Functionalized, biocompatible coating for superparamagnetic nanoparticles by controlled polymerization of a thioglycosidic monomer

Article abstract of DOI:10.1021/bm101325w

It is demonstrated that water-soluble, glucosylated poly(pentafluorostyrene) derivatives revealed favorable coating material properties for magnetic iron oxide nanoparticles. To prepare the coating material in high reproducibility and purity as well as in

Full text of DOI:10.1021/bm101325w

ARTICLE
pubs.acs.org/Biomac
Functionalized, Biocompatible Coating for Superparamagnetic
Nanoparticles by Controlled Polymerization of a
Thioglycosidic Monomer
Krzysztof Babiuch,† Ralf Wyrwa,‡ Kerstin Wagner,‡ Thomas Seemann,‡ Stephanie Hoeppener,†,§
C. Remzi Becer,†,^ Ralf Linke,|| Michael Gottschaldt,†,§ J€urgen Weisser,‡ Matthias Schnabelrauch,‡,§ and
Ulrich S. Schubert*,†,§
†Laboratory of Organic and Macromolecular Chemistry (IOMC) and Jena Center for Soft Matter (JCSM), Friedrich-Schiller University
Jena, Humboldtstr. 10, 07743 Jena, Germany
‡Biomaterials Department, INNOVENT e.V., Pr€ussingstr. 27b, 07745 Jena, Germany
§Dutch Polymer Institute (DPI), John F. Kennedylaan 2, 5612 AB Eindhoven, The Netherlands
^Department of Chemistry, University of Warwick, Library Road, CV4 7AL Coventry, United Kingdom
Surface Engineering Department, INNOVENT e.V., Pr€ussingstr. 27b, 07745 Jena, Germany
S Supporting Information
b
ABSTRACT: It is demonstrated that water-soluble, gluco-
sylated poly(pentafluorostyrene) derivatives revealed favor-
able coating material properties for magnetic iron oxide
nanoparticles. To prepare the coating material in high repro-
ducibility and purity as well as in sufficient amounts, a new
route of synthesis is established. The preparation and char-
acterization of the glucosylated, tetrafluorostyryl monomer,
by thiol-para-fluorine “click” reaction, and its polymerization,
via nitroxide-mediated radical process, is presented in detail.
In addition, the coating material and the resulting particle
properties are investigated by means of XPS, DLS, TGA, TEM, and cryo-TEM as well as flow cytometry. The glycopolymer acts as
an appropriate stabilizing agent for the superparamagnetic nanoparticles by the formation of an approximately 10 nm thick shell, as
shown by the XPS analysis. Furthermore, the application of FITC-labeled glycopolymer yielded fluorescent, superparamagnetic
nanoparticles, which can be used for monitoring cell-carbohydrate interactions, because these particles show no cytotoxicity
toward 3T3 fibroblasts.
’ INTRODUCTION
In general, the shell of the polymer prevents agglomeration by
steric effects as well as electrostatic repulsion, enabling the
stabilization of individual magnetic cores. These biocompatible,
polymer-coated nanoparticles exhibit sufficient stability in diluted
aqueous solution and allow the modification of physical and
chemical properties by varying the structure of the polymer.
In addition, functionalities, such as OH-, NH-, or carboxylic
groups, allow further introduction of functional moieties like
drugs and bioactive ligands, for example, carbohydrates, proteins,
or dyes, enabling drug targeting with directed cell interaction or
usage in bioanalytical applications.13-16 The main drawback for
a broad medical use of magnetic nanoparticles is their insuffi-
cient long-term stability. Although technical ferrofluids possess
extraordinary stability, the coating materials and their synthesis
strategy cannot be applied to iron oxide nanoparticles in aqueous
Superparamagnetic nanoparticles based on iron oxide are of
increasing importance in the fields of bioanalytics and medicine.
The advantage in using iron oxide as magnetic material is its
known low cytotoxicity and high biocompatibility. As a conse-
quence, magnetic nanoparticles can potentially be used in a wide
range of biomedical applications. Some examples include mag-
netic resonance imaging (MRI), cell separation, magnetic drug
targeting, and hyperthermia cancer treatment as well as magneto-
fection.1-6 The advances in the field of magnetic nanoparticle
design for medical purposes have been reviewed recently.7 Shell
materials for magnetic nanoparticles used in these applications
include polysaccharides, for example, dextran or derivatives
thereof, in particular, carboxymethylated dextran, poly(ethyleneimine),
poly(N-isopropyl-acrylamide), poly(ethyleneglycol), or vinyl
alcohol-based polymers among others.8-12 The polymeric ma-
terials that are suitable for biomedical usage and used for coating
and stabilization of iron oxide particles have to fulfill similar require-
ments, that is, water solubility and no or only little cytotoxicity.
Received: November 6, 2010
Revised:
December 17, 2010
Published: January 21, 2011
r
2011 American Chemical Society
681
dx.doi.org/10.1021/bm101325w Biomacromolecules 2011, 12, 681–691
|

Biomacromolecules
ARTICLE
solution since they are not soluble in water. Examples of fre-
quently used coating materials of technical ferrofluids are fatty
acids and polymers, for example, poly(styrene).17,18 To combine
the advantages of polymers used in technical ferrofluids (stability)
with the requirements of medically applicable magnetic nano-
particles, it is necessary to develop water-soluble, noncytotoxic,
polymeric nanoparticle coating materials. In principle, glycopo-
lymers, that is, synthetic polymers carrying pendant sugar moie-
ties, fulfill the above-mentioned requirements. Furthermore,
these polymers have found a wide range of applications in clinical
diagnostics, as targeted drug delivery systems, for affinity separa-
tions, bioassays, and biocapture analysis.19-22 Their therapeutic
applications have been thoroughly reviewed.23 In spite of their
biomedical significance, only very few synthetic glycopolymers
have been applied as coatings of magnetic beads.24-26 To the
best of our knowledge, only one single report of poly(vinylbenzyl-
O-β-D-galacto-pyranosyl-D-gluconamide)-coated iron oxide nano-
particles as a liver-targeting MRI contrast agent exists.27 Recently,
glycopolymers based on poly(pentafluorostyrene) were synthe-
sized using a versatile “click” approach.28,29 They consist of a
carboxylic acid terminated chain and thioglucose substituted,
fluorinated phenyl rings. These linear, saccharide functionalized
polymers show a suitable water solubility mediated by the sugar
moieties. The additional carboxylic function could permit the
electrostatic interaction with the iron oxide surface as known
principle of attachment.2 For reproducible preparation of ferro-
fluids, structurally well-defined glycopolymers are required. For
this purpose, a new route of synthesis was established. In contrast
to our previous work, where the glycopolymers were obtained by
postpolymerization modification of preformed polymeric back-
bones, herein the sugar-modified polymers were obtained by a
direct polymerization of glucosylated monomers. Coupling of
acetylated β-D-thioglucopyranose via a nucleophilic substitution
of para-fluorine of pentafluorostyrene and subsequent deacetyla-
tion with sodium methoxide provided a glycomonomer with an
overall yield of 84%. Its optimized, nitroxide-mediated, radical
polymerization gave a glycopolymer with high monomer conversion
(70%) and low polydispersity index (PDI < 1.2). This glycopolymer
fulfilled all the requirements for preparing stabilized, water disper-
sible, magnetic nanoparticles because it was able to withstand
the harsh conditions used in the coating procedure. Further-
more, for the planned medical use the cytotoxicity of this type
of polymer was investigated. No cytotoxic effects were found.
Herein, the synthesis of a new glycosylated tetrafluorostyrene
monomer, its controlled radical polymerization, labeling and
application as fluorescent, biocompatible coating material for
superparamagnetic nanoparticles based on iron oxide as well as
their detailed characterization are reported.
Characterization. 1H and 13C NMR spectra were recorded on a
Bruker Avance 300 MHz spectrometer and the 19F NMR spectra on a
Bruker Avance 200 MHz spectrometer in deuterated DMF. The chem-
ical shifts were calibrated with respect to DMF residual peaks. Size
exclusion chromatography (SEC) was measured on an Agilent Tech-
nologies 1200 Series gel permeation chromatography system equipped
with a G131A isocratic pump, a G1329A autosampler, a G1362A
refractive index detector, and both a PSS Gram 30 and a PSS Gram
1000 columns in series. LiCl solution (2.1%) in DMA was used as eluent
at 1 mL min-1 flow rate at a column oven temperature of 40 ꢀC. The
3
reported number average molar masses were calculated according to
poly(styrene) standards. For homogenization of the particles a Sonopuls
UW2200 (Bandelin, Germany) device was used. Dynamic light scatter-
ing measurements (DLS) were performed on a Zetasizer device from
Malvern Instruments (Worcestershire, U.K.). The magnetic behavior
was measured with a vibrating sample magnetometer (MicroMagTM,
Princeton Measurement Corp., US, Bmax = 1.6 T). X-ray photoelectron
spectroscopy (XPS) was measured with a Theta Probe system (Thermo
VG Scientific) equipped with monochromatic X-ray Al KR source
(1486.68 eV) using a spot diameter of 400 μm (100 W) and the
analyzer in CAE mode with a pass energy of 150 eV for survey spectra
and 75 eV for elemental region spectra. The spectra were collected with
an angle of 53ꢀ to the surface normal and an angle acceptance of (30ꢀ
and charging compensation by low energy electrons was applied. The
CasaXPS v2.14 software was used for XPS data processing. Electron
binding energies (BE) were corrected by adjusting the low energy
component of the C1s emission to 284.8 eV. Relative sensitivity factors
based on the Scofield cross sections were used for quantification
assuming a homogeneous distribution of the components in the probed
sample volume. Peak positions and contributions of different chemical
states were determined by fit processing. Thermogravimetric analyses
were performed on a Netzsch TG 209 F1 Iris with 10 ꢀC min-1 heating
3
rates from room temperature up to 900 ꢀC, with a two-minute
isothermal step at 450 ꢀC, under nitrogen flow. Flow cytometry was
measured on a Beckmann Coulter Cytomics FC-500 equipped with
Uniphase Argon ionlaser, 488 nm, 20 mW output. Transmission electron
microscopy (TEM) as well as cryo-TEM images were recorded using a
Technai G2 Sphera (FEI) TEM with an acceleration voltage of 200 kV.
Fluorescence of the cells was observed using an Axiovert 25 microscope
(Zeiss AG, Jena, Germany) with filter sets 44 and 14. Photomicrographs
were recorded using a CCD fluorescence imager MP 5000 (Intas,
G€ottingen, Germany). Imaging was supported by the Image-Pro Plus
software (Media Cybernetics, Silver Spring, MD).
Cell Culture. The 3T3 fibroblasts (from the DSMZ German
Collection of Microorganisms and Cell Cultures, Braunschweig, Germany)
were cultured in DMEM supplemented with 10% fetal calf serum,
50 U mL-1 penicillin, and 50 μg mL-1 streptomycin (all components
3
3
from Biochrom, Berlin, Germany), under 5% CO2 atmosphere at 37 ꢀC.
Synthesis of the Glycomonomer
a. Synthesis of 4-(2,3,4,6-Tetra-O-acetyl-1-thio-β-D-glucopyranosido)-
2,3,5,6-tetrafluorostyrene (TFS-GlcAc). 2,3,4,6-Tetra-O-acetyl-1-thio-
β-D-glucopyranose (3.30 g, 9.06 mmol) was dissolved in 90 mL of dry
DMF in a round-bottom flask and cooled in an iced water bath. Penta-
fluorostyrene (1.25 mL, 9.20 mmol) was added dropwise to the stirred
solution. Subsequently, TEA (2.55 mL, 18.30 mmol) was dropped into
the reaction mixture. The reaction was continued for 3 h at room tem-
perature until complete disappearance of the starting sugar, monitored
by TLC in hexane/ethyl acetate (1:1). The solvent and the small excess
of pentafluorostyrene were removed in a rotary evaporator, and the
crude product was purified by column chromatography (hexane/ethyl
acetate (1:1)) on silica gel. The solvent mixture was evaporated and the
’ EXPERIMENTAL SECTION
Materials. 2,3,4,6-Tetra-O-acetyl-1-thio-β-D-glucopyranose was syn-
thesized as previously reported.30 Triethylamine (TEA; for synthesis,
g99%), ferrous chloride tetrahydrate (p.a.), 25% ammonia solution
(p.a.), and potassium permanganate (0.1 N, Titrisol) were purchased from
Merck, N,N-dimethylformamide (DMF; g99.5%), N,N-dimethylaceta-
mide (DMA), sodium thiosulfate solution (0.1 M, Fixanal), and anthrone
(p.a., ACS reagent) from Fluka, fluorescein isothiocyanate isomer I (FITC,
g90%; HPLC) from Sigma, methanol (anhydrous, 99.8%) from Aldrich,
and ferric chloride hexahydrate (p.a., ACS reagent), and 37% hydrochloric
acid (HCl; p.a., ACS reagent) from Roth. Sodium methanolate was
purchased from Fluka and stored under argon prior to use. All other
chemicals were used as received, unless otherwise noted.
product was dried under vacuum to give 4.39 g (90% yield) of a white
3
powder. 1H NMR (300 MHz, DMF-d7, δ): 6.81 (1H, dd, J1 ,2
=
0
0
3
2
0
0
0
0
17.9 Hz, J1 ,3 = 11.9 Hz, CH-vinyl), 6.18 (1H, dd, J2 ,3 = 75.8 Hz,
682
dx.doi.org/10.1021/bm101325w |Biomacromolecules 2011, 12, 681–691

Biomacromolecules
ARTICLE
CH2-vinyl), 5.93 (1H, dd, CH2-vinyl), 5.40 (1H, t, 3J4,3 = 9.4 Hz, H-4),
5.35 (1H, d, 3J1,2 = 10.4 Hz, H-1β), 5.06 (1H, t, 3J3,2 = 9.6 Hz, H-3), 4.99
Synthesis of Iron Oxide Nanoparticles. Superparamagnetic
iron oxide nanoparticles consisting of magnetite and maghemite, with an
average core diameter of 10 nm were prepared as previously described.31
Briefly, aqueous mixtures of iron salts with a molar ratio of 1/2 (FeCl2/
FeCl3) were coprecipitated by adding an excess of 25% aqueous ammonia
solution. The particles were separated magnetically, washed repeatedly
with distilled water, and the pH value was adjusted to 1-2 with HCl.
Particle Coating. A total of 250 mg of the PTFS-GlcOH glyco-
polymer, dissolved in 30 mL of distilled water, was added to a freshly
prepared ferrofluid at 50 ꢀC. Subsequently, the mixture was stirred at 50 ꢀC
for 20 min. A first particle fraction was separated magnetically and
discarded to get rid of larger, uncoated particles. After adjustment of the
pH value of the supernatant to 5, a second fraction was isolated mag-
netically and washed repeatedly with distilled water. Finally, the core-
shell particles were resuspended in water and homogenized by ultrasonic
treatment. The coating with FITC-labeled glycopolymer was carried out
analogously.
Characterization of the Nanoparticles. The iron(II) and
iron(III) contents were determined after dissolution in HCl (37%) by
conventional titration with KMnO4 and Na2S2O3, respectively. The
characterization data of the magnetic behavior (specific saturation
magnetization, δs, coercive field strength, Hc, and relative saturation
remanence, δr/δs) of the nanoparticles (powder from lyophilization as
well as ferrofluid) was obtained from the curve of magnetization, recorded
with a vibrating sample magnetometer. Dynamic light scattering (DLS)
was used to study the size (hydrodynamic diameter, hd), zeta potential
(ζ), and particle polydispersity index (PDIp). Samples were strongly
diluted in double distilled water (5 μL of the ferrofluid in 4 mL of water
for the determination of size and 50 μL in 4 mL of water for the
determination of zeta potential). The size distribution was reported by
intensity.
2
3
(1H, t, H-2), 4.23 (1H, dd, J6 ,6 = 12.8 Hz, J6 ,5 = 6.3 Hz, H-60),
4.14-4.04 (2H, m, H-6, H-5), 2.12, 2.04, 2.01, 2.00 ppm (12H, 4s, CH3-
acetyl). 13C NMR (300 MHz, DMF-d7, δ): 171.0, 170.8, 170.6, 170.5
(CO-acetyl), 150.5-150.2 (m, C-F), 147.3-147.0 (m, C-F),
0
0
4
144.0-143.7 (m, C-F), 126.3 (t, JC,F = 30.2 Hz, CH2-vinyl), 123.2
(CH-vinyl), 119.5 (t, 2JC,F = 55.7 Hz, C-S), 110.0 (t, 2JC,F = 84.6 Hz,
C-C), 85.5 (C1), 76.4 (C5), 74.4 (C4), 71.8 (C2), 69.3 (C3), 63.1
(C6), 21.0, 20.9, 20.8 ppm (CH3-acetyl). 19F NMR (200 MHz, DMF-
d7, δ): -134.2 (2F, m), -144.9 ppm (2F, m).
b. Synthesis of 4-(1-Thio-β-D-glucopyranosido)-2,3,5,6-tetrafluoro-
styrene (TFS-GlcOH). 3.00 g (5.57 mmol) of TFS-GlcAc was added
to 200 mL of dry methanol to give a milky suspension. Under vigorous
stirring, 11.15 mL of a 2 M solution of sodium methanolate was
added dropwise to the reaction mixture. After 15 min, a clear solution
was obtained. The reaction was continued until complete disappearance
of the starting material (1 h), monitored by TLC (silica gel, ethyl
acetate/hexane (1:1) and ethyl acetate/methanol (3:2) to confirm
complete deprotection). Subsequently, the mixture was neutralized with
Dowex 50WX8-200 Hþ-loaded resin, filtered and dried under vacuum.
1
TFS-GlcOH was obtained as a white powder (1.92 g, 93% yield). H
NMR (300 MHz, DMF-d7, δ): 6.77 (1H, dd, 3J1 ,2 = 17.9 Hz, 3J1 ,3
=
0
0
0
0
2
0
0
11.9 Hz, CH-vinyl), 6.12 (1H, dd, J2 ,3 = 78.3 Hz, CH2-vinyl), 5.86
(1H, dd, CH2-vinyl), 5.71 (1H, s, OH), 5.36 (1H, bs, OH), 5.17 (1H, bs,
OH), 4.93 (1H, d, 3J1,2 = 9.5 Hz, H-1), 4.48 (1H, t, 3JOH,CH3 = 5.8 Hz,
OH), 3.79-3.68 (1H, m, H-6), 3.54 (broad signal, H2O and H-60), 3.43
(1H, t, 3J4,3 = 8.3 Hz, H-4), 3.36-3.23 ppm (3H, m, H-2, H-3, H-5). 13
C
NMR (300 MHz, DMF-d7, δ): 150.0 (m, C-F), 146.4 (m, C-F), 143.8
4
(m, C-F), 125.3 (t, JC,F = 30.2 Hz, CH2-vinyl), 123.3 (CH-vinyl),
117.6 (t, 2JC,F = 56.7 Hz, C-S), 112.0 (t, 2JC,F = 85.6 Hz, C-C), 86.8
(C1), 82.9 (C5), 79.8 (C4), 75.9 (C2), 71.6 (C3), 62.7 ppm (C6).
19F NMR (200 MHz, DMF-d7, δ): -136.0 (2F, m), -145.9 ppm
(2F, m). HRMS (ESI, m/z): Calcd for C14H14F4O5SNa [M þ Na]þ,
a. Sugar Titration. The glucose unit, bound to the poly(tetrafluoro-
styrene) backbone, was used for the determination of the shell material
content in an adapted procedure of Dreywood, Gutierrez-Gallego, and
Scott.32-34 In brief, the method is based on the conversion of poly-
saccharides into monosaccharides through acid hydrolysis, and the
conversion of monosaccharide-derived furfurals into an UV/vis-absorb-
ing complex with the tricyclic aromatic hydrocarbon anthrone. The peak
maximum of the anthrone-complex was determined at a wavelength of
λ = 625 nm. The sample was prepared by dissolving the nanoparticles in
HCl (37%) followed by the described procedure. Based on a calibration
curve, the amount of shell material could be determined.
393.0766; found, 393.0390. Anal. Calcd for C14H14F4O5S 2H2O: C,
3
41.38; H, 4.46; S, 7.89. Found: C, 41.54; H, 4.02; S, 7.91.
c. Polymerization of TFSGlcOH. Glucosylated monomer (500 mg,
1.35 mmol), BlocBuilder (from Arkema, 10.3 mg, 0.027 mmol), tetra-
hydrofuran (5 mL), and water (5 mL) were added in a 20 mL pressure-
resistant vial. The mixture was saturated with argon while stirring for 1 h.
The vial was sealed and placed into a preheated oil bath (110 ꢀC). The
reaction was continued for 2 h. Afterward, the reaction vessel was cooled
with tap water and the solution was concentrated in the rotary evaporator
prior to precipitation in cold ethanol. The isolated white powder of PTFS-
GlcOH (350 mg) was dried under vacuum. The conversion (70%) and
the degree of polymerization (DP) of 30 were estimated from 1H NMR
spectra of the mixture before and after polymerization, using THF as the
internal standard. 1H NMR (300 MHz, DMF-d7, δ): 5.62 (m, OH), 5.30
(m, OH), 5.13 (m, OH), 4.77 (m, H-1), 4.30 (m, OH), 3.90-3.14 (broad
signal, H2O, CH2-ethanol and H-2, 3, 4, 5, 6, 60), 2.36-1.88 (broad signal,
CH2-backbone), 1.39-0.8 ppm (broad signal, CH3-initiator and CH3-
ethanol). 13C NMR (300 MHz, DMF-d7, δ): 149.5-142.0 (m, C-F),
123.0-120.0 (m, C-F), 111.8 (m, C-F), 87.0 (C1), 81.4 (C5), 78.5
(C4), 74.8 (C2), 70.2 (C3), 61.5 (C6), 56.9 (CH2-ethanol), 39.6-28.9
(DMF and backbone carbons), 18.2 ppm (CH3-ethanol). 19F NMR (200
MHz, DMF-d7, δ): -132.1 to -136.5 (m), -141.4 to -146.4 ppm (m).
d. FITC-Labeling of PTFS-GlcOH. In an oven-dried, round-bottom
flask, the glycopolymer (451.0 mg, 0.04 mmol) and FITC (7.6 mg, 0.02
mmol) were weighed in and dried for 1 h under vacuum. DMF (10 mL)
was added, and the sealed mixture was stirred for 24 h. The reaction was
quenched and the polymer precipitated by dropping the solution into
ethanol. The precipitated polymer was centrifuged and washed with
ethanol until complete disappearance of fluorescence in the supernatant
(six times, TLC monitoring).
b. TEM and Cryo-TEM Measurements. Dry samples were prepared
by blotting the 10-fold diluted solution onto a carbon coated TEM grid
(Electron Microscopy Science) placed on a filter paper. Samples for
cryo-TEM were prepared using a FEI Vitrobot system. A total of 3 μL of
the sample solution was transferred onto a Quantifoil (R2/2) grid, and
blotting was performed at 3 mm and 3.5 s of blotting time. Samples were
rapidly transferred into liquid ethane and stored in liquid nitrogen until
the measurements were performed using a Gatan cryo holder.
Investigation of Cytotoxicity. The biocompatibility of the mag-
netic nanoparticles was investigated in a live/dead assay using 3T3 cells
and fluorescent dyes: fluorescein diacetate (FDA, labeling live cells) and
ethidium bromide (EtBr, labeling the nuclei of dead cells).35,36 The 3T3
cells were inoculated at 25000 cells cm-2 into a 48-well tissue culture
3
plate (Greiner Bio-One, Frickenhausen, Germany) and cultivated for 6 h
at 37 ꢀC under a 5% CO2 atmosphere. The culture medium (0.5 mL)
was supplemented with 1, 5, and 25 μL of the polymer-coated nano-
particles to give the final concentrations of 0.2, 1, and 5% (v/v),
respectively. The tests were performed in duplicate after 1 and 4 days
with addition of 0.5 mL of the nutrient medium after two days. The live/
dead staining followed the standard protocol. After 1 and 4 days, the
culture medium was withdrawn and the cells were covered with 0.1 mL
of PBS (to avoid the drying of the cells) and with 0.1 mL of 2-fold
683
dx.doi.org/10.1021/bm101325w |Biomacromolecules 2011, 12, 681–691

Biomacromolecules
ARTICLE
Scheme 1. Schematic Representation of the Synthesis of the Glycopolymer
concentrated staining solution realizing the final concentrations of
15 μg mL-1 FDA and 4 μg mL-1 EtBr. The observation of the red
3
3
and green fluorescence started after 1 min by fluorescence microscopy.
The total number of cells and the percentage of living cells were
calculated from the micrographs by counting the orange-fluorescent
nuclei of the dead cells and the green-fluorescent living cells.
’ RESULTS AND DISCUSSION
Glycopolymer Synthesis. To evaluate pentafluorostyrene-
based glycopolymers as potential coating material for super-
paramagnetic iron oxide nanoparticles, well-defined polymers of
high purity and large quantity are required. The methods of glyco-
polymer synthesis have been the subject of many reviews.22,37-43
Basically, sugar-modified polymers can be obtained by two ap-
proaches, namely, the polymerization of glycosylated monomers
or the grafting of sugar moieties onto a preformed polymeric back-
bone. Herein, the first method was employed since the reactants,
pentafluorostyrene and thioglucose tetraacetate, are commer-
cially available, and the applied polymerization technique, the
nitroxide-mediated radical polymerization (NMP), yields pro-
ducts with low polydispersity indices (PDIs) and good conver-
sions for styryl-based monomers.44-46 Furthermore, NMP does
not require any toxic catalyst or heavy metal salt to mediate the
reaction, which represents a major disadvantage for most of the
other polymerization methods and was already previously ap-
plied to obtain poly(styrene)-based glycopolymers.47-55 The
procedure for the synthesis of the glycomonomer and the
subsequent polymerization is illustrated in Scheme 1.
i. Thiol-para-fluorine “Click” Reaction. Significant contribu-
tions have concentrated on the glycomonomer synthesis because
they are usually not commercially available.40,56 The synthesis of
the sugar-bearing monomers most often requires multistep pro-
cedures.41,42 The thiol-para-fluorine reaction was previously re-
ported as an efficient route to functionalize poly(pentafluorostyrene)
with acetylated, thiosugar moieties.28,29 Furthermore, the reac-
tivity of the para-fluorine on the pentafluorophenyl group is
significantly enhanced if soft primary nucleophiles are used.57,58
The potential, versatility, and effectiveness of thiol-halogen nucleo-
philic reactions were recently summarized in an excellent review.59
Moreover, this method results in the formation of materials
with S-glycosidic bound sugar residues, which are, unlike O- and
N-glycosidic bonds, stable against enzymatic degradation.60-62
Herein, 2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranose (GlcAcSH)
was reacted with pentafluorostyrene (PFS) in the presence of
triethylamine in dry dimethylformamide (DMF). The reaction
was carried out at a nearly equimolar ratio of GlcAcSH to PFS. A
slight excess (1 mol %) of pentafluorostyrene was used to ensure
the full conversion of the carbohydrate. The synthesis proceeded
Figure 1. 1H NMR (DMF-d7, 300 MHz) spectra of synthesized
monomers and the glycopolymer.
quantitatively, and after 3 h, GlcAcSH was completely converted
to TFS-GlcAc, as shown by TLC monitoring. The acetylated
monomer was purified from triethylammonium fluoride by
column chromatography and dried to obtain a white powder in
high yield (90%). 1H, 13C, 19F, and HSQC-DEPT NMR spectros-
copy confirmed the structure and the purity of TFS-GlcAc
(Figure 1 and Supporting Information).
The proton spectrum exhibits only the required peaks for the
acetylated β-glucose moiety and the vinyl group. In the 19F
spectrum, only the signals from ortho- and meta-fluorines are
visible (Figure 2).
ii. Carbohydrate Deprotection. The final glycomonomer was
obtained by applying the standard Zemplꢀen procedure for sugar
deprotection as described in the Experimental Section. The purity
1
and structure of TFS-GlcOH was determined by H, 13C, 19F,
and HSQC-DEPT NMR spectroscopy as well as electrospray
ionization mass spectrometry (ESI-MS) and elemental analysis.
The complete deprotection is confirmed by the disappearance of
1
the methyl proton signals of the acetyl groups in the H NMR
spectrum and the appearance of four peaks corresponding to the
hydroxyl groups of the deacetylated β-D-glucose substituent
(Figure 1).
iii. Polymerization of TFS-Glucose. For the polymerization of
the glucosylated tetrafluorostyryl monomer via nitroxide-mediated
controlled, radical polymerization, a β-phosphonylated alkoxya-
mine initiator (BlocBuilder from Arkema, Scheme 1) was used.
The synthesis was carried out in a pressurized vial in a THF/H2O
(1:1) mixture, with a monomer to initiator ratio of 50, at 110 ꢀC
for 2 h. After cooling, the solution was concentrated and precipitated
684
dx.doi.org/10.1021/bm101325w |Biomacromolecules 2011, 12, 681–691

Biomacromolecules
ARTICLE
Figure 2. 19F NMR (DMF-d7, 300 MHz) spectra of synthesized
monomers and the glycopolymer.
Figure 3. SEC traces of the synthesized glycopolymers in N,N-dimethyl-
acetamide with lithium chloride (2.1 g L-1) as eluent.
3
in cold ethanol. The isolated white powder of PTFS-GlcOH
(350 mg, 70% conversion, DP = 30) was characterized by H,
analysis (TGA), flow cytometry (FC), and iron as well as sugar
titrations. Furthermore, their magnetization behavior was examined.
i. Particle Characterization by DLS. For the investigation of
the particles via DLS, the obtained ferrofluids were highly diluted
in double-distilled water (5 μL of the ferrofluid in 4 mL of water
for the determination of size and 50 μL in 4 mL of water for the
zeta potential investigation). For this study, suspensions of naked
and coated with unlabeled PTFS-GlcOH particles were used.
The summary of the obtained results can be found in Table 1.
The noncoated nanoparticles exhibit an average hydrodynamic
diameter value of 140 nm. After the coating procedure, this value
increased to an average of 329 nm indicating successful binding
of the glycopolymer. Furthermore, the change of the zeta potential
(ζ) of the particles from the positive value of 28 mV to negative
(-2.3 mV) provides a confirmation of the presence of the
glycopolymer on the iron oxide core.
1
13C, 19F NMR, and HSQC-DEPT spectroscopic methods as well
as by size exclusion chromatography (SEC). The proton spec-
trum (Figure 1) of the glycopolymer shows the signals of the β-D-
glucose moieties, the polymeric backbone, and the initiator. The
19F NMR spectrum (Figure 2) exhibits two broad signals corre-
sponding to the ortho- (-132.1 to -136.5 ppm) and meta-
fluorines (-141.4 to -146.4 ppm) of the tetrafluorostyryl units.
The broadening of the signals and the local maxima are due to the
different tacticities of the glycopolymer. The SEC trace (Figure 3) of
the obtained polymer shows a narrow, monomodal distribution.
The molar mass of 24000 g mol-1 and the PDI value (1.16)
3
were calculated according to poly(styrene) standards (from PSS).
From the SEC analysis results it can be concluded that the
polymerization was carried out in a controlled fashion, thus a
well-defined glycopolymer was obtained.
ii. Titration Analyses of the Glycopolymer-Coated Nanopar-
ticles. The obtained glycopolymer-coated and uncoated parti-
cles were chemically analyzed in view of the overall iron content
as well as the iron(II) to iron(III) ratio by titration experiments,
following the standard procedures.63 The selective determina-
tion of iron(II) and iron(III) by titration with KMnO4 and
iv. Fluorescence Labeling of the Glycopolymer. For labeling
of PTFS-GlcOH, the glycopolymer (0.04 mmol) and fluorescein
isothiocyanate (FITC, 0.02 mmol) were vacuum-dried and dis-
solved in 10 mL of DMF in a round-bottom flask. After stirring
for 24 h at room temperature, the polymer was precipitated and
purified by repeated washing in ethanol and centrifugation. A
total of 350 mg of yellowish powder were obtained. SEC analysis
of the fluorescent polymer (Figure 3) shows a slight increase,
Na2S2O3, respectively, yielded in 3.4 mg mL-1 of iron(II) and
3
20.0 mg mL-1 of iron(III) for the suspension of the noncoated
3
particles and 1.4 mg mL-1 of iron(II) and 9.8 mg mL-1 of
3
3
from 24000 to 24300 g mol-1 of the molar mass. The low PDI
iron(III) for the one containing the coated particles (Table 1).
To obtain further information of the amount of coating on the
nanoparticles, the amount of glucose units, bound to the poly-
(tetrafluorostyrene) backbone, was quantified by the anthrone
assay as described in the Experimental Section. Based on a
calibration curve, the amount of shell material could be deter-
3
value of 1.16 was maintained for the labeled glycopolymer,
signifying no coupling reactions of the polymeric backbone or
its decomposition.
Synthesis and Characterization of the Glycopolymer-
Coated Superparamagnetic Iron Oxide Nanoparticles. The
superparamagnetic particles were produced by coprecipitation of
iron chlorides with 25% aqueous ammonia as reported in the
literature.31 Coating of the obtained particles with both labeled
and nonlabeled glycopolymer was accomplished in aqueous,
acidic solutions (pH = 1-2) at 50 ꢀC within 20 min. The
shell-core particles were magnetically separated and thoroughly
washed with distilled water. To confirm the success of the coating
procedure and to estimate the quantity of the glycopolymer
attached to the particle, they were investigated by means of dynamic
light scattering (DLS), transmission electron microscopy (TEM),
X-ray photoelectron spectroscopy (XPS), thermogravimetric
mined. The content of 7.5 mg mL-1 of glucose was obtained for
3
the nanoparticle suspension, confirming the presence of the gly-
copolymeric coating material.
iii. Analysis of the Magnetization Behavior. The magnetiza-
tion behavior of noncoated and coated particles was analyzed
using a vibrating sample magnetometer. The fluids as well as the
lyophilized powders were investigated. The results of the anal-
ysis, obtained from the curve of magnetization, are summarized
in Table 1. Both samples revealed superparamagnetism. Further-
more, the magnetization behavior indirectly confirms the pres-
ence of the glycopolymeric material since the specific saturation
685
dx.doi.org/10.1021/bm101325w |Biomacromolecules 2011, 12, 681–691

Biomacromolecules
ARTICLE
Table 1. Characterization Data of the Naked and Glycopolymer-Coated Particlesa
sample
Hc [kA m-1
]
δs [Am2 kg-1
]
δr δs-1
Fe(II) content [mg mL-1
]
Fe(III) content [mg mL-1
]
hd (PDIp) [nm]
ζ [mV]
3
3
3
3
3
powder
naked
0.7
0.3
66.0
32.2
0.02
0.01
coated
suspension
naked
0.8
0.7
1.8
0.4
0.03
0.03
3.4
1.4
20.0
9.8
140 (0.22)
329 (0.24)
28
coated
-2.3
a Hc, coercive field strength; δs, specific saturation magnetization; δr δs-1, relative saturation remanence; hd, hydrodynamic diameter; ζ, zeta potential.
3
agglomerates. The nonhomogenous distribution of the particles
on the TEM grid (Figure 4A) might support this assumption.
However, also drying artifacts will influence the distribution of
the particles. In contrast to the noncoated particles, the presence
of a polymer network is observed in case of coated nanoparticle
assemblies (Figure 4B and 4C). The overall diameter of the
interlinked assemblies is observed to be a few hundred nanome-
ters, which is in agreement with the trend to increased particle
sizes measured by DLS for the coated nanoparticles. This system
was further investigated by means of cryo-TEM investigations
(Figure 3D), which permit the imaging of a solution-like state of
the nanoparticles. The coated nanoparticles also reveal a state of
association here, with assemblies of an average diameter of
100-300 nm, which is in good correlation with DLS as well as
dry TEM measurements.
v. Particle Characterization by XPS. X-ray photoelectron
spectroscopy is a potent tool to characterize surface coatings.
Not only does it provide information about the elemental
composition, it also gives data about the chemical states present
in the analyzed sample as well as the coating thickness.11 There-
fore, the naked and the glycopolymer-coated iron oxide particles
have been investigated by XPS (Figure 5 and Table 2).
In the survey spectrum of the uncoated iron oxide nanopar-
ticles (Figure 5A), mainly O and Fe are found as expected.
Additionally, a small amount of carbon is detected. The C signal
is attributed to the ubiquitous carbon and is always observed on
air exposed samples. The survey spectrum of the pure organic
shell material, PTFS-GlcOH, shows C, O, F, and S as main
components, but no Fe in accordance with its elemental compo-
sition. All the elements mentioned before can be detected in the
sample of the coated particles, indicating that both components,
magnetic nanoparticles as well as shell material, contribute to the
spectrum. The comparison of all survey spectra reveals that the
Fe and O peak intensities of PTFS-GlcOH-coated particles
clearly decreased with respect to the pure nanoparticles, both
in terms of absolute and relative intensity. The change of the
relative C, F, O, and S peak intensities of the coated particles
is much less pronounced compared to the spectrum of the
pure shell material. Therefore, detected elements, relative peak
intensities, and different peak shapes in the elemental region
spectra imply that the magnetic nanoparticles are covered by the
shell polymer. Particularly the background shape adjacent to the
Fe2p emissions (Figure 5B) observed for the coated particles
suggests the presence of magnetic nanoparticles enclosed by
polymeric material rather than a mixture of both components.
The Fe2p spectra show the typical structure for iron oxides with a
broad, main, doublet peak (Fe2p3/2 and Fe2p1/2) and typical
shakeup satellites, particularly well visible in the spectrum of the
pure magnetic nanoparticles. The electron binding energy of the
Figure 4. TEM micrographs of the naked (A) and glycopolymer-coated
(B, C) iron oxide nanoparticles and a cryo-TEM image of the coated
particles (D).
magnetization (δs) is significantly lower for the modified parti-
cles due to the layer of the coating material, consistently to the
results previously obtained for cyclodextrin- and carboxymethyl
dextrane-functionalized iron oxide particles.64,65 Moreover, the
relative saturation remanence (δr/δs) is distinctly lower for the
coated particles, signifying that a stable aqueous suspension of
superparamagnetic particles was obtained.66 The above-men-
tioned properties allow a highly efficient magnetic manipulation
when used in bioseparation or as a drug carrier under relatively
low external magnetic field. In addition, these particles are possible
candidates for application as magnetic resonance contrast agents
in cellular and molecular imaging.67
v. Particle Characterization by TEM. To investigate differ-
ences in shape and architecture of the glycopolymer-coated par-
ticles, the structure of the obtained core-shell, superparamag-
netic particles was investigated. TEM investigations (Figure 4)
were used to determine the diameter of the individual iron nano-
particles, which revealed a typical size of less than 15 nm, both for
noncoated and coated particles. Compared to the typical diam-
eter of the particles measured by DLS, the individual iron
particles appear to be much smaller, indicating that the DLS
measurements provide the hydrodynamic diameter of particle
686
dx.doi.org/10.1021/bm101325w |Biomacromolecules 2011, 12, 681–691

Biomacromolecules
ARTICLE
Figure 5. Selected XPS spectra of the synthesized glycopolymer, coated, and naked particles.
Fe2p3/2 peak maximum of 711.1 eV and the peak shape, lacking
the distinctive low-binding energy (BE) shoulder observed
for Fe3O4, clearly point to Fe(III), thus, Fe2O3.68 Furthermore,
the Fe2p spectra, missing the high-BE shoulder reported for
R-Fe2O3 (hematite), suggest that the outer part of the nanopar-
ticles consist of γ-Fe2O3 (maghemite). This can be explained by
gradual oxidation of the initially formed Fe(II) moieties to
Fe(III). Hence, the spectra reveal the aging of the inorganic
nanoparticle surface. The Fe2p spectral features of the coated
particles are attenuated due to the coating of the particles by the
polymeric material suggesting a shell thickness of slightly below
10 nm. The almost constant Fe2p3/2 BE provides no indication
for a strong chemical interaction between particle and shell that
would result in a change of the chemical state. The C1s spectrum
(Figure 5C) of pure magnetic nanoparticles is typical for minor
surfaces contaminations always present on air-exposed surfaces.
The three discrete states of carbon can be attributed to aliphatic
C, functional groups with C-O single bonds, and to CdO
double bonds.69 The C1s spectrum region of the polymer
component is marked by a clear increase of the peak intensity.
687
dx.doi.org/10.1021/bm101325w |Biomacromolecules 2011, 12, 681–691

Biomacromolecules
ARTICLE
Table 2. Data of XPS Analysis of the Glycopolymer, the Naked, and the Coated Nanoparticles
C1s
O1s
F1s
S2p3/2
eV
Fe2p3/2
C-aliphatic
eV
C-O
C-F
eV
FeOx:
eV
-OH
eV
region
%
%
eV
%
%
%
%
eV
%
%
eV
naked
5.7
284.8
284.8
284.8
1.2
286.5
286.2
286.0
1.2
289.1
287.8
287.4
48.8
0.0
530.4
530.6
9.6
531.7
532.2
533.0
0.0
0.0
4.7
3.5
33.5
0.0
711.0
polymer
coated
23.8
15.7
28.4
24.1
6.8
20.2
16.2
16.2
13.2
687.1
688.1
163.5
163.9
18.0
7.6
1.8
711.1
Also, the intensity distribution for the different chemical states is
significantly altered. It is characterized by an increase of the C-O
intensity becoming the most populous state. It can be attributed
to the hydroxyl groups present in the carbohydrate units. Further-
more, another state appears in the C1s region of the glycopoly-
mer belonging to the C-F bond of the poly(tetrafluorostyryl)
groups. Since the spectrum envelope exhibits only little structure,
it was decided to evaluate the lowest possible number of fitted
states, which are at least indicated by the shape of the emission
peak. The small increase of the background intensity on the high-
BE side of the C1s peak system at about 293 eV is attributed to
shakeup satellites indicating the π-system present in the poly-
mer’s aromatic rings.70 The C1s spectrum of the coated particles
shows a similar shape, however, the relative intensity of the C-F
signal is increased markedly. This could point to a different
orientation or configuration of the polymeric chains in the shell
of the encased nanoparticles compared to the pure glycopolymer,
possibly indicating that the F-containing units are oriented to the
outer sphere of the shell and that the other polymeric units
provide the interaction with the inorganic iron oxide core. The
O1s spectrum (Supporting Information) of the inorganic nano-
particles is characterized by an asymmetric peak that can be
separated into two components by fit. The main component,
state 1, is attributed to O in Fe2O3; the minor component, state 2
on the high-BE side, to hydroxyl groups on the surface. The
presence of the shell polymer encapsulating the nanoparticles
leads in the O1s spectrum to a decrease of state 1 and an increase
of the relative intensity and the BE of state 2, resembling the
symmetric O1s emission of the pure organic shell material. The
F1s BE in the spectrum of the coated particles (Figure 5D) is
typical for F in organic polymers, with an exact value depending
on the degree of fluorination.70 The BE is higher than for the pure
polymeric material as it is observed for the respective O1s, state 2,
and S2p BEs, most likely due to an artifact introduced by
assigning the C1s low-BE component to 284.8 eV. The S2p
peak is only found in the spectra of the coated particles and the
pure glycopolymer (Supporting Information). The BEs clearly
indicate an organosulfidic -S- bond, which is assumed to facilitate
the interaction between the nanoparticles core and the polymeric
shell.69
Figure 6. Traces from the thermogravimetric analysis of the prepared
materials.
backbone, respectively. The analysis curve of the coated particles
shows the same decomposition temperatures as the glycopoly-
mer, thus, proving the presence of the PTFS-GlcOH in the analyzed
sample. From the remaining, nondegraded masses it can be
calculated that the analyzed weight percent ratio between iron
oxide and the coating material is approximately 1:5. This result is
in good correlation with the XPS investigations, where the
particle shell thickness was measured to be 10 nm.
vii. Flow Cytometry Analyses of the Superparamagnetic
Nanoparticles. Flow cytometry represents a powerful tool to
characterize and analyze particles. It provides information about
their size and shape from the forward and the side scattered light
as well as about the fluorescence of particles in the analyzed
sample.71 The naked and coated particles were resuspended by
sonication for 1 min at 10% amplitude, 30 min before analyses.
Subsequently, 50 μL of the ferrofluid was diluted in 2 mL of
Dulbecco’s modified Eagle medium (DMEM) and vigorously
agitated for 1 min. Afterward, the sample was directly analyzed by
flow cytometry. The results can be found in Figure 7 (for the forward
and side scattering plots, see the Supporting Information).
From the forward scattering traces, the size distribution of the
analyzed particles can be estimated. The naked nanoparticles
show a large size distribution of different agglomerates. The size
distribution of the coated particles is much narrower, therefore,
the polymer acts as the stabilizer, preventing agglomeration.
Because the glycopolymer applied in the coating procedure was
fluorescently labeled, the nanoparticles also show fluorescence
thus proving the presence of the polymeric shell. To examine the
stability of the ferrofluid, the coated particles were analyzed after
6 months storage at 8 ꢀC. After resuspension, the flow cytometry
analysis gave the same particle size distribution as for the freshly
prepared nanoparticles (Supporting Information).
vi. Thermogravimetric Analysis of the Glycopolymer-Coated
Nanoparticles. The obtained particle suspensions as well as the
polymeric shell material were dried under vacuum, and their
thermal decomposition behavior was analyzed. The resulting
thermogravimetric curves are presented in Figure 6.
The analysis of uncoated particles shows a total weight loss of
8% up to 900 ꢀC. This behavior can be attributed to the remaining
organic impurities and evaporation of water. The trace of the
thermal decomposition of PTFS-GlcOH has two clear inflection
points: one at 230 ꢀC and a second at 429 ꢀC, corresponding to
the degradation of the glucose moieties and the tetrafluorostyryl
688
dx.doi.org/10.1021/bm101325w |Biomacromolecules 2011, 12, 681–691

Biomacromolecules
ARTICLE
toward 3T3 mouse fibroblasts was examined in a live/dead assay,
using fluorescein diacetate (labeling living cells) and ethidium
bromide (labeling the nuclei of dead cells). As shown in Figure 8,
the addition of increasing amounts of the glycopolymer-coated
nanoparticles, from 0.2% (not shown since uniform to control)
to 1.0 and 5.0% (v/v), to the 3T3 fibroblast cells that were cultured
for 4 days did not elevate the percentage of dead cells, only rarely
observable as orange-stained nuclei, in comparison to a standard
cell culture.
The number of dead cells was below 5% indicating no
cytotoxic effects. The examined cells were adherent and showed
a normal cell morphology. The increase in the cell number from
about 20000-30000 cells cm-2 after 1 day to about 170000-
3
200000 cells cm-2 after 4 days occurred independently from the
3
amount of nanoparticles. Because no remarkable increase in cell
number occurred during the first 24 h after cell seeding and the
differences in adhesion are compensated after 4 h, the slightly
lower cell density of the control, visible in Figure 8a, should result
from random variations of the seeding procedure. This under-
lines that cell proliferation was not influenced by the presence of
the particles. Furthermore, the absence of contaminations, for
example, toxic residues of not converted reactants, catalysts, or
solvents from the synthesis and preparation techniques was
verified.
Figure 7. Results of flow cytometry analysis of the naked (A, C) and
FITC-labeled, glycopolymer-coated particles (B, D): forward scattering
(A, B); fluorescence (C, D).
’ CONCLUSIONS
In summary, these results clearly demonstrate the applicability
of glycopolymers based on poly(pentafluorostyrene) as stabiliz-
ing coating material of magnetic iron oxide nanoparticles. The
synthesis of the new core-shell nanoparticles was performed in
aqueous solution leading to stable ferrofluids. The polymeric
shell was investigated in detail by XPS measurements displaying
characteristic signals of the elements in the shell, and the magnetic
nanoparticles exhibited the expected superparamagnetic beha-
vior. Furthermore, flow cytometry measurements proved the
stability of the obtained ferrofluids and the fluorescence of the
nanoparticles. Investigations of the glycopolymer and the mag-
netic core-shell nanoparticles did not show any cytotoxic
effects, emphasizing their applicability in biology and medicine.
The double-labeled (magnetically and fluorescently) particles
can be used for visualization of cell-carbohydrate interactions
and magnetic separation investigations. In addition, the pre-
sented synthetic approach opens up a new, facile route for
production of well-defined, glycopolymer-based materials of high
purity and in large quantities. Further modifications of penta-
fluorostyryl monomers with various thiosugars and their poly-
merizations as well as copolymerizations are currently under
investigation in our laboratories to introduce various thiosac-
charide moieties into polymeric coatings of magnetic nanopar-
ticles.
Figure 8. Fluorescence microscopy micrographs of FDA/EtBr stained
3T3 cells cultured for 1 day and 4 days in the presence of the
glycopolymer-coated particles (obtained by image overlay of separately
captured red and green fluorescence images). Top: control culture
without particles, middle: 1% (v/v) particle suspension added, bottom:
5% (v/v) particle suspension added.
’ ASSOCIATED CONTENT
Supporting Information. 13C NMR and HSQC-DEPT
S
b
spectra (300 MHz, DMF-d7) of the synthesized monomers and
the glycopolymer. O1s and S2p region XPS spectra of the
analyzed materials. Forward and side scattering plots of the flow
cytometry analysis of FITC-labeled, PTFS-GlcOH-coated parti-
cles. Flow cytometry analysis results of the FITC-PTFS-GlcOH-
coated particles after a 6-month storage. This material is available
free of charge via the Internet at http://pubs.acs.org.
Investigation of Cytotoxicity. Exclusion of any cytotoxic
effects is a primary prerequisite for the application of the obtained
glycopolymer-coated particles in biology and medicine. Even if
the targeted cells are planned to be eliminated, for example,
tumor cells, no negative influence to the surrounding cells has to
be ensured. Therefore, the cytotoxicity of the obtained particles
689
dx.doi.org/10.1021/bm101325w |Biomacromolecules 2011, 12, 681–691

Biomacromolecules
ARTICLE
’ AUTHOR INFORMATION
(26) Sun, X. L.; Cui, W. X.; Haller, C.; Chaikof, E. L. ChemBioChem
2004, 5, 1593–1596.
(27) Yoo, M. K.; Kim, I. Y.; Kim, E. M.; Jeong, H. J.; Lee, C. M.;
Jeong, Y. Y.; Akaike, T.; Cho, C. S. J. Biomed. Biotechnol. 2007, Article ID
94740, 9 pages.
Corresponding Author
*E-mail: ulrich.schubert@uni-jena.de.
(28) Becer, C. R.; Babiuch, K.; Pilz, D.; Hornig, S.; Heinze, T.;
Gottschaldt, M.; Schubert, U. S. Macromolecules 2009, 42, 2387–2394.
(29) Becer, C.; Hoogenboom, R.; Schubert, U. Angew. Chem., Int. Ed.
2009, 48, 4900–4908.
(30) Gottschaldt, M.; Koth, D.; Muller, D.; Klette, I.; Rau, S.; Goerls,
H.; Schafer, B.; Baum, R. P.; Yano, S. Chem. Eur. J. 2007, 13, 10273–
10280.
’ ACKNOWLEDGMENT
We thank the Dutch Polymer Institute (DPI, Technologic
Area HTE) and the German Federal Ministry of Economics
and Technology (Contract Number VF080018) for financial
support.
(31) Wagner, K.; Kautz, A.; Roeder, M.; Schwalbe, M.; Pachmann,
K.; Clement, J. H.; Schnabelrauch, M. Appl. Organomet. Chem. 2004, 18,
514–519.
’ REFERENCES
(1) Winter, P. M.; Caruthers, S. D.; Kassner, A.; Harris, T. D.;
Chinen, L. K.; Allen, J. S.; Lacy, E. K.; Zhang, H.; Robertson, J. D.;
Wickline, S. A.; Lanza, G. M. Cancer Res. 2003, 63, 5838–5843.
(2) Wotschadlo, J.; Liebert, T.; Heinze, T.; Wagner, K.; Schnabelrauch,
M.; Dutz, S.; M€uller, R.; Steiniger, F.; Schwalbe, M.; Kroll, T. C.;
H€offken, K.; Buske, N.; Clement, J. H. J. Magn. Magn. Mater. 2009, 321,
1469–1473.
(3) Alexiou, C.; Jurgons, R.; Schmid, R. J.; Bergemann, C.; Henke,
J.; Erhard, W.; Huenges, E.; Parak, F. J. Drug Targeting 2003, 11,
139–149.
(32) Dreywood, R. Ind. Eng. Chem., Anal. Ed. 1946, 18, 499.
(33) Guiterrez Gallego, R.; Such-Sanmartin, G.; Segura, J. In Recent
Advances in Doping Analysis; Sch€anzer, W., Geyer, H., Gotzmann, A.,
Mareck, U., Eds.; Sport und Buch Strauss: K€oln, 2005; Vol. 13, p 383.
(34) Scott, T. A.; Melvin, E. H. Anal. Chem. 1953, 25, 1656–1661.
(35) Miyamoto, M.; Morimoto, Y.; Nozawa, Y.; Balamurugan, A. N.;
Xu, B. Y.; Inoue, K. Cell Transplant. 2000, 9, 681–686.
(36) Brauer, D. S.; Russel, C.; Vogt, S.; Weisser, J.; Schnabelrauch,
M. J. Biomed. Mater. Res., Part A 2007, 80, 410–420.
(37) Lindhorst, T. K. In Topics in Current Chemistry; Penadꢀes, S., Ed.;
Springer-Verlag: Berlin, Heidelberg, 2002; p 201.
(38) Spain, S. G.; Gibson, M. I.; Cameron, N. R. J. Polym. Sci., Part A:
Polym. Chem. 2007, 45, 2059–2072.
(4) Hergt, R.; Hiergeist, R.; Hilger, I.; Kaiser, W. A.; Lapatnikov, Y.;
Margel, S.; Richter, U. J. Magn. Magn. Mater. 2004, 270, 345–357.
(5) Mykhaylyk, O.; Antequera, Y. S.; Vlaskou, D.; Plank, C. Nat.
Protoc. 2007, 2, 2391–2411.
(39) Wang, Q.; Dordick, J. S.; Linhardt, R. J. Chem. Mater. 2002, 14,
3232–3244.
(40) Ting, S. R. S.; Chen, G.; Stenzel, M. H. Polym. Chem. 2010, 1,
(6) Purushotham, S.; Ramanujan, R. V. Acta Biomater. 2010, 6, 502–510.
(7) Sosnovik, D. E.; Nahrendorf, M.; Weissleder, R. Basic Res.
Cardiol. 2008, 103, 122–130.
(8) Shingel, K. I.; Marchessault, R. H. Iron-polysaccharide compo-
sites for pharmaceutical applications. In Polysaccharides for Drug Delivery
and Pharmaceutical Applications; Marchessault, R. H., Ravenelle, F., Zhu,
X. X., Eds.; American Chemical Society: Washington, DC, 2006; Vol.
934, pp 271-287.
(9) Earhart, C.; Jana, N. R.; Erathodiyil, N.; Ying, J. Y. Langmuir
2008, 24, 6215–6219.
(10) Purushotham, S.; Ramanujan, R. V. J. Appl. Phys. 2010, 107, 9.
(11) Li, G. L.; Zeng, D. L. M.; Wang, L.; Zong, B. Y.; Neoh, K. G.;
Kang, E. T. Macromolecules 2009, 42, 8561–8565.
(12) Mykhaylyk, O.; Steingotter, A.; Perea, H.; Aigner, J.; Botnar, R.;
Plank, C. J. Biomed. Nanotechnol. 2009, 5, 692–706.
(13) Sun, X. X.; Yang, G. L.; Sun, S. S.; Quan, R.; Dai, W. W.; Li, B.;
Chen, C.; Li, Z. Curr. Pharm. Biotechnol. 2009, 10, 753–760.
(14) Pera, N. P.; Kouki, A.; Haataja, S.; Branderhorst, H. M.;
Liskamp, R. M. J.; Visser, G. M.; Finne, J.; Pieters, R. J. Org. Biomol.
Chem. 2010, 8, 2425–9.
1392–1412.
(41) Okada, M. Prog. Polym. Sci. 2001, 26, 67–104.
(42) Voit, B.; Appelhans, D. Macromol. Chem. Phys. 2010, 211, 727–
735.
(43) Slavin, S.; Burns, J.; Haddleton, D. M.; Becer, C. R. Eur. Polym.
J. 2010, DOI: 10.1016/j.eurpolymj.2010.09.019.
(44) Becer, C. R.; Schubert, U. S. Adv. Polym. Sci. 2010, 225, 17–62.
(45) Becer, C. R.; Kokado, K.; Weber, C.; Can, A.; Chujo, Y.;
Schubert, U. S. J. Polym. Sci., Part A: Polym. Chem. 2010, 48, 1278–1286.
(46) Becer, C. R.; Paulus, R. M.; Hoogenboom, R.; Schubert, U. S.
J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 6202–6213.
(47) Ohno, K.; Fukuda, T.; Kitano, H. Macromol. Chem. Phys. 1998,
199, 2193–2197.
(48) Ohno, K.; Tsujii, Y.; Miyamoto, T.; Fukuda, T.; Goto, M.;
Kobayashi, K.; Akaike, T. Macromolecules 1998, 31, 1064–1069.
(49) Ohno, K.; Izu, Y.; Yamamoto, S.; Miyamoto, T.; Fukuda, T.
Macromol. Chem. Phys. 1999, 200, 1619–1625.
(50) Chen, Y.; Wulff, G. Macromol. Chem. Phys. 2001, 202, 3273–
3278.
(15) Wang, X.; Lin, J. M.; Ying, X. T. Anal. Chim. Acta 2007, 598,
261–267.
(51) Chen, Y.; Wulff, G. Macromol. Chem. Phys. 2001, 202, 3426–
3431.
(52) Narumi, A.; Satoh, T.; Kaga, H.; Kakuchi, T. Macromolecules
2002, 35, 699–705.
(53) Narumi, A.; Matsuda, T.; Kaga, H.; Satoh, T.; Kakuchi, T.
Polymer 2002, 43, 4835–4840.
(16) Harrington, P. R.; Vinje, J.; Moe, C. L.; Baric, R. S. J. Virol. 2004,
78, 3035–3045.
(17) Behrens, S.; B€onnemann, H.; Matoussevitch, N.; Dinjus, E.;
Modrow, H.; Palina, N.; Frerichs, M.; Kempter, V.; Maus-Friedrichs, W.;
Heinemann, A.; Kammel, M.; Wiedenmann, A. Z. Phys. Chem.
(Muenchen, Ger.) 2006, 220, 3–40.
(54) Miura, Y.; Koketsu, D.; Kobayashi, K. Polym. Adv. Technol.
2007, 18, 647–651.
(55) Ting, S. R. S.; Min, E. H.; Escale, P.; Save, M.; Billon, L.; Stenzel,
M. H. Macromolecules 2009, 42, 9422–9434.
(56) Wulff, G.; Schmid, J.; Venhoff, T. Macromol. Chem. Phys. 1996,
197, 259–274.
(57) Samaroo, D.; Vinodu, M.; Chen, X.; Drain, C. M. J. Comb.
Chem. 2007, 9, 998–1011.
(58) Pasetto, P.; Chen, X.; Drain, C. M.; Franck, R. W. Chem.
Commun. 2001, 81–82.
(59) Hoyle, C. E.; Lowe, A. B.; Bowman, C. N. Chem. Soc. Rev. 2010,
39, 1355–1387.
(18) van Berkel, K. Y.; Piekarski, A. M.; Kierstead, P. H.; Pressly,
E. D.; Ray, P. C.; Hawker, C. J. Macromolecules 2009, 42, 1425–1427.
(19) Pieters, R. J. Med. Res. Rev. 2007, 27, 796–816.
(20) Miura, Y. J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 5031–
5036.
(21) Coullerez, G.; Seeberger, P. H.; Textor, M. Macromol. Biosci.
2006, 6, 634–647.
(22) Ladmiral, V.; Melia, E.; Haddleton, D. M. Eur. Polym. J. 2004,
40, 431–449.
(23) Spain, S. G.; Cameron, N. R. Polym. Chem. 2010, 2, 60-68.
(24) Rye, P. D. BioTechnology (Rajkot, India) 1996, 14, 155–157.
(25) Rye, P. D.; Bovin, N. V. Glycobiology 1997, 7, 179–182.
690
dx.doi.org/10.1021/bm101325w |Biomacromolecules 2011, 12, 681–691

Biomacromolecules
ARTICLE
(60) Wilson, J. C.; Kiefel, M. J.; Angus, D. I.; von Itzstein, M. Org.
Lett. 1999, 1, 443–446.
(61) Driguez, H. ChemBioChem 2001, 2, 311–318.
(62) Shen, H.; Byers, L. D. Biochem. Biophys. Res. Commun. 2007,
362, 717–720.
(63) Str€ahle, J.; Schweda, E. Jander/Blasius. Einf€uhrung in das
Anorganisch-Chemische Praktikum.; S. Hirzel Verlag: Stuttgart-Leipzig,
1995; Vol. 14.
(64) Cao, H. N.; He, J.; Deng, L.; Gao, X. Q. Appl. Surf. Sci. 2009,
255, 7974–7980.
(65) Wagner, K.; Seemann, T.; Wyrwa, R.; Clement, J. H.; M€uller,
R.; Nietzsche, S.; Schnabelrauch, M. Am. Inst. Phys. Conf. Proc. 2010,
1311, 28–33.
(66) Gossuin, Y.; Gillis, P.; Hocq, A.; Vuong, Q. L.; Roch, A. Wiley
Interdiscip. Rev.-Nanomed. Nanobiotechnol. 2009, 1, 299–310.
(67) Bulte, J. W. M.; Kraitchman, D. L. NMR Biomed. 2004, 17, 484–
499.
(68) Grosvenor, A. P.; Kobe, B. A.; Biesinger, M. C.; McIntyre, N. S.
Surf. Interface Anal. 2004, 36, 1564–1574.
(69) Briggs, D. Surface Analysis of Polymers by XPS and Static SIMS;
Cambridge University Press: New York, 1998.
(70) Beamson, G.; Briggs, D. High Resolution XPS of Organic
Polymers. The Scienta ESCA300 Database; John Wiley and Sons: New
York, 1992.
(71) Siiman, O.; Jitianu, A.; Bele, M.; Grom, P.; Matijevic, E.
J. Colloid Interface Sci. 2007, 309, 8–20.
691
dx.doi.org/10.1021/bm101325w |Biomacromolecules 2011, 12, 681–691