PAPER www.rsc.

org/materials | Journal of Materials Chemistry

Efficiency of singlet oxygen production from self-assembled nanospheres of

molecular micelle-like photosensitizers FC4S
Chi Yu,a Taizoon Canteenwala,b Mohamed E. El-Khouly,c Yasuyuki Araki,c Kenneth Pritzker,a Osamu Ito,c
Brian C. Wilsond and Long Y. Chiang*b
Received 10th January 2005, Accepted 17th March 2005
First published as an Advance Article on the web 29th March 2005
DOI: 10.1039/b500369e

Direct detection of singlet oxygen (1O2) production under irradiation of molecular micelle-like
hexa(sulfo-n-butyl)[60]fullerene (FC4S) self-assembled nanospheres at 500–600 nm was obtained
by the measurement of its near-infrared luminescence at 1270 nm. This photocatalytic effect
makes FC4S a potential alternative sensitizer to TiO2 and feasible for use in the visible region in
addition to its intrinsic high UV efficiency. Despite having a relatively low optical absorption of
FC4S at 600 nm, appreciable 1O2 signal was detected comparable to that of hematoporphyrin
derivatives Photofrin at the same molar concentration, but less than sulfonated aluminium
phthalocyanine, AlS4Pc. The quantum yield of FC4S for the generation of 1O2 in H2O was
roughly estimated to be 0.36 using the relative correlation to that of C60/c-CD. The absolute value
is not available. These results demonstrated efficient triplet energy transfer from 3FC4S* to
molecular oxygen in the nanosphere structure. We also confirmed certain retention between
photocatalytic characteristics of underivatized C60 with that of FC4S, a C60 hexaadduct
containing a single covalent bond between each addend and the fullerene cage, in contrast to other
Bingel-type hexamalonate adducts of C60 in the literature with a low 1O2 yield.

Introduction to other malonic hexaadducts of C60 synthesized by Bingel

High photostability and oxidative power makes titanium
dioxide (TiO2) a good photocatalytic semiconductor for
use as a photodynamic agent in destroying cancer cells and
viruses.1 However, its relatively large band gap of 3.2 eV for the
anatase form makes TiO2 photoactive only at light wave-
lengths shorter than 380 nm which utilizes less than 3% of the
solar irradiance.2
Photoexcitation of C60 and fullerene derivatives induces a
singlet fullerenyl excited state that is transformed to the
corresponding triplet excited state, via intersystem energy
crossing, with nearly quantitative efficiency.3,4 Subsequent
energy transfer from the triplet fullerene derivatives to
molecular oxygen produces singlet molecular oxygen in
aerobic media. This photocatalytic effect becomes one of
the mechanisms in photodynamic treatments using fullerene
derivatives as photosensitizers,5–8 complementary to TiO2.
However, a high degree of functionalizaton on C60 for
enhancement of solubility and compatibility results in a
progressive decrease of the singlet oxygen production quantum
yield [W(1O2)]. Examples were given by Bingel-type malonic
acid, C60[C(COOH)2]n, and malonic ester, C60[C(COOEt)2]n,
[60]fullerene adducts,9 showing a decreasing trend of W(1O2)
as the number of addends (n) increases. When the number
n reaches six for a hexaadduct, its W(1O2) value declines to
only 13% or less of that for C60.10 Our unexpected observation
of relatively high singlet oxygen production quantum yield
of hexa(sulfo-n-butyl)[60]fullerenes (FC4S), as a fullerene
hexaadduct, revealed its unique electronic features in contrast
*Long_Chiang@uml.edu
reaction.
Fullerene molecules are highly hydrophobic. It can be
dispersed into an aqueous medium in a micelle form with the
application of surfactants. For increasing the structural
stability in H2O, we made direct chemical conversion of C60
into its water-soluble derivatives FC4S. The synthesis involves
recently developed hexaanionic C60 (C6026) chemistry for
attaching six surfactant-like sulfobutyl arms on C60 forming a
molecular micelle-like structure.11,12 This facilitates the utility
of fullerenes in vitro showing a free radical scavenging effect in
an aqueous medium. Accordingly, FC4S self-assembled nano-
spheres exhibit biological activities for several disease-related
treatments.13 The use of FC4S nanospheres as photodynamic
therapeutic (PDT) photosensitizers was reported by in vitro
and in vivo antitumoral activities.8 The observed PDT efficacy
was proposed in correlation with the presence of several
reactive oxygen species (ROS) generated photochemically.
One of these, superoxide radical (O22?), was detected in media
containing FC4S and ferricytochrome (Fe+3) under photo-
excitation and confirmed by trapping of it by superoxide
dismutase (SOD) enzyme. The production of O22? in media
increases the optical absorption of ferrocytochrome (Fe+2) at
550 nm. The method can be used for indication of O22?
generation. An electron-transfer mechanism involving the
excited state of FC4S, molecular oxygen, and donor residues
of the cytochrome protein was proposed for the presence of
O22?.8 Here, we report direct evidence of efficient singlet
oxygen (1O2) production under irradiation of the FC4S self-
assembled nanospheres at 500–600 nm by the detection of
1
O2 emission at 1270 nm. The observation allows utilization
of FC4S to harvest light energy extending from UV to

This journal is ß The Royal Society of Chemistry 2005 J. Mater. Chem., 2005, 15, 1857–1864 | 1857

Fig. 1 Chemical composition of hexa(sulfo-n-butyl)[60]fullerene 1
and protonated FC4S 2.
the visible wavelength region for performing photocatalytic
effects.
Materials and methods
General
Photofrin was obtained from QLT Phototherapeutics,
Vancouver, Canada. Tetrasulfonated aluminium phthal-
ocyanine (AlS4Pc) was obtained from Porphyrin Products,
Utah, USA. Phosphate buffered saline (PBS) was purchased
from Sigma, St. Louis. All solvents including methanol,
dimethyl sulfoxide (DMSO), and N,N-dimethylformamide
(DMF) were purchased from Merck, Darmstadt, Germany.
Synthesis of hexa(sulfo-n-butyl)[60]fullerene (FC4S, 1)
A direct one-pot method was used for the preparation of
micelle-like fullerene derivatives. Thus, water-soluble hexa-
(sulfo-n-butyl)[60]fullerene, C60(CH2CH2CH2CH2SO3Na)6 as
shown in Fig. 1, was synthesized at 80–85% yield by treating
C60 in dimethoxyethane (DME) with sodium naphthalenide
(10.0 equiv.) at 25 uC, followed by reacting the resulting
hexaanionic fullerene intermediates, C6026, with an excess
of 1,4-butane sultone (15.0 equiv.).11 After purification by
filtration and repeated reprecipitation in MeOH from an
aqueous solution, the FC4S product gave a single HPLC
peak using a reverse-phase C-18 column eluted with H2O.
Acidification of FC4S with HCl (4 N) gave the corresponding
hexasulfonic acid (FC4SH, 2). The solution of sodium
naphthalenide in DME was titrated by succinic acid prior to
use. The hexaanionic C60 generation reaction was carried out
inside a glove-box conditioned to ,50 ppm O2 to minimize
partial oxidation or oligomerization of fullerenic anions by
dissolved O2. A slight excess of sodium naphthalide was found
to be necessary to achieve the generation of hexaanionic
fullerene intermediates. Similar reaction conditions were also
carried out using a hindered halide instead of 1,4-butane
sultone in reaction with C6026 to yield a highly symmetrical
hexaadduct of C60 with each addend linked on the fullerene
cage by a single covalent bond.{12
Optical absorption measurements
Optical absorption spectra (250–850 nm) were determined in
a quartz cuvette with a UV–vis spectrophotometer (Model
UV160U, Shimadzu, Kyoto, Japan), with the photosensitizers
dissolved in different solvents, including phosphate buffered
{ The structure of one hindered product, denoted emerald green
fullerene EF-6MC4, was identified by the X-ray single crystal
structural analysis and 13C NMR spectroscopic methods to reveal a
highly symmetrical structure and a single covalent bond between each
addend arm and the connecting carbon of the C60 cage.
1858 | J. Mater. Chem., 2005, 15, 1857–1864
saline (PBS), distilled water, methanol, dimethyl sulfoxide, and
dimethylformamide.
Detection of singlet oxygen
Direct measurement of singlet oxygen was carried out by
the detection of its luminescence emission at 1270 nm,
corresponding to 1Dg A 3S2 g transition.
14
The measurement
was accomplished using a photon multiplier tube (PMT)
detector (Model R5509-42, Hamamatsu Corp., Bridgewater,
NJ) with high near-IR sensitivity. The laser instrumentation
and technique were described in detail elsewhere.15 Concisely,
four bandpass filters (1200, 1270, 1300, and 1330 nm) placed
sequentially in front of the detector were used for measure-
ments of the luminescence spectrum. The PMT output was
amplified and converted to a voltage pulse using a high-speed
current preamplifier (Model SR445, Stanford Research
Systems, Sunnyvale, CA). A multichannel scaler (Model
SR430, Stanford) was applied for time-resolved single photon
counting, giving a typical temporal resolution of 80 ns.
Samples of photosensitizer (10 mM) were irradiated by a
tunable pulsed laser system (OPO Rainbow 355, OPOTEK
Inc., Carlsbad, CA), with an irradiation spot size of 3.0 mm
in diameter. The pulse energy applied at the sample was
approximately 1.0 mJ. Emission from the sample at each
detection wavelength was recorded as total photon counts over
2400 laser pulses. Time-resolved measurements were made
using a laser with 20 ns pulse length and 100 ms pulse interval.
Transient absorption measurements
Collection and analysis of nanosecond transient absorption
spectra in the near-IR region (600–1600 nm) were made using
third-harmonic generation (HG, 355 nm) of a Nd–YAG laser
(Spectra-Physics, Quanta-Ray GCR-130, fwhm 6 ns) as an
excitation light source. Light intensity from a pulsed Xe
lamp was monitored and detected with a Ge-APD device
(Hamamatsu Photonics, B2834). For measurement of the
spectrum in the visible region of 400–1000 nm, a Si-PIN
photodiode (Hamamatsu Photonics, S1722-02) was used as the
detector. Triplet lifetime was estimated by using a photo-
multiplier (Hamamatsu Photonics). All the samples in a quartz
cell (1.0 6 1.0 cm) were deaerated by bubbling Ar through the
solution for 15 min.
Results and discussion
Chemical composition of FC4S was determined by various
spectroscopic methods. It was confirmed by elemental analyses
giving a hydrated hexasulfobutylated fullerene sodium salt
product with the weight ratio of each element summarized as
follows. Calculated for C84H88O38S6Na6 as C60(CH2CH2CH2-
CH2SO3Na)6?20H2O: C, 50.45; H, 4.20; O, 28.82; S, 9.60; Na,
6.91, Found: C, 49.56; H, 4.33; S, 9.44; Na, 6.78, where
analysis of sodium was carried out on a Varian Spectro
AA-100 plus analyzer.
Direct mass spectroscopic (MS) measurement of water-
soluble fullerene derivatives was difficult in general with an
extremely low intensity of the molecular mass ion peak
obtainable in most experiments. The use of non-volatile polar
This journal is ß The Royal Society of Chemistry 2005

Fig. 2 Negative ion desorption chemical ionization (DCI2) mass
spectrum of FC4S showing a series of fragmentation groups with up to
six consecutive weight increases of m/z 136, the mass of a sulfo-n-butyl
group, confirming the molecular composition of 1 and 2.
solvent was also problematic due to slow and inefficient
solvent removal after deposition on the probe prior to the MS
measurement. After evaluating various MS detection methods,
we were able to obtain adequate results by desorption chemical
ionization (DCI) and matrix-assisted laser desorption ioniza-
tion (MALDI). The negative ion DCI mass spectrum (DCI2-
MS) of FC4S, as shown in Fig. 2, displayed a maximum
relative intensity at a peak of the C60 ion fragmentation (m/z
720). This was followed by groups of peaks at m/z 776, 832,
887 and 944, having a consecutive weight increase of 56 mass
units that matches the gain of a butyl group to the preceding
ion fragment, corresponding to C60Bu1, C60Bu2, C60Bu3, and
C60Bu4, respectively. In the higher mass region, a series of
fragmentation groups was observed, with a consecutive
weight increase of 136 mass units, corresponding to a
sulfobutyl group (CH2CH2CH2CH2–SO3). The ion fragment
masses agree with those of di-, tri-, tetra-, penta- and hexa-
sulfobutylated fullerenes. In the case of the negative-ion
MALDI mass spectrum (MALDI2-MS) of FC4SH, a mole-
cular ion at m/z 1542 was detected that is conceivably
C60(CH2CH2CH2CH2–SO3H)6. It was followed by two lower
mass groups at m/z 1405 and 1269, corresponding to the ion
mass of C60(CH2CH2CH2CH2–SO3H)5 and C60(CH2CH2CH2-
CH2–SO3H)4, respectively. These data provide evidence of the
chemical compositions of hexa(sulfo-n-butylated) C60 com-
pounds 1 and 2. In addition, conversion of sulfonic moieties
(–SO2–ONa) of 1 to the corresponding hydrophobic diethyl-
sulfamide (–SO2–NEt2) derivatives allowed the detection of a
peak group with the maximum intensity located at m/z 1873
(negative ion DCI-MS) corresponding to the molecular ion
mass of hexa(diethylaminosulfo-n-butyl)[60]fullerene. That
gave further indirect confirmation of the FC4S composition.
Recent success in obtaining the single-crystal molecular
structure of fully symmetrical [6,6] hexasubstituted C60
derivative, synthesized from the same C6026 chemistry under
similar reaction conditions, indirectly suggested a relatively
even distribution of sulfo-n-butyl arms on the C60 cage.{12
This journal is ß The Royal Society of Chemistry 2005
Molecular self-assembly of FC4S for nanosphere formation in
water
Aggregation behaviours of highly water-soluble micelle-like
hexa(sulfo-n-butyl)fullerenes were investigated using small
angle neutron scattering (SANS) in D2O and small angle
X-ray scattering (SAXS) in H2O.16 Molecular self-assemblies
of FC4S resulted in formation of nearly monodisperse
spheroidal nanospheres with the sphere radius of gyration Rg
y 19 Å, where the major axis y29 Å and the minor axis
y21 Å for the ellipsoid-like aggregates, or an estimated long
sphere diameter of 60 Å [the radius 5 (5/3)1/2Rg] for the
aggregates. Interestingly, this radius of gyration was found to
remain relatively constant over a wide concentration range
from 0.35 mM to 26 mM in aqueous solutions. The mean
number of FC4S molecules for the nanosphere was determined
to be 6.5 ¡ 0.7.16
From these data we propose that a strong hydrophobic
interaction between core fullerene cages overcomes loose
charge repulsion at the molecular surface of the micelle-like
FC4S structure. It allows the nanosphere formation at a low
concentration in spite of steric hindrance and high hydro-
philicity arising from six sulfo-n-butyl arms surrounding C60.
Based on the mean FC4S molecule number of slightly more
than six for each small uniform coalescent nanosphere and the
fully stretched molecular length of FC4S being roughly 15 Å,
the dimension of the nanosphere fits well with six cage
molecules packed in close vicinity, perhaps each at the vertex
of an octahedron shape, forming a hydrophobic core region,
as shown in Fig. 3. High hydrophobicity at the central core
results in the bending of butylsulfonate arms outward into the
aqueous phase.
The hydrodynamic volume revealed a large number of
H2O molecules trapped inside the nanosphere. This suggests
possible formation of small hydrophilic domains surrounded
by a main hydrophobic spherical structure, with the C60 cages
composing the latter. Accordingly, a densely-packed nano-
sphere may still allow high singlet oxygen generation by the
fullerene cage. A higher percentage of fullerene cage moieties
located at the interfacial region between the sphere and the
aqueous medium at both internal and external regions are
susceptible to photoexcitation and the photodynamic effect.
Solvation effects on FC4S may play a role in its aggregation
Fig. 3 Schematic representation of a FC4S-derived nanosphere in
aqueous solution based on the aggregation size determined by SANS
and SAXS.
J. Mater. Chem., 2005, 15, 1857–1864 | 1859
 1
behaviour and make the O2 generation non-linear with
concentration.
Singlet oxygen generation by FC4S self-assembled nanospheres
in solution under laser irradiation
Hexafunctionalization of the fullerene cage changes the local
conjugation structure from that of C60 and thus modifies the
corresponding efficacy of singlet oxygen generation, which is a
function of several main factors, including the excited triplet-
state FC4S (3FC4S*) quantum yield, the intermolecular energy
transfer efficiency between 3FC4S* and triplet molecular
oxygen, the stability and lifetime of 3FC4S* in various solvents,
and the competitive thermal decay. Direct measurement of
singlet oxygen produced in situ in the system is the most
reliable method for characterizing this energy transfer process.
Apart from chemical considerations, physical aggregation of
FC4S molecules and the 1O2 lifetime in different solvents are
dominant factors in the overall luminescence signal intensity
measured.
The fullerene cage of FC4S tends to aggregate into
solubilized nanospheres in a solvent-dependent manner as a
function of solubility. Respectively, UV–vis absorption spec-
trum of FC4S in dilute solutions (10 mM) displayed an overall
higher extinction coefficient of FC4S in H2O than in MeOH
(Fig. 4) at the same concentration. The relative peak intensities
correlated well with the solubility of 1 in H2O (high) and
MeOH (low), with those in DMSO and DMF being inter-
mediate, possibly due to the solvent dependence of the
molecular nanosphere size.
The main optical absorption peak is located below 300 nm
with a shoulder around 340 nm. The spectrum profile decreases
monotonically up to 650 nm. Better molecular dispersion of 1
in H2O increased its 350–550 nm optical absorption. In all
solvents, the extended absorption at all wavelengths of 250–
650 nm reveals the existence of fullerenyl olefin conjugation
moieties of various lengths on the C60 cage. Variation of
the olefinic conjugation length, each with a slightly different
energy environment and a different band gap in a number
of resonance forms, leads to effective broadening of the
Fig. 4 Steady state UV–visible absorption spectra of FC4S (10 mM) in
(a) H2O, (b) MeOH, (c) DMSO and (d) DMF.
1860 | J. Mater. Chem., 2005, 15, 1857–1864
Table 1 The molar extinction coefficient of FC4S (10 mM) in different
solvents at 430, 512 and 600 nm
Solvent e430/M21cm21 e512/M21cm21 e600/M21cm21
PBS 13 160 5720 2000
H2O 14 470 5990 1950
MeOH 7930 3460 1230
DMSO 8870 3300 920
DMF 7910 3060 1060
corresponding energy absorption band. A systematic decrease
of the signal intensity at longer wavelengths revealed fewer
domains with a long conjugation length. Table 1 summarizes
the relative absorption extinction coefficients (e) of FC4S in
various solvents at three wavelengths 430, 512, and 600 nm,
applied in photoexcitation studies below. These coefficients
were also used in the calculation of relative 1O2 production
efficiency.
To correlate the singlet oxygen production effectiveness to
the photodynamic tumour treatment,8 a similar concentration
of 10 mM was applied in the study. The time-resolved
luminescence emission from the solution of 1 at 1270 nm
upon the laser excitation at either 512 or 600 nm is shown in
Fig. 5. Even though the optical absorption of FC4S is not high
at these wavelengths, the 1O2 luminescence signal was easily
measurable, indicating an overall high quantum yield and
triplet energy-transfer efficiency of excited FC4S (3FC4S*) to
molecular oxygen. Marked differences were seen in these time-
dependent 1O2 luminescence data between the solvents,
showing decrease in the integrated signal strength in the order
of DMF . DMSO & MeOH . H2O y PBS. In any given
solvent, the peak intensity was roughly 3.6 times higher with
512 nm than with 600 nm excitation, after normalization
for the laser output energy at these two wavelengths. This
correlated well with the corresponding higher (estimated 3.5
times in average) extinction coefficient of FC4S at 512 nm
(Table 1). For example, the relative time-integrated lumines-
cence signals generated by FC4S solution (10 mM) in DMSO
under 512, 600, and 430 nm excitation were found to be 10.2,
2.5, and 1.0, respectively.
The origin of the much lower normalized luminescence
intensity at 430 nm excitation is not clear. The measured 1O2
signal intensity is a function of the quantum yield of FC4S in a
nanosphere structure in solution, the lifetime of singlet oxygen,
and the lifetime of excited FC4S transient triplet state. The
method of curve fitting and the calculation of 3FC4S* and
singlet oxygen lifetimes were described in detail elsewhere.15
Based on curve-fitting of the singlet oxygen peak profiles in
Fig. 5 and the reported O2 concentration in air-saturated
solution, the 1O2 lifetime was calculated to be 18.9, 5.7, and
9.7 ms in DMF, DMSO, and MeOH, respectively (Table 2).
These are comparable to published values of 18–25 ms in
DMF17 and 9.8 ms in MeOH,18 but lower than 30 ms reported
for DMSO.19 The low signal in H2O (Figs. 5Ad and 5Bd) and
PBS (Figs. 5Ae and 5Be) limited accurate lifetime calculation.
However, the value was roughly 2–3 ms in water, which is
similar to the published value of 3.2 ms.18 Lifetime of the
triplet-state of FC4S was calculated as 0.7–0.9, 1.2–1.4 and
0.5–0.7 ms in DMF, DMSO and MeOH, respectively. These
This journal is ß The Royal Society of Chemistry 2005
 600 nm excitation, respectively, in DMSO. As seen in Fig. 4,
the optical absorption of FC4S decreases monotonically from
400 to .700 nm (relative extinction coefficient ratio 5 8.0 and
9.6 for e430/e600 at 430 nm and 3.0 and 3.5 for e512/e600 at 512 nm
in DMF and DMSO, respectively). In principle, the higher
absorption and excitation energy at 430 nm should lead to a
higher production of 1O2 after normalization to the laser
output power. However, this is not the case, unlike the results
at 512 and 600 nm excitation, which showed good correlation
with the relative absorption extinction coefficients. Regardless
of this difference, the observed overall data revealed good
quantum yields of FC4S under excitation over a range of
wavelengths.

Characterization of energy-transfer from triplet 3FC4S* state to

molecular oxygen for 1O2 generation by nanosecond transient
absorption measurements
The transient absorption spectrum observed under nano-
second laser (355 nm, 6.0 ns fwhm) excitation of FC4S in
deaerated DMSO is shown in Fig. 6. The experiment was
carried out at a concentration (0.1 mM) close to that used in
the SAXS and SANS measurements for the nanosphere
characterization. A clear new broad transient absorption band
Fig. 5 Time-resolved 1O2 luminescence emission at 1270 nm pro- with a peak maximum centered at roughly 700 nm was
duced upon laser irradiation of FC4S solution (10 mM) at (A) 512 nm detected. The absorption intensity of this transient band was
and (B) 600 nm in different solvents: (a) DMF, (b) DMSO, (c) MeOH, quenched by the well-known triplet quenchers, such as O2, via
(d) H2O, and (e) PBS in the air-saturated solution.
triplet energy transfer in O2-saturated DMSO (inserted time
profile (c) of Fig. 6). That allowed us to assign it to the triplet-
Table 2 The lifetime of 3FC4S* and singlet oxygen generated from triplet (T–T) transition of FC4S. Although the peak position
FC4S solution (10 mM) upon photoexcitation at (a) 600 nm and (b) of this T–T absorption band is almost the same as those
512 nm in different solvents
(700–740 nm) of pristine C60 and other fullerene derivatives
Solvent Lifetime of 3FC4S*/ms Lifetime of 1O2/ms containing weakly influencing functional groups, the band
(a), DMF 0.7 ¡ 0.1a 17.9 ¡ 0.2a
width is quite broad, probably due to the presence of a number
(a), DMSO 1.2 ¡ 0.1 5.2 ¡ 0.1 of regio-isomers.
(a), MeOH 0.7 ¡ 0.1 9.2 ¡ 0.1 The quantum yield W(1O2) of FC4S in the generation of 1O2
(b), DMF 0.9 ¡ 0.1 18.9 ¡ 0.2
was estimated by the direct comparison of its luminescence
(b), DMSO 1.4 ¡ 0.1 5.7 ¡ 0.3
(b), MeOH 0.5 ¡ 0.0 9.7 ¡ 0.0
a
Data 5 mean ¡ SD (n 5 3), calculated based on the reported
method.18

short lifetimes imply a high energy transfer rate from 3FC4S*

to 3O2.
It is noted that the maximum 1O2 luminescence emission
decreased in the order DMF (11) . DMSO (7.5) & MeOH
(2.5) . H2O y PBS (1.0), as shown in Fig. 5. Hence, the
lowest emission was in water, despite its having the highest
FC4S solubility. Although aggregation characteristics in DMF,
DMSO and MeOH were not determined here, the solvent-
dependent luminescence signal seems to depend more on the
lifetime of 1O2. Taking into account the 1O2 lifetime calculated
in Table 2, and the reported value18 in H2O, the relative 1O2
concentration corresponds roughly to the relative lifetime
ratios, except for DMSO. In any given solvent, the 1O2
luminescence signal can be approximately proportional to the Fig. 6 Transient absorption spectra observed by 355 nm laser
product of laser pulse energy and optical absorption extinction excitation of FC4S solution (1.0 6 1024 M) in Ar-saturated DMSO.
coefficient. We observed the 1O2 luminescence signal of y10 Insert: decay time profiles of triplet 3FC4S* at 700 nm in (a) Ar, (b) air,
and 3.6 times higher at 512 nm excitation than at 430 and and (c) O2-saturated DMSO.

This journal is ß The Royal Society of Chemistry 2005 J. Mater. Chem., 2005, 15, 1857–1864 | 1861
emission characteristics at 1270 nm with that produced from Comparison of singlet oxygen production efficiency of FC4S
c-cyclodextrin encapsulated C60 (C60/c-CD) in water.20 As a nanospheres with Photofrin and AlS4Pc
result, the emission intensity produced from 3FC4S* was found
Photofrin, as hematoporphyrin derivatives,23 and sulfonated
to be roughly a half in value indicating an approximately 50%
aluminium phthalocyanine (AlS2Pc)24 are effective photo-
efficacy of 3FC4S* formation from the corresponding singlet
dynamic therapeutic agents that exhibit photoinduced cyto-
excited state of FC4S. This conclusion is based on the
toxicity associated with the formation of 1O2 and O22?.25,26
arguments of (a) the intersystem crossing process taking place
Regardless of the low absorption of FC4S at 600 nm, 1O2 was
in a nearly quantitative yield for C60/c-CD and (b) the triplet
generated at levels comparable with that of Photofrin, as
energy-transfer rates from 3C60/c-CD* and 3FC4S* to the
shown in Fig. 8A. At the same concentration (2.0 or 10 mM),
molecular oxygen being the same. Accordingly, on the basis of
the luminescence signal from FC4S solution is higher in DMF
W(1O2) being 0.98 for C60 in organic solvent,21 the quantum
than in DMSO, MeOH and H2O, in agreement with the short
yield of FC4S in the generation of 1O2 in H2O was roughly
lifetime of singlet oxygen in H2O. At a concentration of 10 mM
estimated to be 0.36 using this relative correlation method to
that of C60/c-CD.20 The absolute value is not available in the
present study.
Comparison of decay-time profiles (the inset in Fig. 6) of
transient absorption band at 700 nm in Ar-, air- and O2-
saturated DMSO solutions indicated effective quenching of
3
FC4S* by O2. Based on the pseudo-first-order plots, the
triplet-quenching rate constant by O2 [kq(O2)] was calculated
to be 1.68 6 109 M21 s21 in DMSO. The value is in a similar
range to the reported kq(O2) value of the pristine C60 in
benzonitrile [kq(O2) 5 1.4 6 109 M21 s21].22 These kq(O2)
values are slightly smaller than the diffusion controlled limit of
5–6 6 109 M21 s21 in DMSO and benzonitrile. Decay time
profiles of the excited triplet 3FC4S* intermediate in O2-
saturated solutions are shown in Fig. 7. The decay rates
increase in the order of H2O (pH 5 7.0 and pH 5 7.8) ,
DMSO , DMF , MeOH, dependent on the quenching rate
constants and O2 concentrations. By employing saturated O2
concentrations reported in the literature, the quenching rate
constants kq(O2) were calculated as 2.19, 1.68, 1.05, 1.28, and
1.04 6 109 M21 s21 for DMF, DMSO, MeOH, H2O, and
PBS, respectively. The value of these quenching rate constants
may inversely correspond to the lifetime of 3FC4S* in Table 2,
in the same trend as the solvent dependence. In principle, the
energy-transfer rate constant [ken(O2)] can be evaluated by the
equation, ken(O2) 5 kq(O2) 6 quantum yield of energy
transfer. However, the absolute values of the quantum yields
were not obtained in the present study, the rate constants of
the energy transfer can not be determined.

Fig. 7 Decay profiles of triplet state of 1 (3FC4S*) at 700 nm Fig. 8 Time-resolved 1O2 luminescence of (A) Photofrin (10 mM), (B)
observed by 355 nm laser excitation in O2-saturated (a) DMSO, (b) AlS4Pc (2.0 mM), and (C) AlS4Pc (10 mM) at 600 nm excitation in
DMF, and (c) MeOH solutions. different solvents: (a) H2O, (b) MeOH, (c) DMSO, and (d) DMF.

1862 | J. Mater. Chem., 2005, 15, 1857–1864 This journal is ß The Royal Society of Chemistry 2005
Table 3 Relative 1270 nm luminescence intensity (I) of different
photosensitizers at a concentration of 10 mM in DMF
Dye e600 1
O2 luminescence I I/e I/e (rel.)
AlS4Pc 10 680 1138 (600 nm ex.) 0.107 0.29
Photofrin 1780 490 (600 nm ex.) 0.275 0.75
FC4S 1060 389 (600 nm ex.) 0.367 1.00
FC4S 3060a 1150 (512 nm ex.) 0.376 1.02
a
e512 instead.
for all samples, the luminescence level from FC4S in DMF
(Fig. 5Ba) reached more than 79 and 34% of that generated
by Photofrin (Fig. 8Ad) and AlS4Pc (Fig. 8Cd), respectively,
under photoexcitation at 600 nm. This signal intensity was
y80% of that of 2.0 mM AlS4Pc (Fig. 8Bd). In the case of
DMSO (Fig. 5Bb), the luminescence level produced from
10 mM FC4S was nearly equal to that of 2.0 mM AlS4Pc
(Fig. 8Bc). Furthermore, a lower AlS4Pc concentration
(2.0 mM) in different solvents gave reduced luminescence
(Fig. 8B), in general non-proportionally to the concentration
decrease. Higher luminescence per molecule in dilute solution
may be due to the drug being less aggregated.
The luminescence intensity at 600 nm of all three photo-
sensitizers in DMF, relative to their optical absorption, is
summarized in Table 3. This is then a measure of the singlet
oxygen quantum yield relative to that in DMF (assuming that
the longer lifetime of 1O2 in DMF should give a more accurate
measurement of the 1O2 level). High luminescence, corres-
ponding to the optical absorption, was obtained for FC4S
(I/e 5 0.367) and Photofrin (I/e 5 0.275), with a lower
correlated value (I/e 5 0.107) for AlS4Pc aggregates in DMF
due mainly to high optical absorbance of AlS4Pc in this region.
A similar trend was observed in DMSO, showing higher
correlated luminescent efficiency for FC4S. A high I/e value
obtained for FC4S is apparently due to its low optical absorp-
tion intensity (e) in the measured wavelength range. As the
low absorption of FC4S at longer wavelengths is still able to
produce a comparable signal of 1O2, the photon energy
conversion process of triplet AlS4Pc aggregates in the genera-
tion of 1O2 may be interpreted as less effective than the triplet
fullerene nanospheres in both solvents, on the basis of total
light energy involved in the molecular process. In the case of
the aqueous solution, the short lifetime of 1O2 and the low
normalized luminescence intensity made it difficult to derive a
meaningful and accurate peak profile of both FC4S and
Photofrin for comparison.
Conclusion
Singlet oxygen is widely believed to be the major cytotoxic
agent during the PDT processes with many photosensitizers.27
Direct detection of the 1O2 luminescence during irradiation of
FC4S self-assembled nanospheres at 500–600 nm has sub-
stantiated its photocatalytic property in the visible region. The
results also provide evidence of the efficient energy transfer
from the excited 3FC4S* to triplet molecular oxygen at a low
concentration of 0.1 mM which is close to that used in the
SAXS and SANS measurements for the nanosphere charac-
terization. We concluded that functionalization of C60 to its
This journal is ß The Royal Society of Chemistry 2005
molecular micelle-like derivative FC4S with six hydrophilic
addends, each being bound on the C60 cage by a single
covalent bond, does not appear to markedly alter the photo-
excitation process, triplet state formation, and energy transfer
efficiency of the fullerene cage in the form of a nanosphere.
That agrees with a quantum yield of FC4S for the generation
of 1O2 in H2O estimated to be roughly 0.36, using the relative
correlation to that of C60/c-CD. Despite having relatively low
optical absorptions at 600 nm, FC4S nanospheres produce
appreciable 1O2 emission signal in solution comparable to
that of the hematoporphyrin derivative Photofrin at the same
molar concentration. Unlike Photofrin, FC4S is photostable.
This photocatalytic effect makes FC4S a potential alternative
sensitizer to TiO2 and feasible for use in the visible region in
addition to its intrinsic high UV efficiency.
Acknowledgements
The authors thank M. Niedre and R. Weersink for their
assistance on singlet oxygen measurements. Development of
the singlet oxygen technique was supported by the Canadian
Cancer Society under a grant from the National Cancer
Institute of Canada. Transient absorption measurements were
supported by the Ministry of Education, Culture, Sports,
Science and Technology, a Grant-in-Aid for the Center of
Excellence (COE) Project, Giant Molecules and Complex
Systems, 2004.
Chi Yu,a Taizoon Canteenwala,b Mohamed E. El-Khouly,c
Yasuyuki Araki,c Kenneth Pritzker,a Osamu Ito,c Brian C. Wilsond and
Long Y. Chiang*b
a
Pathology and Laboratory Medicine, Mount Sinai Hospital and
University of Toronto, Canada. E-mail: kpritzker@mtsinai.on.ca;
Fax: (416) 586 8589; Tel: (416) 586 4453
b
Department of Chemistry, Institute of Nanoscience and Engineering
Technology, University of Massachusetts, Lowell, MA, USA 01854.
E-mail: Long_Chiang@uml.edu; Fax: (978) 934 3013;
Tel: (978) 934 3663
c
Institute of Multidisciplinary Research for Advanced Materials, Tohoku
University, Katahira, Aoba-ku, Sendai, 980-8577, Japan.
E-mail: ito@tagen.tohoku.ac.jp; Fax: (81) 22 617 5608;
Tel: (81) 22 617 5608
d
Department of Medical Biophysics, Ontario Cancer Institute and
University of Toronto, Ontario, Canada.
E-mail: wilson@uhnres.utoronto.ca; Fax: (416) 946 6529;
Tel: (416) 946 2952
References
1 D. M. Blake, P. Maness, Z. Huang, E. J. Wolfrum, J. Huang and
W. A. Jacoby, Sep. Purif. Methods, 1999, 28, 1.
2 A. Shiga, A. Tsujiko, S. Yae and Y. Nakato, Bull. Chem. Soc. Jpn.,
1998, 71, 2119.
3 R. C. Dunn, J. L. Anderson, C. S. Foote and J. D. Simon, J. Am.
Chem. Soc., 1993, 115, 5307. For a review: D. M. Guldi and
M. Prato, Acc. Chem. Res., 2000, 33, 695.
4 J. L. Anderson, Y. Z. An, Y. Rubin and C. S. Foote, J. Am. Chem.
Soc., 1994, 116, 9763; C. Taliani, G. Ruani, R. Zamboni,
R. Danieli, S. Rossini, V. N. Denisov, V. M. Buriakov, F. Negri,
G. Oriandi and F. Zerbetto, J. Chem. Soc., Chem. Commun., 1993,
220.
5 D. M. Guldi and P. V. Kamat, in Fullerenes, Chemistry, Physics
and Technology, ed. K. M. Kadish and R. S. Ruoff, Wiely-
Interscience, New York, 2000, pp. 225–281; M. Maggini and
D. M. Guldi, in Molecular and Supramolecular Photochemistry, ed.
V. Ramamurthy and K. S. Schanze, Marcel Dekker, New York,
2000, vol. 4, pp. 149–196.
J. Mater. Chem., 2005, 15, 1857–1864 | 1863
 6 E. Nakamura and H. Isobe, Acc. Chem. Res., 2004, 36, 807;
I. Nakanishi, S. Fukuzuami, T. Konishi, K. Ohkubo, M. Fujitsuka,
O. Ito and N. Miyata, J. Phys. Chem. B, 2002, 106, 2372.
7 J. Hirayama, H. Abe, N. Kamo, T. Shinbo, Y. Ohnishi-Yamada,
S. Kurosawa, K. Ikebuchi and S. Sekiguchi, Biol. Pharm. Bull.,
1999, 22, 1106; J. P. Kamat, T. P. A. Devasagayam, H. Mohan,
L. Y. Chiang and J. P. Mittal, Fullerene Sci. Technol., 1998, 6, 663.
8 C. Yu, T. Canteenwala, H. H. C. Chen, B. J. Chen,
M. Canteenwala and L. Y. Chiang, Proc.–Electrochem. Soc.,
1999, 12, 234.
9 D. M. Guldi and K. D. Asmus, J. Phys. Chem., 1997, 101, 1472.
10 R. V. Bensasson, M. N. Berberan-Santos, M. Bretreich,
J. Frederiksen, H. Göttinger, A. Hirsch, E. J. Land, S. Leach,
D. J. McGarvey, H. Schönberger and C. Schröder, Phys. Chem.
Chem. Phys., 2001, 3, 4679.
11 J. B. Bhonsle, C. Yu, J. P. Huang, J. Shiea, B. J. Chen and
L. Y. Chiang, Chem. Lett., 1998, 465.
12 T. Canteenwala, P. A. Padmawar and L. Y. Chiang, J. Am. Chem.
Soc., 2005, 127, 26.
13 Y. T. Lee, L. Y. Chiang, W. J. Chen and H. C. Hsu, Proc. Soc.
Exp. Biol. Med., 2000, 224, 69; H. C. Hsu, L. Y. Chiang, W. J. Chen
and Y. T. Lee, J. Cardiovasc. Pharmacol., 2000, 36, 423;
S. S. Huang, S. K. Tsai, C. L. Chih, Y. L. Chiang, H. M. Hsieh,
C. M. Teng and M. C. Tsai, Free Radical Biol. Med., 2001, 30, 643;
S. S. Huang, L. H. Chih, C. H. Lin, L. Y. Chiang, T. Mashino,
M. Mochizuki, K. Okuda, T. Hirota and M. C. Tsai, Fullerene Sci.
Technol., 2001, 9, 375; Y. L. Lai, W. Y. Chiou, F. J. Lu and
L. Y. Chiang, Br. J. Pharmacol., 1999, 126, 778; S. C. Chueh,
M. K. Lai, M. S. Lee, L. Y. Chiang, T. I. Ho and S. C. Chen,
Transplant Proc., 1999, 31, 1976; M. J. Chen, L. Y. Chiang and
Y. L. Lai, Toxicol. Appl. Pharmacol., 2001, 171, 165.
14 A. U. Khan, Chem. Phys. Lett., 1980, 72, 112.
15 M. Niedre, M. S. Patterson and B. C. Wilson, Photochem.
Photobiol., 2002, 75, 382.
1864 | J. Mater. Chem., 2005, 15, 1857–1864
16 U. S. Jeng, T. L. Lin, C. S. Tsao, C. H. Lee, T. Canteenwala,
L. Y. Wang, L. Y. Chiang and C. C. Han, J. Phys. Chem. B, 1999,
103, 1059; U. Jeng, T.-L. Lin, W.-J. Liu, C.-S. Tsao, T. Canteenwala,
L. Y. Chiang, L. P. Sung and C. C. Han, Physica A, 2002, 304, 191.
17 P. R. Ogilby and J. Sanetra, J. Phys. Chem., 1993, 97, 4689;
B. Aveline, O. Delgado and D. Brault, J. Chem. Soc., Faraday
Trans., 1992, 88, 1971.
18 M. S. Patterson, S. J. Madsen and B. Wilson, J. Photochem.
Photobiol. B, 1990, 15, 69.
19 R. Nilsson and D. R. Kearns, J. Phys. Chem., 1974, 78, 1681.
20 T. Konishi, M. Fujitsuka, H. Luo, Y. Araki, O. Ito and L. Y. Chiang,
Fullerenes, Nanotubes, Carbon Nanostruct., 2003, 11, 237.
21 J. W. Arbogast, A. P. Darmanyan, C. S. Foote, Y. Rubin,
F. N. Diederich, M. M. Alvarez, S. J. Anz and R. L. Whetten,
J. Phys. Chem., 1991, 95, 11; M. Terazima, H. Noburo,
H. Shinohara and Y. Saito, J. Phys. Chem., 1991, 95, 9080;
R. Stackow, G. Schick, T. Jarrosson, Y. Rubin and C. S. Foote,
J. Phys. Chem. B, 2000, 104, 7914.
22 S. D.-M. Islam, M. Fujitsuka, O. Ito, A. Ikeda, T. Hatano and
S. Shinkai, Chem. Lett., 2000, 78.
23 T. J. Dougherty, G. B. Grindey, R. Fiel, K. R. Weishaupt and
D. G. Boyle, J. Natl. Cancer Inst., 1975, 55, 115.
24 S. G. Bown, C. J. Tralau, P. D. Coleridge Smith, D. Akdemis and
T. J. Wieman, Br. J. Cancer, 1986, 54, 43; E. Ben-Hur and
I. Rosenthal, Int. J. Radiat. Biol., 1985, 47, 145; W. S. Chan,
R. Svensen, D. Phillips and I. R. Hart, Br. J. Cancer, 1986, 53, 255;
N. Brasseur, H. Ali, D. Autenrieth, R. Langlois and J. E. van Lier,
Photochem. Photobiol., 1985, 42, 515.
25 J. Moan, E. O. Pettersen and T. Christensen, Br. J. Cancer, 1979,
39, 398.
26 B. W. Henderson and T. J. Dougherty, Photochem. Photobiol.,
1992, 55, 145.
27 K. R. Weishaupt, C. J. Gomer and T. J. Dougherty, Cancer Res.,
1976, 36, 2326.
This journal is ß The Royal Society of Chemistry 2005