Nanostructured thermosensitive polymers with radical scavenging ability

Article abstract of DOI:10.1002/chem.200600590

The thermosensitive [60]fullerene end-capped poly(N-isopropylacrylamide) was successfully synthesized by the reaction of C₆₀ with dithiobenzoate-terminated poly(N-isopropylacrylamide), which was prepared by reversible addition-fragmentation cha

Full text of DOI:10.1002/chem.200600590

FULL PAPER
DOI: 10.1002/chem.200600590
Nanostructured Thermosensitive Polymers with Radical Scavenging Ability
Guangchang Zhou,[a] Issifu I. Harruna,[a] Weilie L. Zhou,[b] Wilhelm K. Aicher,[c] and
Kurt E. Geckeler*[d]
Abstract:
The
thermosensitive
mined by FTIR, UV/Vis, and carbon
and proton NMR spectroscopy as well
as by size exclusion chromatography
(SEC). The novel fullerenated polymer
retained the thermosensitivity of
poly(N-isopropylacryl
amide).
More-
[60]fullerene end-capped poly(N-iso-
propylacrylamide) was successfully syn-
thesized by the reaction of C60 with di-
over, it is soluble in water and most of
the common organic solvents. Interest-
ingly, it was able to form nanoparticle
clusters in methanol and exhibited sig-
nificant radical scavenging ability in
cell viability and metabolic activity
tests with fibroblasts and NOR-3 radi-
cals.
thiobenzoate-terminated
poly(N-iso-
propylacrylamide), which was prepared
by reversible addition-fragmentation
chain-transfer (RAFT) polymerization
in the presence of azobisisobutyroni-
trile (AIBN). Its structure was deter-
Keywords: fullerenes · nanostruc-
tures · polymerization · radical scav-
enging
Introduction
Therefore, they have to be chemically modified and a solu-
bilizing appendage covalently attached.[5] Several ap-
proaches have been explored for the introduction of ful-
lerenes into an aqueous or polar environment.[3,4] For exam-
ple, water-soluble fullerene carboxylic acid derivatives, ful-
One of the most promising applications of the fullerenes is
based on their biological properties. Very exciting findings
pioneered by Wudl,[1] Nakamura,[2] and co-workers have
stimulated much interest and generated further successful
investigations.[3,4] From a practical point of view, a funda-
mental issue that must be addressed when dealing with ful-
lerenes is the solubilization of the candidate molecule in
polar media that are necessary for biological tests.[3,4] Ful-
lerenes are not soluble in water or in polar organic solvents.
A
tives have been prepared and used in studying the
biochemical and medicinal activities of fullerenes.[3–7] In par-
ticular, the introduction of C60 into water-soluble polymers
is one of the simple and useful methods for solubilizing the
C60 in water. Therefore, some C60-containing water-soluble
polymers have been prepared for potential biomedical appli-
cations.[8]
[a] Dr. G. Zhou, Prof. Dr. I. I. Harruna
Poly(N-isopropylacrylamide) (PNIPAM) is a representa-
tive temperature-responsive polymer that exhibits a rapid
and reversible hydration–dehydration change in response to
small temperature cycles around its lower critical solution
temperature (LCST; 328C) in aqueous media.[9–13] In recent
years, considerable research attention has thus been focused
on PNIPAM-based polymers due to their potential biomedi-
cal and pharmaceutical applications, including controlled
drug delivery,[9] molecular separation,[10] tissue culture sub-
strates,[11] enzyme activity controlling systems,[12] and materi-
als for improved biocompatibility.[13] However, to the best of
our knowledge, there are no reports regarding C60-contain-
ing PNIPAM thermosensitive polymers. Therefore, here we
report the synthesis, characterization, thermosensitivity, col-
loidal behavior, and radical scavenging properties of
PNIPAM polymers with [60]fullerene end-caps.
Department of Chemistry, Clark Atlanta University
223 James P. Brawley DR SW, Atlanta, GA 30314 (USA)
[b] Dr. W. L. Zhou
Advanced Materials Research Institute
University of New Orleans, New Orleans, LA 70148 (USA)
[c] Prof. Dr. W. K. Aicher
Basic Science Research Laboratories
Center of Orthopaedic Surgery
Eberhard Karls University Tübingen, ZMF
Waldhoernle Strasse 22, 72072, Tübingen (Germany)
[d] Prof. Dr. K. E. Geckeler
Department of Materials Science and Engineering
Gwangju Institute of Science and Technology (GIST)
1 Oryong-dong, Buk-gu, Gwangju 500-712 (South Korea)
Fax : (+82)62-970-2338
E-mail: keg@gist.ac.kr
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2007, 13, 569 – 573
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Results and Discussion
Thus, the reaction between C60 and the dithiobenzoate-ter-
minated PNIPAM was carried out in such a mixed solvent
system at 808C in the presence of the initiator AIBN for
48 h. The resulting reaction mixture was separated to give
the [60]fullerene end-capped poly(N-isopropylacrylamide)
polymer (PNIPAM-C60). PNIPAM-C60 dissolves in water
and methanol, in addition to some organic solvents such as
THF.
The UV/Vis absorption spectrum (Figure 1A) of the aque-
ous solution of PNIPAM-C60 is clearly different from that of
the starting PNIPAM. A new absorption peak at 260 nm,
which is a characteristic absorption for C60, appeared in the
spectrum of PNIPAM-C60. This implies that a covalent link
has been formed between the PNIPAM and the C60. No
other specific band could be detected apart from a new very
long tail stretching over several 100 nm. The FTIR spectrum
(Figure 1B) of PNIPAM-C60 showed the appearance of two
new weak peaks at 2240 and 527 cmÀ1, attributed to the
characteristic peaks of the 2-cyano-2-propyl group and the
functionalized C60 cage, respectively. The remaining peaks
are essentially the same as those of the parent PNIPAM. In
the 13C NMR spectrum (Figure S1) of PNIPAM-C60, a new
broad band at d=135–160 ppm (fullerene aromatic carbons)
appeared. However, its 1H NMR spectrum (Figure S2) is
very similar to that of the parent PNIPAM.
Reversible addition-fragmentation chain-transfer (RAFT)
polymerization is a radical polymerization technique that
controls molecular weight and molecular-weight distribution
of polymers. Interestingly, it has been demonstrated that the
RAFT-prepared polymers bear functional end groups (-S-
C(S)-Ph)[14,15] that are able to further lead to polymerization
of monomers in the presence of small amounts of initiators.
More recently, Gilbert[14] and Müller[15] independently pre-
pared PNIPAM with different molecular weights by means
of RAFT polymerization and confirmed that each PNIPAM
chain bears a (-S-C(S)-Ph)-functional endgroup by MALDI-
TOF-MS. Similarly, we synthesized PNIPAM homopolymers
using benzyl dithiobenzoate as the chain-transfer agent and
azobisisobutyronitrile (AIBN) as the initiator by means of
RAFT polymerization. Thus, it can be deduced that our pre-
pared PNIPAM polymer also bears an active functional
group (-S-C(S)-Ph) at one end of the chains.
It is known that the addition reaction between C60 and
free radicals is easily carried out to form fullerenated poly-
mers.[16] According to the RAFT polymerization mechanism
elucidated by the MALDI-TOF-MS technique,[14,15] the ad-
dition of 2-cyano-2-propyl radicals formed by the decompo-
sition of the initiator AIBN to the C=S part of the end-
group (-S-C(S)-Ph) will result in the formation of macromo-
lecular PNIPAM radicals. The macromolecular radicals
easily add to the molecules of C60 to give the less active C60-
PNIPAM adduct radical. The new adduct radical will be ter-
minated by combination with the other free radicals and/or
abstracting a hydrogen atom. The proposed reaction mecha-
nism is shown in Scheme 1.
The size exclusion chromatographic (SEC) trace (Fig-
ure 1C) of PNIPAM-C60 recorded by refractive index (RI) is
almost the same as that of the parent PNIPAM, the only dif-
ference being that the elution time of PNIPAM-C60 is a little
shorter than that of PNIPAM. This also indirectly confirmed
that the C60 moiety was incorporated into one end of the po-
lymer chains. The number-average molar mass and polydis-
We found that an N,N-dimethylformamide–chlorobenzene
mixture is a good solvent system for both C60 and PNIPAM.
persity index of PNIPAM-C60 resulted in Mn,SEC
12400 gmolÀ1 and Mw/Mn =1.22. However, as observed in a
previous work,[17] there is
=
a
marked deviation between the
SEC-determined and theoreti-
cal
molecular
weights
(Mn,theory =23590 gmolÀ1). The
main cause for this discrepancy
is that polystyrene standards
were used for calibration.
The temperature-dependent
transmittance changes of aque-
ous solutions of PNIPAM-C60
and PNIPAM are shown in
Figure 2 (left). The aqueous so-
lution of PNIPAM-C60 was
transparent at lower tempera-
ture, while the transmittance of
the solution changed drastically
from transparent to turbid
above characteristic tempera-
tures. The PNIPAM-C60 solu-
tion exhibited the temperature-
induced phase transition be-
tween the hydrated and dehy-
Scheme 1. Proposed mechanism for the reaction of dithiobenzaote-terminated poly(N-isopropylacrylamide)
homopolymer with [60]fullerene.
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Chem. Eur. J. 2007, 13, 569 – 573

Fullerenes
FULL PAPER
drated states of the polymer. As can be seen in Figure 2, the
lower critical solution temperature (LCST) of PNIPAM-C60
is the same as that of the parent PNIPAM.
The temperature-dependent amphiphilic PNIPAM-C60 po-
lymer can undergo self-association by a hydrophobic C60
cage, leading to the formation of a micellar structure in an
aqueous phase at temperatures lower than its LCST. The
characteristics of the PNIPAM-C60 polymer micelles in the
aqueous phase were investigated by using dynamic light-
scattering measurements. The particle-size distribution is
presented in Figure 2 (right). Interestingly, the PNIPAM-C60
polymer is able to form nanoparticle clusters in methanol.
Figure 3 shows the TEM image of particles formed in meth-
anol.
Figure 1. A) UV/Vis spectra, B) FTIR spectra, and C) SEC curves of the
[60]fullerene end-capped PNIPAM (a), and the dithiobenzoate-terminat-
ed PNIPAM (b).
Figure 3. Transmission electron micrograph of the particles formed by
PNIPAM-C60 in methanol.
PNIPAM-C60 could have potential biomedical applications
including the treatment of acute or chronic inflammation.
CÀ
Different cells are known to produce radicals, such as NO
CÀ
and O2 , which harm biomolecules and affect the viability,
replication, and the regulatory and biosynthetic functions of
cells. However, fullerenes and their derivatives are known
to have a radical scavenging ability.[7] The effects of radicals
on the cellular metabolism and viability were mimicked by
CÀ
the addition of NOR-3, a potent NO radical donor, to
NIH3T3 murine fibroblasts according to previous experi-
mental procedures.[18] As observed in Figure 4, the NOR-3
radicals significantly reduced the cell viability and metabolic
activity (p=0.001). The addition of PNIPAM-C60 ameliorat-
ed the NO effects and significantly increased the cell via-
bility and metabolic activity (p<0.02). Also, the addition of
Figure 2. Temperature dependence of optical transmittance changes for
PNIPAM-C60 and dithiobenzaote-terminated PNIPAM polymers (all
sample solutions contained 10À2 wt% polymers in water) (left), and size
distribution of the particles formed by [60]fullerene end-capped
PNIPAM in water at room temperature (the diameter is plotted on a log-
arithmic scale) (right).
CÀ
PNIPAM-C60 did not reduce the cell viability or metabolic
Chem. Eur. J. 2007, 13, 569 – 573
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571

K. E. Geckeler et al.
mixture was poured into a large excess of diethyl ether to precipitate the
resulting polymer. The polymer was purified by reprecipitation from 1,4-
dioxane into a large excess of diethyl ether three times, and then dried
under vacuum at room temperature to yield 6.368 g of an orange poly-
mer. The conversion of the monomer styrene was determined to be
42.45% by gravimetry. The molecular weight of the dithiobenzoate-ter-
minated homopoly(N-isopropylacrylamide) polymer was determined by
SEC. Also, its theoretical molecular weight was calculated according to
the equation:
M
n,theory =([M]i/[CTA]i)fM0 +MCTA, in which [M]i and
A
[CTA]i are the initial concentrations of the monomer NIPAM and the
transfer agent BTB, respectively, f is the fractional conversion, and M0
and MCTA are the molecular weights of the monomer NIPAM and the
used RAFT agent BTB. SEC: Mn,SEC =11500, Mw =14500, Mw/Mn =1.26;
M
n,theory =23590.
Synthesis of [60]fullerene end-capped poly(N-isopropylacrylamide)
(PNIPAM-C60): Dithiobenzoate-terminated PNIPAM (500.0 mg, 2.12”
Figure 4. Viability index (vi, y axis) and metabolic activity of fibroblasts
in the presence of PNIPAM-C60 and/or radical NOR-3 (controls repre-
sent the addition of neither PNIPAM-C60 nor NOR-3). The data show
the mean value Æ standard deviations (n=8 individual samples) of one
representative experiment. For statistical analysis a two-sided T-test was
performed (MSExcelꢂ) and p values <0.05, <0.01, or <0.001 were con-
sidered to be significantly different and marked in the artwork according-
ly (*, **, ***).
10À2 mnmol based on the molecular weight of Mn,theory =23590 gmolÀ1
,
Mw/Mn =1.26), C60 (150.0 mg, 20.81”10À2 mmol), and AIBN (30.0 mg,
18.27”10À2 mmol) were dissolved in the mixed solvent (90/10, v/v%) of
chlorobenzene and N,N-dimethylformamide (50 mL). The reaction mix-
ture was degassed through four freeze–thaw–evacuate cycles, sealed
under vacuum, and then heated at 808C for 48 h. The resulting mixture
was precipitated into a large excess of diethyl ether to give a crude prod-
uct (0.590 g). The crude product was added into an Erlenmeyer flask
with methanol (1000 mL). The methanolic mixture was stirred at room
temperature for 2 h and was left to stand overnight. The insoluble solid
was filtered off (the unreacted C60 and adduct of C60 with 2-cyano-2-
propyl radicals). The filtrate was evaporated under reduced pressure to
dryness to give a methanol-soluble crude product (0.500 g). The metha-
nol-soluble crude product was dissolved in of THF (125 mL) to give an
unclear solution, and then the solution was centrifuged to remove trace
insoluble residue, and a clear solution was obtained. After removing
THF under reduced pressure, the methanol-soluble crude product
(0.474 g) was obtained and further purified according to the following
typical procedure. A specific weight ratio cw =1.226 in a methanol–water
mixture at room temperature was first determined under which the pre-
cursor PNIPAM homopolymer is just soluble, then the crude product was
dissolved in MeOH in advance to give a solution, then a suitable amount
of water was added dropwise into the polymer solution (the amount of
water added can be calculated using cw and the amount of MeOH used,
in this case, 2.00 g of MeOH vs. 1.600 g of H2O). The methanol–water
mixture solution was vigorously shaken, and centrifuged to give two
phases. The upper phase, which contained unreacted PNIPAM, was re-
moved, and then the same amounts of MeOH and water were added in
turn into the brown lower phase, and shaken and centrifuged again. The
procedure was repeated three times. The lower brown phase was dried to
give 0.275 g of pure product. Yield: 53%.
activity; the changes in comparison to nontreated controls
were statistically not significant. Although PNIPAM-C60
shows the capability to enhance significantly the cell viabili-
ty and metabolic activity, its full pharmacological potentials
as well as the general toxic effects are under investigation.
Conclusion
The thermosensitive [60]fullerene end-capped poly(N-iso-
propylacrylamide) polymer was successfully prepared by
means of reversible addition-fragmentation chain-transfer
(RAFT) polymerization and further characterized by FTIR,
UV/Vis, and NMR spectroscopy, and by size exclusion chro-
matography. The new fullerenated polymer retains the ther-
mosensitivity of the poly(N-isopropylacrylamide). More-
over, it dissolves in common solvents such as water and
methanol. It is able to form nanoparticle clusters in metha-
nol and also exhibits a significant radical scavenging ability
as demonstrated by cell viability and metabolic activity tests
with fibroblasts and NOR-3 radicals.
Characterization: 1H and 13C NMR spectra were recorded on a Varian
Unity Inova instrument (500 MHz) in CDCl3, with tetramethylsilane as
the internal reference. FTIR spectra were recorded on KBr slide in trans-
mission mode on a Nicolet Impact 400 spectrometer. UV/Vis spectra and
optical density measurements were conducted on a Cary UV/Vis spectro-
photometer (Cary Varian Optical Spectroscopy Instruments). Size exclu-
sion chromatography (SEC) was carried out on a Viscotek SEC assembly
consisting of a model P1000 pump, a model T60 dual detector, a model
LR40 laser refractometer, and three mixed-bed columns (10 cm) by using
THF as eluent with a flow rate of 0.70 mLminÀ1. Polymer concentrations
Experimental Section
Materials: 2,2’-Azobis(isobutyronitrile) (AIBN; 97%, Aldrich) was puri-
fied by recrystallization from methanol. N-Isopropylacrylamide (NIPAM;
97%, Aldrich), was purified by multiple recrystallization from a mixture
(60/40, v/v%) of toluene and hexane. The chain transfer agent, benzyl di-
thiobenzoate (BTB), was prepared by the ester interchange reaction be-
tween carboxymethyl dithiobenzoate [s-(thiobenzoyl)thioglycolic acid]
and benzyl mercaptane.[19]
for SEC experiments were prepared in
a concentration of about
3 mgmLÀ1. The SEC system was calibrated by using narrow standards
prior to use. The polystyrene standards were purchased from American
Polymer Standards Corp. The particle size and distribution were deter-
mined using laser scattering technique (DynaPro 99 Molecular Sizing In-
strument). TEM measurements were performed on a Philips CM12 trans-
mission electron microscope operated at 100 kV. Samples for TEM were
deposited on Cu-grid and allowed to air dry.
Synthesis of dithiobenzoate-terminated homopoly(N-isopropylacryl-
amide) by RAFT polymerization: NIPAM (14.847 g, 131.2 mmol), AIBN
(20.0 mg, 0.122 mmol), and BTB (65.9 mg, 0.270 mmol) were dissolved in
1,4-dioxane (32 mL) to give a clear solution. The solution was transferred
into an ampoule and degassed through five freeze–thaw–evacuate cycles,
sealed under vacuum, and heated at 608C for 24 h. The polymerization
Cell viability and metabolic activity tests: The NIH3T3 fibroblasts were
inoculated in 96-well plates at a starting density of 3000 cells per well in
Dulbeccoꢁs modified Eagle medium (DMEM) enriched with 10% bovine
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Fullerenes
FULL PAPER
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1.25 mgmLÀ1), NOR-3 solution (Calbiochem, in DMSO ad 250 mm) or
both were added to the cells and mixed. Nontreated cells served as con-
trols. The cells were incubated in a cell incubator (humidified controlled
atmosphere, 10% CO2) overnight and the metabolic activity was mea-
sured by the MTT assay as described.[18] The data show the mean value
Æ standard deviations (n=8 individual samples) of one representative
experiment. For statistical analysis a two-sided T-test was performed
(MSExcelꢂ) and p values <0.05, <0.01, or <0.001 were considered to be
significantly different and marked in the artwork accordingly (*, **, ***
in Figure 4).
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Received: April 28, 2006
Published online: October 10, 2006
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