Stability of polyelectrolyte-coated iron nanoparticles for T2-weighted magnetic resonance imaging

Article abstract of DOI:10.1016/j.jmmm.2017.04.026

Iron nanoparticles are highly-effective magnetic nanoparticles for T₂ magnetic resonance imaging (MRI). However, the stability of their magnetic properties is dependent on good protection of the iron core from oxidation in aqueous media. Here we report the synthesis of custom-synthesized phosphonate-grafted polyelectrolytes (PolyM3) of various chain lengths, for efficient coating of iron nanoparticles with a native iron oxide shell. The size of the nanoparticle-polyelectrolyte assemblies was investigated by transmission electron microscopy and dynamic light scattering, while surface attachment was confirmed by Fourier transform infrared spectroscopy. Low cytotoxicity was observed for each of the nanoparticle-polyelectrolyte (“Fe-PolyM3”) assemblies, with good cell viability (>80%) remaining up to 100?μg?mL^?1 Fe in HeLa cells. When applied in T₂-weighted MRI, corresponding T₂ relaxivities (r₂) of the Fe-PolyM3 assemblies were found to be dependent on the chain length of the polyelectrolyte. A significant increase in contrast was observed when polyelectrolyte chain length was increased from 6 to 65 repeating units, implying a critical chain length required for stabilization of the α-Fe nanoparticle core.

Full text of DOI:10.1016/j.jmmm.2017.04.026

Journal of Magnetism and Magnetic Materials 439 (2017) 251–258
Contents lists available at ScienceDirect
Journal of Magnetism and Magnetic Materials
journal homepage: www.elsevier.com/locate/jmmm
Research articles
Stability of polyelectrolyte-coated iron nanoparticles for T -weighted
2
magnetic resonance imaging
a,
⇑
b
a
c
b
Andrew J. McGrath , Ciaran Dolan , Soshan Cheong , David A.J. Herman , Briar Naysmith ,
c
c
d
d
b
Fangrong Zong , Petrik Galvosas , Kathryn J. Farrand , Ian F. Hermans , Margaret Brimble ,
David E. Williams , Jianyong Jin , Richard D. Tilley
b
b
a,e
a
School of Chemistry, University of New South Wales, Sydney, NSW 2052, Australia
School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
School of Chemical and Physical Sciences and the MacDiarmid Institute for Advanced Materials and Nanotechnology, Victoria University of Wellington, Wellington 6012, New Zealand
Malaghan Institute of Medical Research, P.O. Box 7060, Wellington 6012, New Zealand
Australian Centre for Nanomedicine, University of New South Wales, Sydney, NSW 2052, Australia
b
c
d
e
a r t i c l e i n f o
a b s t r a c t
Article history:
Iron nanoparticles are highly-effective magnetic nanoparticles for T magnetic resonance imaging (MRI).
However, the stability of their magnetic properties is dependent on good protection of the iron core from
2
Received 24 March 2017
Received in revised form 12 April 2017
Accepted 12 April 2017
oxidation in aqueous media. Here we report the synthesis of custom-synthesized phosphonate-grafted
polyelectrolytes (PolyM3) of various chain lengths, for efficient coating of iron nanoparticles with a native
iron oxide shell. The size of the nanoparticle-polyelectrolyte assemblies was investigated by transmission
electron microscopy and dynamic light scattering, while surface attachment was confirmed by Fourier
transform infrared spectroscopy. Low cytotoxicity was observed for each of the nanoparticle-
Available online 10 May 2017
Keywords:
Iron nanoparticles
Magnetic nanoparticles
MRI
ꢀ1
polyelectrolyte (‘‘Fe-PolyM3”) assemblies, with good cell viability (>80%) remaining up to 100
lg mL
Fe in HeLa cells. When applied in T -weighted MRI, corresponding T relaxivities (r ) of the Fe-PolyM3
2
2
2
assemblies were found to be dependent on the chain length of the polyelectrolyte. A significant increase
in contrast was observed when polyelectrolyte chain length was increased from 6 to 65 repeating units,
Polyelectrolyte
Coating
implying a critical chain length required for stabilization of the
a-Fe nanoparticle core.
Ó 2017 Elsevier B.V. All rights reserved.
1
. Introduction
aqueous and physiological media through surface ligation chem-
istry known for iron oxide nanoparticles. A considerable challenge
lies in performing the phase transfer of iron nanoparticles from
organic to aqueous media while avoiding oxidation of the highly
Interfacing inorganic nanoparticles with biological systems is
an ongoing field of intense research [1–3]. With effective surface
modification and core protection, nanoparticles have a great
potential to be utilized as novel and non-invasive biomedical
agents in the clinic [4–6]. One particular area of biomedicine which
has benefited from the emergence of nanotechnology is magnetic
resonance imaging (MRI) of early-stage cancers via the localization
of magnetic nanoparticles into tumour tissue [7–9]. Metallic iron
nanoparticles are of high interest as MRI agents, due to their high
reactive
a-Fe core [14,15]. When the nanoparticle is exposed to
conditions of air and moisture, the iron atoms in the core diffuse
to the surface and oxidize through the Kirkendall effect [16], caus-
ing voids to form in the core and significantly reducing the mass
magnetization of the nanoparticles. One strategy currently used
for protection of iron nanoparticles toward oxidation is functional-
ization of the iron oxide surface with dimercaptosuccinic acid
(DMSA) [10–12,14,17], which gives the iron nanoparticles good
hydrophilicity as well as protection against oxidation via reductive
linkage of adjacent thiol groups [18]. However, concerns have been
raised in the literature regarding the efficient cell internalization of
DMSA possibly resulting in overloading of such nanoparticles
within cells, inducing cytotoxicity [19]. Developing alternative
strategies for making biocompatible nanoparticle composites
remain an area of extensive research.
2
saturation magnetization that allows for highly-enriched T con-
trast at biocompatible concentrations [10–14]. Though often syn-
thesized in hydrophobic solvents, upon exposure to ambient
conditions iron nanoparticles oxidize on the surface to form iron
oxide shells, which then allows them to be rendered stable in
⇑
Corresponding author.
E-mail address: a.mcgrath@unsw.edu.au (A.J. McGrath).
http://dx.doi.org/10.1016/j.jmmm.2017.04.026
304-8853/Ó 2017 Elsevier B.V. All rights reserved.
0

2
52
A.J. McGrath et al. / Journal of Magnetism and Magnetic Materials 439 (2017) 251–258
Nanoparticles stabilized with polyelectrolytes have proven to
broadband decoupled mode, and assignments were determined
using distortionless enhancement by polarization transfer
(DEPT) and heteronuclear single quantum coherence (HSQC)
sequences. Matrix-assisted laser desorption/ionization time-of-
flight (MALDI-ToF) mass spectrometry measurements were per-
formed on an Applied Biosystems Voyager DE-Pro MALDI-ToF mass
spectrometer using 1,8,9-anthracenetriol (dithranol) as the matrix.
All experiments were carried out using an accelerating voltage of
20 kV and the resulting spectra were accumulated from 500 shots.
Sample solutions for MALDI-ToF were prepared by making stock
solutions of dithranol (80 mg/mL) and polymer (10 mg/mL) in
tetrahydrofuran (THF). Aliquots of these stock solutions were then
combined in a weight ratio of 8: 1 (dithranol: polymer), immedi-
ately spotted on an assay sample plate, and dried at room temper-
ature for analysis. Gel-permeation chromatography (GPC) analysis
was performed on a Viscotek VE2001 GPC Max module with a
Viscotek TDA 305 detector. Polystyrene standards (95 kDa and
235 kDa) were used as calibration standards for the GPC and all
samples were filtered prior to analysis (0.22 mm filter). THF was
used as eluent with a flow rate of 1.0 mL/min.
be highly effective biomedical imaging agents in vivo. Block poly-
electrolytes with a high density of anchoring groups per molecule
have been widely used to stabilize nanoparticles of a variety of
compositions, and have been widely used to transfer magnetic
2
nanoparticles from organic to aqueous phase for T MRI applica-
tions [20–24]. Due to each anchoring group being covalently linked
at the polyelectrolyte backbone, a second cross-linking step to
encapsulate nanoparticles is not required post-ligand exchange.
By polymerizing monomer species containing selected groups,
the physicochemical properties of these polyelectrolytes can be
tailored accordingly [25]. This polyelectrolyte coating strategy
has not been previously used for coating of iron nanoparticles,
therefore questions regarding the potential instability of iron
nanoparticles using this approach remain unanswered. Previous
studies have suggested that the molecular weight, and therefore
the chain length of block polymers, plays a significant role in pro-
tecting and stabilizing nanoparticles in solution [26–28]. It was
predicted that the chain length of polyelectrolyte should have a
significant effect on its ability to stabilize iron nanoparticles
accordingly, and by extension an effect on the long-term T
performance of the nanoparticle-polymer assembly.
2
MRI
Samples for transmission electron microscopy (TEM) were pre-
pared by drop-casting a solution of nanoparticles suspended in
toluene (as-synthesized) or in methanol (ligand exchanged with
Among the different polymerization methods, reversible addi-
tion fragmentation chain transfer (RAFT) polymerization has been
successfully used to synthesize phosphonate-grafted polyelec-
trolytes with good control over polyelectrolyte chain lengths and
polydispersity, which is a concern for large-scale polyelectrolyte
synthesis [29]. This study reports the RAFT synthesis of a
phosphonate-containing polyelectrolyte poly(2-(acrylamido)ethyl
phosphonic acid) (herein denoted as PolyM3), in which the phos-
phonate group is utilized to give the polyelectrolyte high
hydrophilicity [30] and at the same time a high affinity for the sur-
face of iron nanoparticles [31]. The functionalization of nanoparti-
cles occurs via a facile, one-step process involving sonication of a
suspension of nanoparticles and the polyelectrolyte. The biocom-
x
PolyM3-n ) onto a carbon-coated copper grid. Images were taken
on a JEOL 2010 microscope operating at an acceleration voltage
of 200 kV. For dynamic light scattering measurements, a portion
of the Fe-PolyM3 nanoparticle conjugates was suspended in dis-
ꢀ1
tilled water at a concentration of ꢁ0.1 mg mL . The aqueous solu-
Ò
tion was filtered through a 0.45 mm filter (Millipore Millex -HV,
polyvinylidene fluoride membrane) to remove aggregates prior to
analysis by dynamic light scattering (DLS). Measurements were
taken on a Malvern Instruments Zetasizer instrument. For Fourier
transform infrared (FTIR) spectroscopic analysis, samples were
prepared by drying a portion of the sample under vacuum. The
dried sample was ground using a mortar and pestle with dried
KBr, and the powder mixture ground into a disc. The solid sample
was analysed using a Perkin Elmer FTIR spectrometer. For mag-
netic measurements, a dispersion of the nanoparticles was dried
under vacuum and the powder transferred to a gelatin capsule
under ambient conditions. The capsule was sealed, and the sample
inserted into a Quantum Design Superconducting Quantum
Interference Device instrument. All measurements were taken at
300 K.
2
patibility, magnetic properties and T contrast and relaxivities of
the iron nanoparticles coated with PolyM3 were examined. The
results from this study have implications for the rational design
of polyelectrolytes for functionalization of air-sensitive
nanoparticles.
2
. Materials & methods
2.1. Materials and instrumentation
2.2. Synthesis of diethyl (2-acrylamidoethyl)phosphonate M3
All solvents and reagents were of technical or analytical grade
0
and were used as received unless otherwise stated. 2.2 -Azobio
Diethyl (2-aminoethyl)phosphonate (3.93 g, 21.70 mmol) was
(
isobutyronitrile) (AIBN) was recrystallized from ethanol and used
synthesized via known methods (see Supporting Information for
details) [32,33], then dissolved in CHCl (35 mL), at which point tri-
ethylamine (Et N) (4.54 mL, 32.55 mmol) was added. The mixture
was cooled to 0 °C and stirred vigorously under a nitrogen atmo-
sphere. Next, acryloyl chloride (2.28 mL, 28.21 mmol) in CHCl
(5 mL) was added dropwise to the reaction over 1 h. The mixture
was warmed to room temperature and stirred for a further 18 h
under a nitrogen atmosphere. The solvent was then evaporated
as an initiator. Dimethylformamide (DMF) was used after purifica-
tion using an LC Technologies Solutions Inc. solvent purifier. Ana-
lytical thin-layer chromatography (TLC) was performed using
Kieselgel F254 0.2 mm (Merck) silica plates with visualisation by
ultraviolet irradiation (254 nm) followed by staining with vanillin
or potassium permanganate. Silica gel flash chromatography was
performed with Davisil silica gel (LC60A 40–63 micron) stationary
phase. H nuclear magnetic resonance (NMR) and C NMR spectra
were performed on a Brucker AC 400 MHz using CDCl or D O as
solvent, as stated. Chemical shifts were referenced to d 7.26 and
d 77.0 ppm from chloroform for 1H and C, respectively, d 2.50
and d 39.52 from dimethylsulfoxide (DMSO) or d 3.31 and d
3
3
3
1
13
2
and the residue diluted with Et O (30 mL) and filtered through
celite. The filtrate was concentrated under reduced pressure and
the crude product was purified by silica gel flash chromatography
3
2
13
(10% MeOH/CH
a pale yellow oil. H NMR (400 MHz, CDCl
2
Cl
2
1
) to give the title compound M3 (2.96 g, 58%) as
) d (ppm): 6.67 (br s,
3
H
1
13
4
9.0 ppm from methanol for H and C, respectively, as stated.
1H), 6.26 (dd, J = 1.5, 17.1, 1H), 6.09 (dd, J = 10.3, 17.1, 1H), 5.63
(dd, J = 1.5, 10.4, 1H), 4.10 (m, 4H), 3.63 (m, 2H), 2.01 (m, 2H),
1
The multiplicities of H signals are designated by the following
abbreviations: s = singlet; d = doublet; t = triplet; dd = doublet of
doublets; m = multiplet; br = broad. All coupling constants J are
1.32 (t, J = 7.0, 6H); d
C
(100 MHz; CDCl
= 5), 25.3 (J = 142), 16.2 ( J
data were in good agreement with those previously reported [34].
3
) 165.5, 130.7, 126.0, 61.7
2
2
3
( J
P
= 7), 33.4 ( J
P
P
P
= 6). Spectroscopic
13
reported in hertz. All
C NMR spectra were acquired using

A.J. McGrath et al. / Journal of Magnetism and Magnetic Materials 439 (2017) 251–258
253
2.3. General procedure for the RAFT polymerization of diethyl
2.6. Ligand exchange of OLA with PolyM3
(
2-acrylamidoethyl)phosphonate) to synthesize P2
5
mg of dried, washed NPs was suspended in 1 mL toluene. To
Diethyl
.13 mmol),
(2-acrylamidoethyl)phosphonate
2-methyl-2[(dodecylsulfanylthiocarbonyl)sulfanyl]
M3
(500 mg,
this NP solution, 0.2 mL of PolyM3 solution in methanol
ꢀ1
2
(50 mg mL ) was added, and the sample vial left to sonicate for
ꢀ1
propanoic acid (65 mg, 0.177 mmol) and AIBN (10 mg,
.058 mmol) in DMF (3 mL) were added to a Schlenk tube. The
mixture was then degassed thoroughly by bubbling with nitrogen
for at least 1 h. The reaction vessel was then evacuated and back-
filled with nitrogen (3 times) and left stirring at 80 °C for 20 h.
The reaction was quenched by rapid cooling in liquid nitrogen
1 h (for PolyM3 where n = 65, 1 mL of 10 mg mL polymer solu-
tion was added, due to the low solubility of the polymer in metha-
nol). The nanoparticles were recovered via centrifugation at
14,000 rpm, and washed once with methanol and then three
0
2
times with distilled H O. The nanoparticles were suspended in
Dulbecco’s phosphate buffered saline (PBS, 10 mM).
1
and a small sample taken for H NMR analysis to ensure polymer-
ization had occurred. DMF was then removed by high vacuum and
the residue dissolved in a small aliquot of CHCl , and cold hexane
3
2.7. Cytotoxicity tests
(
ꢁ100 mL) added to precipitate the product. This process was
HeLa carcinoma cells were cultured in an incubation chamber in
% CO at 37 °C, in Roswell Park Memorial Institute medium
2
repeated four times to give the title polymer P2 (324 mg, 65%
based on monomer weight conversion) as a yellow gum. H NMR
5
(
1
RPMI) supplemented with 10% fetal bovine serum. Cells were
(
400 MHz, CDCl
and CH -CH), 1.59 (CH
d (ppm): 29.2. MALDI-ToF (m/z – highest intensity mass peak):
715.00 (n = 10). Spectroscopic data closely matched previously
reported data [34].
3
) d
H
(ppm): 4.08 (OCH
2
); 3.43 (CH
2
N), 2.03 (CH
2
P
3
)
5
seeded at a concentration of 1 ꢂ 10 per well in a 24-well plate
in 300 L of RPMI media, and incubated for 6 h in a 5% CO2 cham-
ber at 37 °C to achieve 70–80% confluency. Medium containing
Fe-PolyM3-n was then added to the cells, to a total media volume
of 500 L per well, and the plates containing the cells were
31
2
2
-CH), 1.31 (CH
3
). P NMR (162 MHz, CDCl
l
2
x
l
incubated for a further 16 h. The medium was then removed via
carefully pipetting off the supernatant from the cells, and
tripsinization and staining agents were added to the cells. The
addition of trypsinization agent cleaves the cells from the plates,
and through careful pipetting the cells were re-suspended back
into solution. Cell viability after 24 h was measured via the Trypan
2
.4. General procedure for the deprotection of P2
Polymer P2 (315 mg, 0.116 mmol) was dissolved in dry CHCl
3
(
15 mL). Bromotrimethylsilane (0.7 mL, 5.3 mmol) was added
dropwise and the reaction was left stirring at room temperature
for 20 h. The solvent was evaporated under reduced pressure and
the residue dissolved in MeOH (20 mL) and stirred for a further
blue vital stain method. A 10
pipetted into a secondary 96-well plate, and 10
dye added. 10 L of cell/dye solution was pipetted onto a Neubauer
l
L aliquot of the cell solution was
lL of Trypan Blue
l
4
h. The solvent was then evaporated to dryness and the residue
chamber (hemocytometer). The total number of alive/dead cells
was calculated via counting the unstained (live) cells in the hemo-
cytometer via an optical microscope, and scaling the cells counted
freeze dried to give the title compound (302 mg, 96%) as a yellow
1
solid. H NMR (400 MHz, D
and CH -CH), 1.53 (CH -CH). P NMR (162 MHz, D
6.5. Spectroscopic data closely matched previously reported data
34]. The product from this step is hereafter referred to as
‘PolyM3”.
2
O) d
H
(ppm): 3.43 (CH
2
N), 2.01 (CH
2
P
3
1
2
2
2
O) d (ppm):
H
in 10
lL to the total cell solution volume.
2
[
‘
2.8. Preparation of agar gels for MRI
For T
2 x
MRI measurements, Fe-PolyM3-n were suspended in
ꢀ1
1
0 mM Dulbecco’s PBS at concentrations of 20, 10, 4 and 2 lg mL
2.5. Synthesis of iron nanoparticles
Fe. Separately, a 1% w/w agar solution was made up, and 100
agar solution added to 100
PBS, for final Fe mass concentrations of 10, 5, 2 and 1
For control wells, 100 L of agar was added to 100
tion. The gels/PBS solutions were left to set at room temperature.
l
L of
l
L of stock Fe-PolyM3-nx solutions in
Iron nanoparticles were synthesized via an adaptation of an
ꢀ1
l
g mL .
lL of PBS solu-
existing method [10]. The iron precursor Fe(C
thesized via known methods. After purification via sublimation,
.3 g (1.5 mmol) of the iron precursor was dissolved in 6 mL mesi-
tylene, along with 1.5 mL (4.5 mmol) of oleylamine (OLA, 80–90%,
Acros Organics) was added. The solution was degassed under N
5 5 6 7
H )(C H ) was syn-
l
0
2.9. T
2
-weighted MRI measurements
2
,
and transferred to a Fischer Porter bottle. The bottle was flushed
with 1 bar hydrogen gas three times, and then pressurized with
MRI was performed using a Bruker Instruments AVANCE400
nuclear magnetic resonance (NMR) spectrometer, equipped with
a Bruker Micro 2.5 imaging module. MR images were acquired at
3
1
bar hydrogen gas. The bottle was placed in an oven set at
10 °C and left for three days. The bottle was then removed,
9
.4 T using a 2D multi-slice multi-echo sequence, at room temper-
ature, with the following parameters: echo time (TE) = 16 ms,
repetition time (TR) = 3000 ms, pixel size = 98 m, slice
allowed to cool to room temperature, and then opened to air. A
mL aliquot of this solution was immediately transferred to a sam-
ple vial fitted with a septum, and kept under a blanket of N flow
2
l
m ꢂ 98
l
2
thickness = 1 mm, number of echoes = 32, 4 averages, total experi-
on the Schlenk line. This step is done as quickly as possible in order
to prevent oxidation of the reactive iron nanoparticle surface.
Separately, a precursor solution was made up containing 0.3 g
ment time = 16 min.
(
1.5 mmol) Fe(C
mesitylene. The precursor solution was degassed under vacuum
and N , and added to the seed solution previously aliquoted out.
5
H
5
)(C
6
H
7
), 0.5 mL (1.5 mmol) OLA and 6 mL
3. Results & discussion
2
The synthesis of PolyM3, as outlined in Scheme 1, follows a
slight moderation of a synthetic method previously published
[29]. PolyM3 is formed from acrylamide-functionalized monomers,
which are known to be more stable than their ester counterparts,
potentially resulting in enhanced chemical stability of the
materials [29]. The synthesis of the key phosphonate-containing
The seed/precursor solution was then left to react under 3 bar
hydrogen gas at 110 °C for one day further. Nanoparticles were
retrieved and washed via centrifugation at 14,000 rpm using a
1
:1 toluene:methanol co-solvent, and re-dispersed in toluene for
TEM analysis.

2
54
A.J. McGrath et al. / Journal of Magnetism and Magnetic Materials 439 (2017) 251–258
Scheme 1. Synthesis of M3 monomer and subsequent RAFT polymerization yielding PolyM3 polymers.
ꢀ
1
acrylamide monomer M3 was accomplished in two steps from
diethyl (2-azidoethyl)phosphonate 1. Reduction of 1 to give the
corresponding diethyl (2-aminoethyl)phosphonate 2 was achieved
in quantitative yields following stirring with 10% Pd/C under a
hydrogen environment for 18 h. Finally, the synthesis of acry-
lamide phosphonate monomer M3 was achieved following the
reaction of 2 with acryloyl chloride with triethylamine in dichlor-
omethane which afforded the monomer in a 58% yield.
15,647 g mol
of 21,864 g mol
w
(n = 65), weight average molecular weight (M )
ꢀ1
w n
and a polydispersity index (M /M ) of 1.40.
The characteristic properties of all polyelectrolytes synthesized
are summarized in Table 1.
The polyelectrolytes were conjugated to the surface of iron
nanoparticles by a simple ligand exchange process. Iron nanoparti-
cles with an average size of 11 ± 2 nm were synthesized by a ther-
mal decomposition process (Fig. S4). Upon exposure to air and
multiple washing cycles, the surface of the nanoparticles oxidized
to form a passivating iron oxide shell, as observed by TEM and con-
sistent with previous reports [35]. In a simple one-step ligand
exchange process, a solution of the polyelectrolyte in methanol
2
-Methyl-2[(dodecylsulfanylthiocarbonyl)sulfanyl] propanoic
acid was chosen as RAFT/chain transfer agent (CTA) due to its com-
patibility with acrylamide-containing monomers [29]. The molar
ratio of monomer:RAFT agent was adjusted accordingly for the
desired polyelectrolyte chain length. RAFT polymerization was
achieved using the favored equivalence of M3 monomer, 1 equiv-
alent of RAFT agent and 0.3 equivalents of AIBNinitiator in DMF
ꢀ1
(50 mg mL ) was added to a solution of OLA-coated iron/iron
ꢀ1
oxide nanoparticles in toluene (5 mg mL ), and sonicated for 1 h
at room temperature. The nanoparticles were purified via a series
of centrifugation and washing steps, firstly in methanol and then
three times in distilled water. The nanoparticles dispersed readily
into aqueous media.
x
at 80 °C for 20 h to give polyelectrolytes PolyM3-n , where x = 6,
1
0 or 65. The formation of all the polyelectrolytes was confirmed
1
by H NMR spectroscopy (Fig. S1), which showed broadening of
all the monomeric peaks and the complete disappearance of the
The Fe-PolyM3 assembly structure was investigated using
transmission electron microscopy (TEM). Fig. 1A shows a single
H
monomer olefinic protons at d 5.5–6.5 ppm. The polyelectrolytes
were then purified by repeated precipitation (3 times) in cold hex-
ane and were isolated in good yields of 52%, 65% and 69% (Table 1)
based on their monomer weight conversion, respectively.
Fe-PolyM3-n
6
assembly, of size ꢁ100 nm. Several voided-out
nanoparticle cores can be observed within the structure. When
polyelectrolyte chain length increases to 10 (Fig. 1B), the size of
the Fe-PolyM3 increases to ꢁ 180 nm, and remains at about this
size even when polyelectrolyte chain length is increased to
n = 65 (Fig. 1C). This observation indicates that the size of the Fe-
PolyM3 nanoparticle assemblies reaches a maximum at chain
length near n = 10, and does not significantly increase with an
increasing polyelectrolyte size. In individual nanostructures in
Figs. 1A-C, several nanoparticles can be observed within larger
organic structures. This observation agrees with that reported for
the ‘‘bridging flocculation” mechanism of polyelectrolyte grafting
to nanoparticles, where polyelectrolyte chains may bind to more
than one nanoparticle surface [25,36]. A minor TEM size increase
x
For polyelectrolytes PolyM3-n where x = 6 or 10, whose molec-
ular weight reached the lowest end of the measurement limit of gel
permeation chromatography (GPC) column, MALDI-ToF mass spec-
trometry analysis was employed and showed a very good corre-
spondence between the experimentally determined highest
intensity molecular mass peaks and those of the targeted polyelec-
6
trolytes (Fig. S2). PolyM3-n was isolated with a highest intensity
ꢀ1
molecular mass peak of 1,773 g mol (n = 6) while PolyM3-n10
was found to have a highest intensity molecular mass peak at
ꢀ
1
2
,715 g mol
(n = 10). GPC results (Fig. S3) indicated that
n
PolyM3-n65 had a number average molecular weight (M ) of
Table 1.
Characteristic properties of synthetic polymers P1-P3.
, targeta) [g molꢀ1
]
Monomer conversion [wt.%]
M
, exp. [g mol
ꢀ1
]
M /M
w n
Average n
Polymer
Monomer:RAFT ratio
M
n
n
P1
P2
P3
6:1
12:1
100:1
1,773
3,190
23,887
52
65
69
1,803b)
n/a
n/a
1.40
6
10
65
b)
2,715
c)
c)
15,647
a
n n
The target number-average molar mass (M ,target) was calculated using the following equation; M ,target = MRAFT + Mmonomer ([Monomer]/[RAFT]). Where, MRAFT and
Mmonomer are the molar masses of RAFT agent and monomer, respectively. The contribution of the molar mass of the chains initiated by AIBN was neglected and calculations
are based on 100% monomer conversion.
b
Average molecular weight determined by MALDI-ToF mass spectrometry. The average mass was taken as the highest intensity peak from the spectrum.
Number average molecular weight/polydispersity index estimated by GPC, using polystyrene as standards (TSK-gel standards A500 through F128).
c

A.J. McGrath et al. / Journal of Magnetism and Magnetic Materials 439 (2017) 251–258
255
the reactive a-Fe core [14,15]. Based on the TEM images observed
for each Fe-PolyM3 nanoparticle assembly, a simplified ligand
exchange mechanism is proposed in Scheme 2.
The interaction between the phosphonate-grafted polyelec-
trolytes and the iron nanoparticles was investigated further using
FTIR spectroscopy (Fig. 2). The spectrum taken for oleylamine-
coated Fe nanoparticles (Fe-OLA) nanoparticles is shown in Fig. 2A,
ꢀ
1
showing peak signals at 2924 and 2956 cm corresponding to
ꢀ
1
alkyl CAH stretches, a shoulder peak at 3018 cm corresponding
ꢀ1
to @CAH stretches, and at 1465 cm
corresponding to C-N
stretches. The spectra taken for Fe-PolyM3 assemblies are illus-
trated for comparison (Fig. 2B-D). Most notably, the peaks at
ꢀ
1
ꢁ
1078 cm (indicated by the dashed line) are assigned to the
characteristic Fe-O-P stretches [37], confirming the presence of
the phosphonate-containing polymeric coating.
Zeta potential measurements of the Fe-PolyM3 assemblies
showed a highly negative surface charge for each Fe-PolyM3 type,
with
35.1 ± 5.51 mV for Fe-PolyM3-n
Fig. S6). This suggested the presence of exposed phosphonate
values
of
ꢀ35.0 ± 3.48 mV,
ꢀ36.8 ± 4.48 mV
and
ꢀ
6
,
n
10 and 65 respectively
n
(
groups on the surface of the Fe-PolyM3 assemblies, which provide
strong electrostatic stabilization of the assemblies in solution and
prevent aggregation.
The cytotoxicity of the Fe-PolyM3 assemblies is a primary
concern for their application in biomedicine. Fig. 3 shows the cell
viability for HeLa carcinoma cells cultured in the presence of
Fe-PolyM3, for polyelectrolyte chain lengths of 6, 10 and 65 repeat-
ing units, with increasing Fe concentrations. The Fe content of each
sample was confirmed by atomic absorption spectroscopy. Cell via-
bilities are expressed as a percentage value of the control group, i.e.
when HeLa cells are incubated with no nanoparticles present. For
each Fe-PolyM3 type, the number of tests at each concentration
(N) = 4. ANOVA statistical analysis was used to compare the statis-
tical significance between groups of data. These results show
excellent cell viability (>95% within error) for iron nanoparticles
at Fe concentrations at and exceeding those previously utilized
ꢀ
1
for iron nanoparticles in MRI (<20 mg mL , or 360
[
l
M Fe)
ꢀ
1
10,38]. At Fe concentrations up to 100 mg mL , good cell viability
at >80% (within error) is observed for all PolyM3-coated nanopar-
ticles. Specifically, near-100% cell viability is well maintained for
Fe-PolyM3 with chain length of 6 repeating units at all Fe
concentrations tested, which may be a result of the smaller size
of the Fe-PolyM3 assemblies. Phosphonate groups which remain
exposed on the surface of the Fe-PolyM3 assemblies may provide
an electrostatic stabilization in aqueous media, which is consistent
Fig. 1. A–C) TEM images of the Fe-PolyM3-n
x
assemblies with x = 6 (A), 10 (B) and
6
5 (C) respectively. Inset in A) shows a higher-magnification of an area within the
Fe-PolyM3 assembly where voided regions can be observed within individual
nanoparticles (scale bar corresponds to 10 nm). D-F) SAED patterns corresponding
to the Fe-PolyM3-n
JCPDS #04-014-0360) and the iron oxides (‘‘FeOx”) (JCPDS #00-019-0629)
accordingly.
x
assemblies from A-C, respectively. Signals are indexed to a-Fe
(
is observed for Fe-PolyM3 assemblies with increasing chain
lengths of PolyM3. DLS measurements (Fig. S5) showed a
slight increase in hydrodynamic diameter (D
z
) compared to
TEM-measured size for Fe-PolyM3-n assemblies, up to 167 nm,
6
indicating some aggregation of the assemblies in aqueous solution.
However, no significant aggregation of the other Fe-PolyM3 assem-
blies was observed from DLS measurements, with average D
sured as 177 and 180 nm for Fe-PolyM3-n where x = 10 and 65,
respectively. Selected area electron diffraction (SAED) patterns
Figs. 1D–F) confirm the presence of the -Fe core for each sample,
however for Fe-PolyM3-n the presence of voids between many
z
mea-
x
(
a
6
Scheme 2. Schematic diagram illustrating PolyM3 structure, and simplified scheme
of the ligand exchange process, where OLA on the surface of the iron nanoparticles
is exchanged with PolyM3.
nanoparticles’ cores and shells can be observed (magnified in the
inset in Fig. 1A), which is indicative of insufficient protection of

2
56
A.J. McGrath et al. / Journal of Magnetism and Magnetic Materials 439 (2017) 251–258
Fig. 2. FTIR spectra taken from powder samples of iron nanoparticles stabilized
with OLA (A) and with PolyM3 with chain length (n) of 6 (B), 10 (C) and 65 (D)
repeating units.
with zeta potential measurements for each type of Fe-PolyM3
showing a highly negative surface charge in each case. This data
illustrates excellent non-cytotoxicity for Fe-PolyM3 and suggests
that the PolyM3 polymer backbone is an excellent candidate for
further functionalization toward specific site targeting of cells.
As the biosafety of the Fe-PolyM3 nanoparticles had been estab-
lished up to and beyond concentrations required for biomedical
applications, their efficacy as T
examined. Magnetic measurements and T
2
MRI contrast agents was also
MRI tests of iron
2
Fig. 4. A) Magnetization (M) with applied magnetic field (H) of Fe-PolyM3
assemblies with different chain lengths of PolyM3. B) T -weighted MRI images of
Fe-PolyM3 assemblies with polymer chain lengths as indicated, resolved by Fe
concentration. C) Comparative r values for iron nanoparticles stabilized with
PolyM3 of different chain lengths, as indicated.
nanoparticles were conducted several days post-ligand exchange
with PolyM3, during which time the Fe-PolyM3 nanoparticle
assemblies were stored in distilled water. The results obtained
are thus considered a fair indicator of the long-term magnetic sta-
bility of the Fe-PolyM3 assemblies under biologically relevant con-
ditions. As for cell viability assays, the Fe content of each sample
was confirmed by atomic absorption spectroscopy. Fig. 4A shows
magnetization versus external field data for the OLA stabilized
nanoparticles and the Fe-PolyM3 assemblies with PolyM3 of differ-
2
2
ꢀ
1
M
the
s
value falls to 83 emu g Fe
,
indicating further oxidation of
value
increases to 88 emu g . When the chain length increases to 65
a
-Fe nanoparticle core. For Fe-PolyM3-n10, the M
s
ꢀ1
ꢀ1
repeating units, the M
that of the OLA-stabilized nanoparticles, indicating complete pro-
tection of the -Fe cores in the nanoparticles. This is also indicative
that oxidation of the -Fe nanoparticle core does not occur under
the conditions utilized for ligand exchange.
s
value is 101 emu g Fe, which approaches
ent chain lengths (Fe-PolyM3-n
x
). The saturation magnetization of
the iron nanoparticles as-synthesized was measured as
a
ꢀ
1
1
03 emu g Fe. For nanoparticles stabilized with PolyM3 with 6
a
repeating units in the polyelectrolyte chain (Fe-PolyM3-n ), the
6
Fig. 3. Cytotoxicity data for nanoparticles coated with PolyM3 of varying molecular weights (N = 4 for each group), at varying Fe concentrations. ***P < 0.001, two way ANOVA
with Bonferroni multiple comparisons.

A.J. McGrath et al. / Journal of Magnetism and Magnetic Materials 439 (2017) 251–258
257
The magnetic data showed a variance between M
each Fe-PolyM3 nanoparticle type. In order to measure the impli-
cations of this for nanoparticles’ performance in T -weighted
MRI, the Fe-PolyM3 nanoparticle assemblies with the highest and
lowest magnetizations were analyzed for MRI performance. Fe-
s
values for
of the Fe-PolyM3 assemblies. The research here shows the impor-
tance of polyelectrolyte size when designing coatings for the stabi-
lization of magnetic nanoparticles containing reactive particle
cores.
2
6
PolyM3-n and n65 were prepared for analysis of MRI performance
Acknowledgements
by suspending in agar gels. Fig. 4B demonstrates the contrast
induced in agar gels at a range of Fe concentrations (determined
by flame atomic absorption spectroscopy), under a magnetic field
of 9.4 T. Compared to the control wells (1:1 agar gel:PBS v/v), the
wells containing the Fe-PolyM3 assemblies each show unambigu-
All authors acknowledge the New Zealand Ministry of Business,
Innovation and Employment for funding.
2 6
ous, Fe concentration-dependent T contrasts. For Fe-PolyM3-n ,
the contrast induced is much lower per mM of Fe than for iron
nanoparticles stabilized with PolyM3-n65. This is thought be dueto
further oxidation of the a-Fe nanoparticle core when n = 6 due to
insufficient protection of the core from PolyM3, and is consistent
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jmmm.2017.04.
0
26.
with the data obtained from magnetic measurements showing
reduced M
PolyM3 assemblies. The r
of the linear fit to the T
the corresponding particles as shown in Fig. S7. The r
s
. Fig. 4C shows values calculated for r
values are calculated from the gradient
deduction of different concentrations in
value for
and this value increases to
for PolyM3-n65. For PolyM3-n65, this value com-
pares favorably with those of commercial T MRI agents such as
) [38]. When the chain length n = 65,
the nanoparticles are sufficiently protected that a high r value is
2
of the Fe-
2
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