicates
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S Mahajan et al
that better negative contrast enhancement may be obtained
in MRI with lower concentrations of the agent. These
nanoconstructs have potential for use as therapeutic contrast
enhancers for folate-receptor overexpressing cancers. ATRP
was used to prepare polymers in a reproducible and scalable
manner. Low CMC values indicate that micelles carrying
the SPION will be stable on injection and not lose their
payload upon dilution. Cell uptake studies in HeLa cervical
carcinoma cells, which overexpress the folate receptor,
reveal that folate-conjugated SPION–micelle formulations
are internalized to a markedly higher concentration than the
unconjugated micelles. Thus, this system shows promise as a
T2 -weighted MRI contrast agent for cancer imaging, and has
further scope for active drug targeting and adjuvant therapy
(radiofrequency hyperthermia).
Acknowledgment
SM acknowledges the Council for Scientific and Industrial
Research, India, for a senior research fellowship.
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