The stabilization and bio-functionalization of iron oxide nanoparticles using heterotelechelic polymers

Article abstract of DOI:10.1039/b815202k

Iron oxide nanoparticles (IONPs) are important tools for nanobiotechnology applications. However, aqueous instability and non-specific biodistribution problems limit the applications of IONPs. Considering this, α-phosphonic acid, ω-dithiopyridine function

Full text of DOI:10.1039/b815202k

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PAPER
www.rsc.org/materials | Journal of Materials Chemistry
The stabilization and bio-functionalization of iron oxide nanoparticles using
heterotelechelic polymers†
a
Cyrille Boyer, Volga Bulmus, Priyanto Priyanto, Wey Yang Teoh, Rose Amal and Thomas P. Davis
b
a
c
c
a
*
*
Received 1st September 2008, Accepted 30th September 2008
First published as an Advance Article on the web 11th November 2008
DOI: 10.1039/b815202k
Iron oxide nanoparticles (IONPs) are important tools for nanobiotechnology applications. However,
aqueous instability and non-specific biodistribution problems limit the applications of IONPs.
Considering this, a-phosphonic acid, u-dithiopyridine functionalized polymers were synthesized via the
reversible addition–fragmentation chain transfer (RAFT) polymerization and used for stabilizing and
biofunctionalizing IONPs. A new trithiocarbonate RAFT agent bearing dimethyl phosphonate group
was utilized in the synthesis of well-defined telechelic polymers of styrene, oligoethylene glycol acrylate
(OEG-A) and N-isopropylacrylamide (NIPAAm). IONPs were grafted with a-phosphonic acid,
u-dithiopyridine functionalized poly(OEG-A) through the a-chain end of the polymer, as evidenced by
FTIR-ATR, XPS and zeta potential measurements. Using TGA results, the grafting density of the
polymer chains was calculated between 0.12 and 0.23 chains/nm2 particle depending on the molecular
weight of the polymer. DLS measurements indicated that the particles grafted with poly(OEG-A) larger
than 10 000 g/mol were stable in water for several days and the mean diameter of the particles was
between 40 and 130 nm depending on the molecular weight of the polymer. Moreover, particles
stabilized with poly(OEG-A) with a Mn ¼ 62 000 g/mol were stable in phosphate buffer (pH 6.5, 0.1 M)
containing varying concentrations of BSA. Polymer-stabilized IONPs were successfully functionalized
with two different peptides, i.e. reduced glutathione as a model peptide and NGR motif as a tumor-
targeting peptide through the u-dithiopyridine functionality of the polymer, as measured by XPS and
zeta potential analysis. Poly(OEG-A)-stabilized IONPs were also found to be resistant to protein
adsorption.
Besides small molecular surfactants,18–20 biocompatible and
hydrophilic polymers that can stabilize the particles in aqueous
media and also provide functional groups for conjugation of
biological molecules have been attractive materials. Polymers
used for this purpose are required to have more than one
functional group for both interaction with IONPs and
biofunctionalization. The portfolio of polymers used to date has
been limited mostly to dextran and other poly(saccharide)s,
poly(vinyl alcohol), telechelic PEGs, poly(hydroxyethyl meth-
acrylate) and poly(acrylic acid).21–26 The widespread utilization
of these polymers arises not only from their hydrophilicity,
biocompatibility25 and possession of functional groups, but also
from their readily-availability to nanotechnologists and
biomedical researchers through commercial sources. Thus there
is a need to enlarge the portfolio of IONP stabilizing and func-
tionalizing polymers especially with vinyl polymers which are
relatively easy to generate under laboratory conditions
commonly accessible to polymer non-specialists. In addition,
commercially available polymers used to date in general do not
possess reactive groups (besides the groups binding to the iron
oxide surface) necessary for more sophisticated bioconjugations
with high selectivity and reactivity. For example, most polymers
used to date contain hydroxyl, amino or carboxylic acid groups,
for both interaction with iron oxide and conjugation with
biological moieties such as cell targeting molecules, bioaffinity
ligands, etc. To incorporate functional groups with higher reac-
tivity and selectivity to polymer coated IONPs, additional
Introduction
Magnetic nanoparticles have become leading tools for nano-
biotechnology applications.1–4 In particular, iron oxide nano-
particles (IONPs), because of their superparamagnetic properties
and general low toxicity,5 have been increasingly used as contrast
agents for magnetic resonance imaging,6–9 as vectors for gene
therapy,10,11 in non-invasive local drug delivery,12,13 clinical
diagnosis14 and bioseparations.15 In most biomedical applica-
tions, iron oxide nanoparticles are required to have uniform size
below a critical value which is typically below 50 nm,16 good
dispersibility and biocompatibility in physiological medium, and
at the same time to be (multi)functionalizable with biological
molecules like targeting agents, affinity ligands and drug
molecules.17
aCentre for Advanced Macromolecular Design (CAMD), School of
Chemical Sciences and Engineering, The University of New South Wales,
Sydney, NSW, 2052, Australia. E-mail: t.davis@unsw.edu.au
bSchool of Biotechnology and Biomolecular Sciences (BABS), The
University of New South Wales, Sydney, NSW, 2052, Australia. E-mail:
vbulmus@unsw.edu.au
cARC Centre of Excellence for Functional Nanomaterials, School of
Chemical Sciences and Engineering, The University of New South Wales,
Sydney, NSW, 2052, Australia
† Electronic supplementary information (ESI) available: Complement
experimental part, RAFT agent synthesis, 1H NMR of RAFT agent
and equations. See DOI: 10.1039/b815202k
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modification steps are inevitable. Also, the common functional
groups (i.e. carboxylic acid, hydroxyl, amine)27–34 that the
commercially available polymers possess may not provide strong
attachment between the polymer coating and iron oxide, result-
ing in the leaching of the polymer chains from IONPs under
physiological conditions. To ensure that the polymer coating on
IONPs does not leach, polymers with functional groups
providing stronger chelating with iron oxide need to be
generated.
was filtered through an alumina column to remove the inhibitor
prior to use. Styrene was distilled over CaH2 prior to use. The
characteristics of the reagents and materials used are given in the
ESI.† Iron oxide nanoparticles in the form of maghemite were
synthesized rapidly via a one-step flame spray pyrolysis (FSP) as
similarly reported in our previous publications.60,61 Briefly,
a liquid precursor consisting of 0.5 M of iron 2-ethylhexanote
(52% in mineral spirit, Alfa Aeser) in xylene (Riedel de Haen)
was delivered to the FSP nozzle at 5 and 10 ml minꢁ1 where it was
dispersed with 5 L minꢁ1 and ignited by a supporting oxy-
methane flame. Additional 5 L minꢁ1 O2 was issued as sheath gas.
NGR peptide (sequence: GNGRGC, purity 95%) was
purchased from AUSPEP company (PO Box 806, Parkville 3052,
Australia). Bovine serum albumin (BSA, purity 90%), Bradford
assay and reduced glutathione (95%) were purchased from
Aldrich.
In this context, living radical polymerization (LRP) tech-
niques,35–38 such as reversible addition–fragmentation chain
transfer polymerization (RAFT), offer an excellent platform for
the fabrication of polymer coatings on IONPs via grafting-from
and -to approaches. It is possible to generate a variety of func-
tional and water-soluble polymers with well-defined properties
such as designed end-group functionalities, controlled molecular
weights and narrow molecular weight distributions with these
techniques.34 Among these techniques, RAFT does not
require special reagents and conditions for efficient polymeriza-
tions except the use of chain transfer agents,39–42 tolerant to
functional groups and impurities to some extent,43–46 and
provides well-defined hetero-functional telechelic polymers
which eliminates the need for post-polymerization end-group
modifications.47–54 Nevertheless, the potential of the RAFT
technique in stabilization and biofunctionalization of IONPs has
yet to be fully explored.
RAFT agent synthesis
The synthesis of the RAFT agent is described in ESI.†
RAFT polymerizations
Styrene polymerization. RAFT agent (50.0 mg, 9.7 ꢂ 10ꢁ2
mmol) (bearing phosphonate and carboxylic groups) and styrene
(1.04 g, 1 mmol) were dissolved in acetonitrile (8 mL) in septa
sealed vials. The vials were placed in an ice bath and the solutions
were purged with nitrogen for 30 minutes. Deoxygenated solu-
tions of the initiator (i.e. AIBN, 1.2 mL from 15.2 mM in
acetonitrile (1.8 ꢂ 10ꢁ2 mmol)) were introduced into the reaction
mixture at 0 ꢀC via a syringe. The final concentrations of the
monomer, RAFT agent and the initiator in the polymerization
medium were: 1.00, 9.7 ꢂ 10ꢁ3, 1.78 ꢂ 10ꢁ3 M, respectively
([Monomer]0:[RAFT]0:[Initiator]0 ¼ 103.3:1.0:0.2). The solu-
tions were further deoxygenated at 0 ꢀC for 15 min and then
Herein, we report a new method for efficient stabilization and
biofunctionalization of IONPs using hetero-telechelic polymers
generated by RAFT polymerization. A new trithiocarbonate
RAFT agent bearing a dimethyl phosphonate group was utilized
in the synthesis of a number of telechelic polymers including
water soluble polymers, poly(oligoethyleneglycol acrylate) and
poly(N-isopropyl acrylamide). Following the post-polymeriza-
tion modifications, IONPs were grafted with a-phosphonic acid,
u-dithiopyridine functionalized water-soluble polymers to yield
nanoparticles stable in aqueous solutions and reactive toward
thiol groups. While the phosphonic acid end group provides
strong iron oxide chelation properties for stabilization of the
particles,32,55 the pyridyl disulfide end group makes the site-
selective conjugation of biomolecules to the nanoparticles
possible via selective thiol–pyridyldisulfide exchange reac-
tion.56,57 We also show the results of the surface adsorption of
heterotelechelic polymers to IONPs, physicochemical properties
of the polymer coated particles and biofunctionalization with
two different peptides, i.e. reduced glutathione as a model
peptide and NGR motif,58,59 being a potential tumor-targeting
peptide.
ꢀ
placed in an oil bath at 70 C. Aliquots (0.5 ml) were taken at
predetermined time intervals and quenched via rapid cooling and
exposure to oxygen. Two or three drops of hydroquinone solu-
tion (5%-wt) were added. These samples were directly analyzed
by 1H NMR and THF GPC to determine the monomer
conversion and the molecular weight. The polymer was
concentrated by partial evaporation of acetonitrile, and the
polymer was precipitated in cold methanol (in an ice bath) two
times to remove the non-reacted monomer and RAFT agent.
Post-filtration, the solid was dried in vacuum at 40 ꢀC for 4 hrs to
give a yellow-orange solid. The samples were further analyzed by
1
THF GPC and H, 31P NMR.
NIPAAm and OEG-A polymerizations. Similar procedures
were followed for the polymerizations of NIPAAm and OEG-A
except for changes in the concentration of the components
used. For polymerizations with NIPAAm, the final concentra-
tions of NIPAAm, RAFT agent and the initiator were 1.0, 10.09
ꢂ 10ꢁ3, 2.02 ꢂ 10ꢁ3 M, respectively, yielding a ratio of [Mono-
mer]0:[RAFT]0:[Initiator]0 of 100.0:1.0:0.2. For polymerizations
with OEG-A, the final concentrations of OEG-A, RAFT agent
and the initiator were 0.51, 20.27 ꢂ 10ꢁ3, 4.06 ꢂ 10ꢁ3 M,
respectively, yielding a ratio of [monomer]0:[RAFT]0:[Initiator]0
of 20.0:1.0:0.2. Both NIPAAm and OEG-A polymerizations
Experimental
Material
All reagents were purchased from Aldrich at the highest available
purity, and used without further purification unless indicated.
N-Isopropylacrylamide (NIPAAm) (97%) was recrystallized
twice from hexane prior to use (mp ¼ 64 ꢀC). 2,20-Azobisiso-
butyronitrile (AIBN) was recrystallized twice from methanol
prior to use. Oligoethylene glycol acrylate (OEG-A) (number
average molecular weight Mn ¼ 450 g/mol, PDI ¼ 1.04, Aldrich)
112 | J. Mater. Chem., 2009, 19, 111–123
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were performed at 65 ꢀC. The samples obtained during the
polymerizations of NIPAAm and OEG-A were directly analyzed
by 1H NMR and DMAc GPC to determine the monomer
conversions and the molecular weights. The polymer samples
were concentrated by partial evaporation of acetonitrile, and the
poly(NIPAAm) was precipitated in cold diethyl ether (in an ice
bath) two times to remove the non-reacted NIPAAm and the
RAFT agent, while the poly(OEG-A) was dialyzed using a dial-
ysis membrane with a molecular weight cutoff (MWCO) of 3 500
Da in a mixture of ethanol and water (50/50 v/v). After filtration,
the solids were dried in vacuum at 40 ꢀC for 24 hrs to give
a yellow-orange solid. The purified products were further
nanoparticles (10 mg), (diameter 8 nm) were dispersed in water (9
mL). The solution was sonicated for 1 min at 12 watts with
a sonicator 3000 Misonix. Poly(OEG-A) (20 mg), (Mn DMAc
GPC ¼ 6 200 g/mol, PDI ¼ 1.22) was dissolved in water (1 mL).
The solution of poly(OEG-A) was added slowly to the dispersion
of iron oxide nanoparticles, and the resulting solution/dispersion
was sonicated for 2 mins (power ¼ 30 W). The solutions were
filtered and centrifuged (using a Eppendorf Centrifuge 5804) for
10 mins (1 112 relative centrifugal force) to remove the unsta-
bilized particles. Subsequently, the particles were again centri-
fuged at high speed for 1 h at 4 468 rcf/min to separate the
particles from the supernatant. The iron oxide nanoparticles
were isolated from the base of the centrifuge tube. The solutions
were decanted and the particles were again dispersed in 20 mL of
ethanol. The solutions were placed in the centrifuge one more
time and the process was repeated 3 times. Finally, the iron oxide
nanoparticles were dialyzed (MWCO ¼ 50 000 Da) against water
for 2 days and then freeze-dried (8 mg of product). Iron oxide
nanoparticles were subsequently analyzed by TGA, XPS, TEM
and DLS.
1
analyzed by H NMR, DMAc GPC and by FT-IR. The molec-
ular weights assessed by DMAc GPC for poly(NIPAAm) and
poly(OEG-A) were 9 000 g/mol (PDI ¼ 1.16) and 6 500 g/mol
(PDI ¼ 1.22), respectively.
Cleavage of dimethylphosphonate into phosphonic acid by bro-
mosilane. A similar procedure was used for all polymers. For
example, poly(styrene) (0.36 g, 5.0 ꢂ 10ꢁ5 mol) (Mn, THF GPC
¼ 7 200 g/mol) was dissolved in dichloromethane (5 mL) in
a vial at room temperature. The solution was degassed for
2 mins to remove traces of moisture and the flask was sealed by
a septum. A solution of bromosilane (0.5 g (3.2 ꢂ 10ꢁ3 mol) in
1 mL) was slowly added at 0 ꢀC. The solution was stirred for
4 hrs at room temperature. At the end of the reaction, the large
excesses of bromosilane and dichloromethane were removed by
evaporation under low pressure. After the total elimination of
bromosilane, ethanol (50 mL) was added to the flask. The
solution was stirred at room temperature for 1 hr to generate
phosphonic acid and ethanol was removed under vacuum. The
Conjugation of model peptides (glutathione and NGR peptide) to
a-phosphonic acid, u-dithiopyridine functionalized poly(OEG-A).
Reduced glutathione and NGR peptide were conjugated to iron
oxide nanoparticles coated with polymer (e.g. a-phosphonic
acid, u-dithiopyridine functionalized poly(OEG-A) (Mn: 5 100
g/mol by NMR and 6 200 g/mol by GPC, PDI: 1.22, conver-
sion 70%). In a typical conjugation experiment, iron oxide
nanoparticles (5 mg) were incubated with the peptide (3.44 ꢂ
10ꢁ6 mol) in a phosphate buffer (5 mL) at pH 7 for 6 hours at
room temperature. Purifications of the peptide conjugated
nanoparticles were performed by centrifugation, as described
previously. The particles were also purified via dialysis, over
3 days, against water, using a membrane with a MWCO of 50
000 Da. Peptide conjugation was quantified by monitoring the
release of pyridine-2-thione (present in the solution
after centrifugation) via a UV-vis spectrophotometer at 343 nm
(3 ¼ 8080 Mꢁ1cmꢁ1 in water).62,63 The purified nanoparticle
samples were further characterized by XPS, zeta potential
measurements and TEM.
1
product was analyzed by H and 31P NMRs, by ESI-MS (for
low molecular weights), and by FT-IR. The yield was evaluated
by 31P NMR, indicating 100%, consistent with a total shift of
signal at 35 ppm to 40 ppm.
Chemical modification of trithiocarbonate into pyridyl disulfide
group. The trithiocarbonate was removed by aminolysis to
generate a thiol. The thiol was modified in-situ to give a pyridyl
disulfide group in the presence of dithiopyridine. A typical
reaction is described. Poly(NIPAAm) (0.9 g, 10ꢁ4 mol) (i.e. Mn,
DMAc GPC ¼ 9 000 g/mol) and dithiopyridine (0.1 g, 4.5 ꢂ 10ꢁ4
mol) were dissolved in dichloromethane (10 mL) in a reaction
flask. The flask was sealed by a septum and the solution was
purged with nitrogen for 30 minutes to remove traces of oxygen.
A degassed solution of aminoethoxyethanol (0.025 g, 2.4 ꢂ 10ꢁ4
mol diluted in 1 mL of dichloromethane) was added slowly. The
solution was stirred at room temperature for 4 hrs, undergoing
a color change from yellow-orange to yellow-green. Dichloro-
methane was removed. The product was dissolved in ethanol and
dialyzed (MWCO ¼ 1 000 Da) for 3 days in a mixture of water/
ethanol (50/50 v/v) to remove the by-product pyridithione and
the excess of dithiopyridine and aminoethoxyethanol. The
solvent was evaporated to leave a colorless product (yield 80%).
The product was analyzed by 1H NMR, UV-visible, ESI-MS (for
the low molecular weights) and by DMAc GPC.
Protein adsorption. Magnetic particles (1.0 g/L) were
suspended in a series of phosphate buffers (pH ¼ 6.5, similar
results were obtained at 7.4) with initial bovine serum albumin
(BSA) concentrations from 0.05 g/L to 16.00 g/L. The samples
were then placed on a shaker at room temperature for 3 h to
reach adsorption equilibrium. Iron oxide nanoparticles were
removed by using a magnet and the supernatant analyzed. The
particles were redispersed in a buffer solution at pH ¼ 6.5.
The supernatant BSA concentrations were determined
following the Bradford method64 using a UV-visible spec-
trometer (Varian Cary 300 scan). Supernatant (10 mL) was
added to Bradford’s reactant (3 mL), and mixed for 2 mins at
room temperature (the solution becomes blue). The concen-
tration of BSA was measured by adsorption at 595 nm. The
test was repeated three times for each sample. The average of
the replicate measures was taken to determine the BSA
concentration at equilibrium. The amount of BSA adsorbed
was calculated by mass balance.
Grafting of a-phosphonic acid, u-dithiopyridine functionalized
poly(OEG-A) to the iron oxide nanoparticles. Iron oxide
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radicals.67 However, in the present case of 11-undecen-1-ol,
a large quantity of by-products was observed by THF GPC, i.e.
diadduct and higher adducts. To minimize the formation of these
by-products, an excess of dialkyl hydrogenophosphonate was
used. Nonetheless, trace amounts of diadduct and triadduct were
still detected by ESI-MS and by THF GPC. To avoid the
formation of these by-products, the hydroxyl group was capped
by acetyl chloride. Indeed, it is known that the addition of
alcohols onto enes can occur in radical medium and by Michael’s
addition catalyzed by the presence of phosphine. The elimination
of these by-products was achieved using a silica column. 1H and
31P NMR confirmed the successful synthesis of 11-dimethyl-
phophonate decan-1-ol. Subsequently, the hydroxyl function
was reacted with bromopropionic bromide introducing
a bromine atom. A new NMR signal (CH–Br) appears at
4.1 ppm, while the signal of CH2–O is shifted from 3.5 to 4.0 ppm
after esterification. Finally, the terminal bromine atom was
exploited to introduce a trithiocarbonate group by nucleophilic
substitution by a dithiocarboxysulfanyl propionic acid salt. The
NMR signal of CH- is shifted from 4.1 ppm to 4.8 ppm
(attributed to CH–S–(C]S)–) (Fig. S1 in the ESI†).
[BSA]adsor. ¼ [BSA]0 ꢁ [BSA]eq.
where [BSA]0 and [BSA]eq. correspond to BSA concentrations
measured by Bradford’s assay without iron oxide nanoparticles
and BSA concentrations in the presence of iron oxide nano-
particles at equilibrium, respectively.
Results and discussion
Synthesis of a-phosphonate, u-carboxylic acid RAFT agent
A new RAFT agent bearing a dialkyl phosphonate group, i.e.
dimethyl phosphonate or diethylphosphonate, and a carboxylic
acid group as the R- and Z-fragments, respectively, was
synthesized and characterized (Scheme 1 and see ESI†). The
introduction of a phosphonate group to the RAFT agent was
achieved using radical addition of dialkyl hydro-
genophosphonate groups onto the double bond of 11-undecen-
1-ol at 135 ꢀC. This approach has been described previously for
radical addition to 11-undecenoic acid, resulting in a selective
monoaddition,65,66 as the P–H bond is easily cleavable by
Polymerizations of OEG-A, NIPAAm and styrene
The new RAFT agent was used to mediate the radical poly-
merizations of styrene, N-isopropylacrylamide (NIPAAm) and
oligo(ethylene glycol)-acrylate (OEG-A) in the presence of AIBN
(Scheme 2). NIPAAm and OEG-A were used as hydrophilic
monomers with potential utility for stabilizing iron oxide nano-
particles. Styrene was used, as a conventional, model monomer,
to ensure the living nature of the RAFT polymerization and to
study the subsequent chemical transformation of the dialkyl
phosphonate group to phosphonic acid. Different polymeriza-
tions were performed with a typical [RAFT]0/[Initiator]0 ratio,
i.e. 5/1, to confirm the efficacy of the new RAFT agent. The
experimental conditions and results obtained for the polymeri-
zations are summarized in Table 1. The targeted and experi-
mental molecular weights were in good agreement for all three
monomers, confirming the utility of this RAFT agent.
For all three monomers, the pseudo first-order kinetic plots are
linear, indicative of constant radical concentrations. Short inhi-
bition periods were observed. Inhibition periods in RAFT are
commonplace and have been ascribed to a number of possible
origins including slow fragmentation,68 initiation69,70 and
Scheme 1 Synthesis of the trithiocarbonate RAFT agent bearing
a di(methyl)phosphonate and a carboxylic acid group at R- and
Z-fragments, respectively.
Scheme 2 RAFT-mediated synthesis of heterotelechelic polymers.
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Table 1 RAFT polymerization recipes and the properties of some polymers generateda
Mn (g/mol)
Run
Monomer
T (ꢀC)
[M] (M)
[M]0/[RAFT]0/[I]0
Conv.(%)
Theoretical
GPC
NMR
PDI
f
1
2
3
4
5
5
6
7
Styrene
80
60
60
60
60
60
60
60
1.00
1.00
1.00
1.00
1.00
1.02
1.02
1.02
100.0/1.0/0.2
20.0/1.0/0.2
50.0/1.0/0.2
100.0/1.0/0.2
200.0/1.0/0.2
20.0/1.0/0.2
50.0/1.0/0.2
100.0/1.0/0.2
70
55
65
72
70
65
74
70
7 500
4 800
14 000
32 000
63 000
1 700
7 500
6 000
13 000
36 000
62 000
3 200
8100
—
—
—
—
3600
4400
8500
1.12
1.22
1.21
1.22
1.24
1.08
1.12
1.08
0.90
0.90
0.85
—
OEG-A
OEG-A
OEG-A
OEG-A
NIPAAm
NIPAAm
NIPAAm
—
0.95
0.90
—
4 900
8 900
4 500
7 500
a
Note: [M], [RAFT], [I] are the feed concentrations of monomer, RAFT agent and initiator, respectively. Conversion and PDI are monomer conversion
and polydispersity index, respectively. All polymerizations were performed in acetonitrile. Theoretical molecular weight was calculated by Mn
¼
Conversion (%) ꢂ [M]0/[RAFT]0 ꢂ MW(monomer) + MWRAFT. GPC molecular weights were determined by GPC (mobile phases: DMAc for OEG-A
and NIPAAm; THF for styrene), f is the ratio between dimethyl phosphonate group and carboxylic group.
impurities.71 Any of these explanations could be applied here. As
expected, styrene polymerization displays the slowest rate, in
contrast to NIPAAm and OEG-A.
(data not shown). As expected, polymer chains terminated with
AIBN fragments were also present as a sub-population.
The linear evolution of molecular weights (determined by
GPC) with increasing monomer conversions indicates behaviour
consistent with the known features of living radical polymeri-
zation. The attainment of low polydispersities up to 70%
conversion (Fig. 1 for OEG-A) furnishes evidence that the
di(methyl)phosphonate functionality is benign in radical poly-
merizations.
Cleavage of the dimethyl phosphonate into phosphonic acid group
Two established methods are known to facilitate the cleavage of
dimethyl phosphonate: the first method uses sodium iodide at 60
ꢀC in acetone for 14 hrs and the second method uses bromosilane
at room temperature for a couple of hours. These two methods
result in different product structures. The use of sodium iodide
generates a monophosphonic acid,65 whereas the use of bromo-
silane results in a diphosphonic acid.72 As our intention was to use
the phosphonic acid to stabilize iron oxide particles we decided to
maximise the phosphonic acid functionality by adopting bromo-
silane to effect the total cleavageof dialkyl phosphonate. 1H NMR
and 31P NMR spectroscopy both confirmed a total conversion
into phosphonic acid by the disappearance of the CH3 signal at
3.6 ppm and by a shift of the P signal from 35 ppm to 39 ppm.
Moreover, the presence of a signal at 2.6 ppm after treatment
(which corresponds to CH2–COH) confirmed that the bromosi-
lane treatment did not alter the integrity of the RAFT function-
ality. However, we caution that a prolonged reaction in the
presence of bromosilane (>14 hrs) was observed to cause some
degradation to the RAFT agent, characterized by a colour change
from yellow-orange to dark orange. Finally, the presence of a new
broad absorbance band at 3100 cmꢁ1 observed by ATR confirms
the formation of phosphonic acid (P–OH) for both poly(OEG-A)
(Fig. 3) and poly(NIPAAm).
Experimental conditions: [OEG-A]0/[RAFT]0/[Initiator]0 ¼ 50.0/
ꢀ
1.0/0.2 and 100.0/1.0/0.2, initiated by AIBN at 60 C.
Subsequent 1H NMR analyses of the purified polymers
confirmed the presence of the dimethyl phosphonate and
carboxylic acid groups on the polymer chains by signals at 3.60
ppm and 2.60 ppm (see Fig. 2 for poly(NIPAAm), NMR). 31P
NMR provided additional confirmation of the presence of
phosphonate group, with a characteristic signal at 35 ppm.
Molecular weights calculated from 1H NMR, assuming one
di(methyl)phosphonate group per polymer chain (Mn, 1H NMR
Ð
Ð
at 4.0
at 3.6
¼
CH
ppm/ (CH3
ppm)/6), were also in good
agreement with the theoretical molecular weights in the case of
poly(NIPAAm) (Table 1). The telechelic stoichiometry (i.e. mole
ratio of the u-carboxylic acid to the a-dimethyl phosphonate
end-group) for NIPAAm polymers (up to 70% conversion),
calculated from 1H NMR spectra, was above 0.85–0.90 (Table 1),
consistent with the generation of well-defined heterotelechelic
polymers. As expected, the stoichiometry decreases slowly with
monomer conversion (probably the result of conventional
Chemical transformation of the trithiocarbonate functionality
radical
termination
and
transfer
processes).
The
The trithiocarbonate functionality is retained in the polymer
after the RAFT polymerisation. This functionality is often used
to control subsequent block copolymerizations. However, in an
atom-efficient synthetic approach, it can also be used to intro-
duce additional functionality. Aminolysis of the trithiocarbonate
group can be exploited to generate a thiol, as described by
others.73–78 Optimization of this reaction, to avoid oxidation of
the thiol to disulfide, requires protection of the thiol by using
theoretical proportion of non-bifunctional polymer can be esti-
mated by f ¼ [RAFT]0/([RAFT]0 + [Init.]0 ꢂ (1 ꢁ exp(kd.t))),
where [RAFT]0 and [Init.]0 are the initial RAFT and initiator
concentrations, kd and t are the initiator decomposition constant
and polymerization time respectively.
Additional evidence from ESI-MS was accrued to confirm the
successful synthesis of telechelic polymers. Short-chain poly
(NIPAAm)s were synthesized by RAFT polymerization and the
ESI-MS analyses confirmed the expected heterotelechelic poly-
mer structure as the principal population (centered at 906.5 m/z)
DTT
(DL-Dithiothreitol)
and
TCEP
(Tris(2-carbox-
yethyl)phosphine hydrochloride). The thiol can be easily
protected by reaction with dithiopyridyl disulfide (Scheme 3).
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Fig. 2 1H and 31P NMR of poly(NIPAAm) obtained by RAFT poly-
merization in the presence of RAFT agent bearing a phosphonate and
a carboxylic group. (Bottom spectrum: before cleavage of phosphonate
group; top spectrum: after cleavage of di(methyl) phosphonate group
into phosphonic acid).
Fig. 3 FT-IR spectra of poly(OEG-A) (A) obtained by RAFT poly-
merization in the presence of a RAFT agent bearing a di(methyl)-
phosphonate group, and (B) after cleavage of di(methyl)phosphonate
group into phosphonic acid group.
appearance of signals at 8.4, 7.6 and 7.2 ppm (dithiopyridyl ring),
and the disappearance of the signal at 2.6 ppm. Moreover,
ESI-MS spectra confirmed the expected product structure for
poly(NIPAAm) (data not shown). XPS also showed the disap-
pearance of the trithiocarbonate group (signal at 162–163.2 eV
from C]S bond and at 163.6–164.8 eV for C–S bonds) and the
appearance of a new signal attributed to disulfide bond at
163.4–164.6 eV54 (Fig. S2 in ESI†).
Fig. 1 Polymerization results of oligo(ethylene glycol)-acrylate (OEG-
A) in the presence of RAFT agent bearing a di(methyl)phosphonate
group and a carboxylic acid group. A: evolution of Ln ([M]0/[M]) with
polymerization time, B: evolution of Mn and PDI with OEG-acrylate
conversion, C: evolution of DMAc GPC distribution with OEG-A
conversion.
Experimental
conditions:
(red
symbol)
[M]/
[RAFT]:[I] ¼ 100/1/0.2 and (blue symbol) [M]/[RAFT]:[I] ¼ 50/1/0.2.
This synthetic approach was adopted for different poly(OEG-
A) and poly(NIPAAm) polymers. The presence of pyridyl
disulfide was confirmed by 1H NMR spectroscopy, with the
To confirm the presence and reactivity of the pyridyl disulfide
functionality we performed a model reaction with thioglycolic
acid using poly(OEG-A) and poly(NIPAAm). UV-visible
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Fig. 4 X-Ray diffraction spectra of IONPs 1 and 2 prepared by flame
spray pyrolysis.
in higher flame temperature as well as its residence time (longer
flame). These conditions including the higher metal concentra-
tions are favorable for the coalescence and sintering of the parti-
cles whilst in the flame.79,80 Based on this, IONPs of two different
sizes were synthesized. This is clearly evident from Fig. 4 where
Bragg peak broadening was observed for IONPs synthesized at
5 ml minꢁ1 liquid feed flow rate (IONPs 1) as compared to those at
10 ml minꢁ1 (IONPs 2). Further estimation of the crystallite size by
the Scherrer formula81 found dXRD of 9 and 15 nm, respectively.
These values are in good agreement with the specific surface area
derived sizes, dBET, of 8 and 13 nm, respectively. The slightly
smaller (dBET<dXRD) but otherwise comparable sizes were typical
of the higher surface area to volume ratio of the single crystallite
and non-porous hexagonal IONP platelets.60,61
Scheme 3 Synthesis of a,u-hetero functional polymer by modification
of di(methyl)phosphonate group into phosphonic acid group and
trithiocarbonate into ethylpyridyl disulfide end-group.
spectroscopic analysis of pyridine-2-thione release indicated
a reaction yield of 80% for both polymers (data not shown).
Grafting of telechelic poly(OEG-A) onto iron oxide
nanoparticles
Synthesis of iron oxide nanoparticles (IONPs)
Two different iron oxide nanoparticles in the form of maghemite
(g-Fe2O3) having diameter of 8 and 13 nm (Table 2 summarizes
the physical characteristics of the iron oxide particles) were
synthesized in a rapid one-step flame spray pyrolysis.60,61 Flame
spray pyrolysis (FSP) of 0.5 M iron 2-ethylhexanoate in mineral
spirit/xylene mixture resulted in IONPs consisting of g-Fe2O3
maghemite phase (Fig. 4). This is in agreement with our earlier
findings using an acetylacetonate based precursor.60,61 During the
one-step FSP, increasing the liquid feed flow rates from 5 to 10 ml
minꢁ1 directly increases the rate of combustion enthalpy as well as
the metal concentrations in the flame. The former in turn resulted
Confirmation of particle surface modification was attained via
a number of analytical approaches, namely FTIR-ATR and XPS
(Fig. 5) and TGA. FTIR-ATR results confirmed the presence of
poly(OEG-A) on the surface by bands at 1730 cmꢁ1 (Fig. 5A).
However, ATR cannot prove polymer attachment to the surface.
Therefore, XPS analysis (Fig. 5B and C) was performed
confirming the presence of the P–O–Fe bond at 113.2 eV (Fig. S3
in the ESI†), while before attachment P–OH appears at 112.4 eV
in agreement with a previous study82 before attachment. More-
over, characteristic signals of C and O of poly(OEG-A) were also
observed (Table 3 and Fig. S4 in the ESI†). The quantification of
Table 2 Summary of physical properties of iron oxide nanoparticles used for this studya
BET (N2 adsorption)
IONPs
dXRD (nm)
Isotherm type
SSA (m2/g)
dBET (nm)
IONPs 1
IONPs 2
9
15
II
II
147
94
8
13
a
Note: There is a slight hysteresis arising from pores between the non-porous particles.
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Table 3 Binding energies and assignments of the component peaks
Envelope Binding Energy (eV) Assignment
P2p
P2p
S2p
S2p
S2p
C1s
C1s
132.6 ꢃ 0.1
133.5 ꢃ 0.1
162.0–163.2
163.0–164.2
163.5–164.7
285 ꢃ 0.1
–P(O)–(OH)2
–P(O)–(O–Fe)2
C]S
C–S in –C–S–S–C– group
C–S in –C–S–(C]S)–S–C– group
Non-functional carbon, sp3, sp2
Non-functional carbon adjacent to
–COOR
285.6 ꢃ 0.05
C1s
286.7 ꢃ 0.1
Carbon linked to oxygen via
a simple bond, i.e. C–O–C
Carbon in COOR
N pyridinyl
N in amide group
N in imine group
Carbonyl of amide group
Carbonyl of ester group
O in C–O–C group
C1s
N1s
N1s
N1s
O1s
O1s
O1s
288.1 ꢃ 0.1
399.2 ꢃ 0.1
399.9 ꢃ 0.1
400.5 ꢃ 0.1
531.4 ꢃ 0.1
532.2 ꢃ 0.1
533.5 ꢃ 0.1
Fig. 5 Characterization of the iron oxide nanoparticle surface. A: ATR of
surface of iron oxide nanoparticles after attachment of phosphonic acid
functionalized poly(OEG-A). A-1: surface of iron oxide nanoparticles; A-2:
surface of iron oxide nanoparticles coated with poly(OEG-A) with
a molecular weight of 13 000 g/mol; B and C: XPS results of iron oxide
nanoparticles (B) before and (C) after attachment of polymer on the surface.
Fig. 6 TGA analysis of iron oxide nanoparticles (8 nm) coated with
poly(OEG-A)s having varying molecular weights (experimental condi-
tions: 5 ꢀC/min under air).
these elements confirmed the attachment of poly(OEG-A) on the
surface.
Thermogravimetric analysis (TGA) was applied to determine
the amount of polymer on the surface of the iron oxide. The
results for different molecular weights of polymer grafted onto
the surface of the iron oxide nanoparticles are shown in Fig. 6. In
the absence of a polymer coating, the weight loss is less than 4%
(Fig. 6), attributed to the desorption of water from the surface.
The weight loss from polymer-stabilized particles is much more
substantial, ranging from 10%–60%, depending on the polymer
molecular weights and the nanoparticle sizes. It is possible to
correlate the weight loss (see equations in the ESI†) with the
number of chain per particle and thus determine the grafting
density. It is noteworthy that the number of chains per particle
decreases concomitantly with the molecular weight of the poly-
mer chains (Table 4). It may be surmised that steric hindrance
limits the accessibility of the end group, i.e. phosphonic acid to
the surface of iron oxide. It may be also expected that mushroom
type assembly of the polymer chains onto the surface is favoured
with the increase in the polymer chain length, a phenomenon
widely observed in polymer adsorption to surfaces via a grafting-
to approach.83
Table 4 Characterization of purified iron oxide nanoparticles after
grafting with phosphonic acid functionalized poly(OEG-A) polymer
Particle
Number
Run diameter (nm) Mn (g/mol) PDI Wt-(%) of chainsa Densityb
0
1
2
3
4
5
6
8
8
8
8
8
0
6 000
—
04.0
—
186
—
1.22 18.0
1.21 35.0
1.22 44.6
1.26 55.6
1.22 12.0
1.21 17.2
0.23
0.23
0.18
0.14
0.21
0.12
13 000
36 000
62 000
13 000
36 000
180
144
115
2200
1600
13
13
a
Number of chains per particle. b Density of chains per nm2.
Characterization of polymer coated iron oxide nanoparticles
After the treatment of iron oxide nanoparticles with varying
telechelic poly(OEG-A)s, the physico-chemical properties of the
nanoparticles were totally changed. The particles treated with
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poly(OEG-A) with a Mn above 10 000 g/mol could be dispersed
easily in water. The dispersions obtained were stable for several
days. In contrast to particles without polymer treatment, fast
aggregation was observed after 5 mins. A control experiment was
carried out by incubating iron oxide particles with PEG (Mn
from 5 000 to 20 000 g/mol). In this case we observed the
formation of a large aggregate after 1 hr as also observed by DLS
(Fig. 7C). This was to rule out the possibility of particle stabili-
zation through adsorption of PEG. Another control experiment
was performed with non-functional poly(OEG-A) obtained by
free radical polymerization, no significant improvement of the
stability was observed.
observed, viz., for polymers of a similar molecular weight, the
size of the stabilized particles was independent of the size of the
original iron nanoparticles (8 nm and 13 nm).
DLS was conducted on samples as they were allowed to age, to
assess the level of stability with time (Fig. 8). After 14 days in
water, the average particle size changed when poly(OEG-A)s
with an Mn > 10 000 g/mol were used for stabilization. In
contrast, when polymers of Mn < 10 000 g/mol were used for
stabilization, the particles showed some aggregation after 72 hrs.
The stability of the nanoparticles was also examined in phos-
phate buffer (pH 6.5, 0.1 M) containing BSA (0.1 g/L) (Fig. 8B),
to mimic the physiological conditions. Only the particles stabi-
lized with poly(OEG-A) with a Mn ¼ 62 000 g/mol were stable
for 48 h under these conditions (without any precipitation and
any significant variation of size determined by DLS). When
incubating IONPs (stabilized with poly(OEG-A) of 62 000 g/
mol) in buffer containing BSA at increasing concentrations (0.05
g/L–1.6 g/L) for 5 h, the size was not affected by BSA
Dynamic light scattering (DLS) was used to determine the size
of the stabilized nanoparticles in water. The results indicated that
the size of the nanoparticles increased with increasing molecular
weight of the grafted polymer (Fig. 7). In the case of
a non-functional PEG, the particles were unstable and formed
aggregates larger than 1 mm (Fig. 7C). An unexpected result was
Fig. 7 Physical properties of iron oxide nanoparticles (IONPs) coated
with poly(OEG-A). A: Picture of IONP solutions: 1 and 2: IONPs
(13 nm) stabilized with poly(OEG-A) (Mn ¼ 30 000 g/mol, PDI ¼ 1.22) in
water at 2 mg/mL and 0.1 mg/mL concentration, respectively; 3: IONPs
(13 nm) without a polymer coating, in water; 4: IONPs (8 nm) without
a polymer coating, in water; 5 and 6: IONPs (8 nm) stabilized with
poly(OEG-A) (Mn ¼ 30 000 g/mol, PDI ¼ 1.22), in water at 0.5 mg/mL
and 2 mg/mL concentration, respectively. B: Size distribution of IONPs
(8 nm) stabilized with poly(OEG-A)s having varying molecular weights,
assessed by DLS. C: Evolution of the size of IONPs (with two different
original diameters: C 13 nm; - 8 nm) with the molecular weight of
poly(OEG-A); : IONPs (13 nm) after treatment with a non-functional
PEG at varying molecular weights.
Fig. 8 Particle size and stability of iron oxide nanoparticles (IONPs)
after coating with poly(OEG-A). A: Pictures of IONPs (original particle
diameter: 8 nm, Mn of stabilizing poly(OEG-A): 62 000 g/mol) after
incubation in phosphate buffer (concentration 0.1 M, pH ¼ 6.5) con-
taining BSA (0.1 g/L) for varying times. Tube 1 contains the IONPs
without polymer after 15 mins. Concentration of IOPNs was fixed at
0.5 mg/mL for all the experiments. B: Evolution of the size of IONPs with
time: IONPs (diameter 8 nm, Mn of stabilizing poly(OEG-A): 62 000
g/mol) : in phosphate buffer containing BSA and - in water. Arrow
shows the time when the precipitation occurs. C: Evolution of the particle
size in phosphate buffer (at pH 6.5) containing BSA with increasing BSA
concentrations (g/L) for : IONPs (diameter 8 nm, Mn of polymer: 62 000
g/mol) and - IONPs (diameter 13 nm, Mn of polymer: 62 000 g/mol).
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concentration in the studied range (Fig. 8C). The effective
stability of the particles was maintained for 72 hours (Fig. 8C).
However, after 7 days, the formation of a small precipitate was
observed. When lower molecular weights of poly(OEG-A) were
used, some precipitation occurred after 1 day, consistent with
DLS measurements indicating the formation of aggregates.
Evolution of particle sizes and zeta potentials were also
measured as a function of pH as shown in Fig. 9. For IONPs
without any polymer coating, the charges vary from positive
(+30 mV) to negative (ꢁ30 mV) with increasing pH. In contrast,
the charges of IONPs with poly(OEG-A) grafted on their
surfaces remain constant, close to neutral zeta potential
(Fig. 9B). This result confirms the presence of a polymer shell on
the surface of IONPs, as poly(OEG-A) is an uncharged polymer.
Moreover, the particles stabilized with a polymer shell retain
dimensional stability, independent of pH, confirming the efficacy
of the phosphonate attachment (Fig. 9A).
SEM was used to evaluate the size and shape of the polymer-
stabilized nanoparticles. In the absence of polymer, the particles
(sampled from aqueous solution, 0.1 mg/mL) were observed to
form large aggregates (size > 1 mm) (Fig. 10-A1). In contrast,
with polymer, the particle sizes were around 140 nm (Fig. 10-A2).
TEM also confirmed the improved dispersion with polymer
(Fig. 10-B1 and B2). TEM was also applied together with
a contrast agent to stain the polymer layer. Thus, the polymer
image appeared grey around the iron oxide nanoparticles (see the
Fig. 10 Representative SEM (A) and TEM (B) images of iron oxide
nanoparticles. A-1: IONPs dispersed in water without polymer
coating, A-2: with a polymer coating (poly(OEG-A); Mn ¼ 62 000 g/mol);
B-1: IONPs without polymer coating, B-2: with a polymer coating
(poly(OEG-A); Mn ¼ 62 000 g/mol), B-3 and B-4: TEM images of
polymer coated IONPs treated with a contrast agent (phosphotungstic
acid) demonstrating the polymer coating (poly(OEG-A); Mn ¼ 36 000
g/mol) (the polymer appears in grey) (B-3: IONPs with an original
diameter of 8 nm, B-4: IONPs with an original diameter of 13 nm).
micrographs in Fig. 10-B3 and B4). The morphologies vary
depending on the sizes of the original iron oxide nanoparticles.
Several small nanoparticles (8 nm) clustered within each polymer
shell (Fig. 10-B3), whereas the larger nanoparticles (13 nm) were
largely isolated within each polymer shell (Fig. 10-B4). This
observation is in good agreement with the DLS results and
explains well why the size of the stabilized nanoparticles was
observed to be independent of the size of the original iron oxide
nanoparticles.
Attachment of peptides onto the polymer coated nanoparticles
The polymer coated IONPs decorated by pyridyl disulfide groups
were reacted with the free thiol group of reduced glutathione, as
a model peptide, and NGR motive containing peptide, as
a tumor-specific targeting peptide.58,59 The conjugation reaction
yields a reversible disulfide bond between the polymer end group
and the peptide, and a by-product, pyridine-2-thione.49 The
formation of pyridine-2-thione in the conjugation reaction
supernatant was measured by a UV-Vis spectrometer (3 ¼ 8080
Mꢁ1 cmꢁ1 at 343 nm).84 The conjugation yield was then evaluated
using the measured concentration of pyridine-2-thione and the
Fig. 9 Effect of pH on (A) particle size (nm) of iron oxide nanoparticles
with a polymer coating, and (B) zeta potential (mV) of iron oxide
nanoparticles with (C) and without (-) a polymer coating (poly(OEG-
A) Mn ¼ 62 000 g/mol).
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Fig. 12 BSA adsorption isotherms of iron oxide nanoparticles (IONPs)
(1 g/L): 8nm in phosphate buffer (0.1 M, pH ¼ 6.5). (C) IONPs without
a polymer coating, (-) IONPs with a poly(OEG-A) coating (Mn ¼ 62
000 g/mol). Note: Each dot is an average of three measurements.
the BSA adsorption conforms to a Langmuir isotherm (as
previously observed for different surfaces, such as silica, titania,
zirconia, and alumina89). The saturation adsorption values were
found to agree with previous results reported in the literature,60,88
indicative of a BSA monolayer. However, for polymer-stabilized
iron oxide nanoparticles, low protein adsorption (<30 mg/g
nanoparticles) was observed over a wide range of BSA concen-
tration (0.05 to 10 g/L) which can be explained by the presence of
a hydrophilic and neutral polymer layer on the iron oxide
particle. The presence of a well-known protein-repellent poly-
mer, OEG, on the surface lowers the protein adsorption to the
iron oxide particles, making them compatible with physiological
fluids.54,60,90–92
Fig. 11 XPS results of IONPs before and after attachment of peptide
(NGR).
amount of polymer chains estimated from TGA measurement
330
(yield ¼ (Ab nm/3pyridine-2-thione)/nPolymer, with nPolymer present on
IONPs surface). The yield was 70 ꢃ 10% for both peptides (moles
polymer). The peptide attached nanoparticles were also analyzed
by XPS (Fig. 11 and S4†). After attachment of NGR peptide, the
intensity of the peaks associated with C1s of carbonyl ((C]O)–
NH and (C]O)–O) at 288.1 and 289.0 eV increased significantly
(Fig. S4 in the ESI†). In the case of N1s, the presence of a new
signal at 400.5 eV, attributed to imine group85,86 present in the
peptide structure, was also observed (Fig. 11). Moreover, the
relative amount of nitrogen increases from 0.2 to 0.8 atomic%,
consistent with successful peptide grafting. Finally, the presence
of disulfide was also observed by the S2p of disulfide bond at
163.6–164.8 eV87 confirming the attachment of the peptide on the
nanoparticles (Fig. 11).
Conclusions
In this report, we described a new synthetic approach to a,u-
telechelic polymers suitable for stabilizing and biofunctionalizing
iron oxide nanoparticles. We have shown that the stability of the
iron oxide nanoparticles is significantly enhanced, even in
conditions similar to the physiological medium. Further, we have
shown that we can decorate the stabilised nanoparticles with
specific peptides that potentially could be used for targeting
purposes. Our future efforts will be focused on investigations of
the cellular uptake of the stabilised nanoparticles.
The peptide functional nanoparticles were then analysed by
DLS and zeta potential. The size of the nanoparticles remained
virtually unchanged as expected. However, the charge on the
nanoparticles was measured to be positive (+0.2 mV) at pH ¼ 7,
consistent with peptide functionalization. In contrast, in the
absence of peptide the poly(OEG-A)-stabilized particles are
uncharged or slightly negatively charged (ꢁ0.1 mV).
Acknowledgements
The authors thank the Australian Research Council for Research
Grants and a Federation Fellowship Award for T.P.D. We also
acknowledge the Bioanalytical Mass and NMR Spectrometry
Facilities at UNSW.
Anti-fouling properties of polymer coated nanoparticles
Protein adsorption studies were also performed on the polymer-
stabilised IONPs. It is already known that IONPs exhibit surface
charge that facilitates surface adsorption of proteins.17,88,89 The
adsorption of BSA onto the particles was investigated, with the
results shown in Fig. 12. In the absence of polymer stabilization,
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