Synthesis, spectroscopic properties and photodynamic activity of a fulleropyrrolidine bearing a basic amino group and its dicationic analog against Staphylococcus aureus

Article abstract of DOI:10.1016/j.jphotochem.2015.05.022

N-Methyl-2-[4-(3-N,N-dimethylaminopropoxy)phenyl]fulleropyrrolidine (MPC₆₀) was synthesized by 1,3-dipolar cycloaddition reaction of 4-(3-N,N-dimethylaminopropoxy)benzaldehyde, N-methylglycine and fullerene C₆₀ in 43% yield. The amine groups of MPC₆₀ were methylated with dimethyl sulfate to obtain a dicationic fullerene DPC₆₀²⁺ in 96 % yield. Absorption spectra of these fullerenes in N,N-dimethylformamide (DMF) and toluene/sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/water reverse micelles showed strong absorptions in the UV region, with a peak at 430 nm and broader range of absorption up to 710 nm. Fluorescence quantum yields of about 10^-4 were calculated for these compounds in DMF. A higher singlet molecular oxygen, O₂(¹Δ_g), generation was found for MPC₆₀ than DPC₆₀²⁺ in DMF. The photodynamic activity of these photosensitizers remained high in a simple biomimetic AOT system. Also, the formation of superoxide anion radical induced by MPC₆₀ and DPC₆₀²⁺ was detected in presence of NADH. Decomposition of l-tryptophan in DMF mediated by both fullerenes indicated a possible contribution of type I photoprocess. Photosensitized inactivation of Staphylococcus aureus was investigated using different conditions. Cell suspensions of 10⁸ cells/mL incubated with 0.5 μM fullerene and irradiated for 30 min exhibited a 4.4 and 5.0 log decrease of cell survival by MPC₆₀ and DPC₆₀²⁺, respectively. Therefore, these fullerene derivatives can be used as effective photosensitizers for the photodynamic inactivation of S. aureus cells.

Full text of DOI:10.1016/j.jphotochem.2015.05.022

Accepted Manuscript
Title: Synthesis, spectroscopic properties and photodynamic
activity of a fulleropyrrolidine bearing a basic amino group
and its dicationic analog against Staphylococcus aureus
Author: Maximiliano L. Agazzi Mariana B. Spesia Natalia S.
Gsponer M.Elisa Milanesio Edgardo N. Durantini
PII:
S1010-6030(15)00187-2
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jphotochem.2015.05.022
JPC 9910
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
3-1-2015
30-4-2015
17-5-2015
Please cite this article as: Maximiliano L.Agazzi, Mariana B.Spesia, Natalia S.Gsponer,
M.Elisa Milanesio, Edgardo N.Durantini, Synthesis, spectroscopic properties and
photodynamic activity of a fulleropyrrolidine bearing a basic amino group and its
dicationic analog against Staphylococcus aureus, Journal of Photochemistry and
Photobiology A: Chemistry http://dx.doi.org/10.1016/j.jphotochem.2015.05.022
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Synthesis, spectroscopic properties and photodynamic activity of a fulleropyrrolidine bearing
a basic amino group and its dicationic analog against Staphylococcus aureus
Maximiliano L. Agazzi, Mariana B. Spesia, Natalia S. Gsponer, M. Elisa Milanesio, Edgardo N.
*
Durantini edurantini@exa.unrc.edu.ar
Departamento de Química, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad
Nacional de Río Cuarto, Agencia Postal Nro 3, X5804BYA Río Cuarto, Córdoba, Argentina.
*
Corresponding author. Tel.: +54 358 4676157; fax: +54 358 4676233.
1

Graphical abstract
Spectroscopic and photodynamic properties of novel fullerene derivatives were studied in different
media and in Staphylococcus aureus.
R1
R : NCH
1
3
R2
MPC60
R :
O
O
N
2
+
R : N (CH )
1
3 2
DPC602+
R :
N+
2
2

Highlights
A novel fulleropyrrolidine derivative was synthesized by 1,3-dipolar cycloaddition.
A dicationic fulleropyrrolidinium was obtained by exhaustive methylation.
Fullerene derivatives produce efficiently singlet molecular oxygen.
Fullerenes photosensitize the formation of superoxide anion radical in presence of NADH.
A high photoinactivation of Staphylococcus aureus was found for the dicationic fullerene.
3

Abstract
N-Methyl-2-[4-(3-N,N-dimethylaminopropoxy)phenyl]fulleropyrrolidine (MPC ) was synthesized
6
0
by 1,3-dipolar cycloaddition reaction of 4-(3-N,N-dimethylaminopropoxy)benzaldehyde, N-
methylglycine and fullerene C in 43 % yield. The amine groups of MPC were methylated with
60
60
2
+
dimethyl sulfate to obtain a dicationic fullerene DPC60 in 96 % yield. Absorption spectra of these
fullerenes in N,N-dimethylformamide (DMF) and toluene/sodium bis(2-ethylhexyl)sulfosuccinate
(AOT)/water reverse micelles showed strong absorptions in the UV region, with a peak at 430 nm
-4
and broader range of absorption up to 710 nm. Fluorescence quantum yields of about 10 were
1
calculated for these compounds in DMF. A higher singlet molecular oxygen, O (  ), generation
2
g
2+
was found for MPC than DPC
in DMF. The photodynamic activity of these photosensitizers
remained high in a simple biomimetic AOT system. Also, the formation of superoxide anion radical
60
60
2+
induced by MPC60 and DPC60 was detected in presence of NADH. Decomposition of L-
tryptophan in DMF mediated by both fullerenes indicated a possible contribution of type I
photoprocess. Photosensitized inactivation of Staphylococcus aureus was investigated using
8
different conditions. Cell suspensions of 10 cells/mL incubated with 0.5 M fullerene and
2+
irradiated for 30 min exhibited a 4.4 and 5.0 log decrease of cell survival by MPC and DPC ,
60
6
0
respectively. Therefore, these fullerene derivatives can be used as effective photosensitizers for the
photodynamic inactivation of S. aureus cells.
Keywords
fullerene; photosensitizer; photodynamic inactivation; bacteria; Staphylococcus aureus
4

1
. Introduction
In recent years, new approaches to the treatment of microbial infections have become
necessary due to the changing patterns of infectious diseases and the emergence of antibiotic-
resistant microbes. This is mainly because of the inappropriate prescription of antibiotics, the
application in prophylaxis and the systemic use that also affect the normal flora and the failure of
patients to complete the treatments. The antibiotic era was perhaps largely expected to eliminate
Staphylococcus aureus and other bacterial pathogens as a leading cause of human infections [1].
However, S. aureus has extraordinary ability to develop resistance to antibiotics, which have been
the impetus for waves of antimicrobial resistance over the past 60 years [2]. In this sense, new
alternative therapies have been proposed [3]. In particular, photodynamic inactivation (PDI) of
microorganisms has been proposed to controlling bacterial infections [4]. PDI involves the
administration of a photosensitizer that is accumulated in the microbial cells and the subsequent
irradiation with visible light. In the presence of oxygen, the photodynamic activity produces cell
inactivation. The main advantages of this approach are: i) fast eradication of microorganisms, ii)
double selectivity, specific accumulation of photosensitizer in microbial cells and light delivered
only to affected area and iii) similar photoinactivation regardless of the antibiotic resistance and
lack of induction of resistance to photodynamic treatments [5].
Appropriate photosensitizers have specific chemical and biological properties. Thus, the
combination of visible light absorption and a long lifetime of triplet excited state allow fullerenes to
act as photosensitizers [6]. However, fullerenes are hydrophobic molecules with low solubility in
polar solvents and consequently fullerenes form aggregates in aqueous solutions [7]. Thus, the lack
of solubility in biological environments is the major obstacle in the development of this field.
Different approaches have been developed for the transfer of fullerenes to water. Perhaps, the most
versatile methodology to resolve this problem is based on the chemical modification of the fullerene
by the attachment of functional hydrophilic cationic groups. These amphiphilic fullerenes can
produce an increase in the biological activity and therefore act as efficient photosensitizer to
5

inactivate microbial cells. The development of covalent chemistry of C has opened the possibility
6
0
to attach this sphere structure with several groups, which allows increase in the biological activity
8]. Thus, chemical modification of the fullerene can be used to attach functional hydrophilic
[
cationic groups. These amphiphilic fullerenes can be efficient photosensitizer in biological media.
Interesting results using cationic fullerenes as photosensitizers have been found to photoinactivate
microorganisms [9-11].
3
*
Under aerobic conditions, the triplet excited state of fullerene ( C ) can interact with
6
0
ground state molecular oxygen to form reactive oxygen species (ROS). This process can occur by
3
*
1
energy transfer from the C60 to produce singlet molecular oxygen, O (  ) or by electron transfer
2
g
.
-
1
to form superoxide anion radical (O ) [12]. Fullerenes are extremely efficient O (  ) generators
2
2
g
with a quantum yield that is near unity. On the other hand, fullerenes can be easily reduced to C60
.
-
.-
3
*
radical anion (C ) by electron transfer. Thus, the C
60
or C60 can transfer an electron to
60
.
-
1
molecular oxygen forming O . In contrast to O (  ) generation, the electron transfer type of
2
2
g
reaction preferentially occurs in polar solvents, particularly in the presence of reducing agents such
.
-
1
as NADH. These pathways, yielding O (  ) and O , are analogous to the two main photochemical
2
g
2
reaction types known as type II and type I mechanisms, respectively [6].
In the present work, a novel fulleropyrrolidine C60 derivative (MPC ) was synthetized,
60
which contains an aliphatic chain with an amino group at the end (Scheme 1). The nitrogen atoms in
2
+
the amine groups were used to obtain a dicationic fulleropyrrolidinium (DPC60 ). The formation of
cationic amphiphilic photosensitizers has several interesting features that make these compounds
attractive photosensitizers for a variety of biological systems [6]. The spectroscopic and
photodynamic properties of these fullerenes were studied in organic solution and in a simple
biomimetic medium formed by reverse micellar system. Also, photodynamic activity mediated by
these photosensitizers was evaluated in vitro for inactivation of S. aureus cells.
2
. Materials and methods
6

2
.1. General
Proton nuclear magnetic resonance spectra were performed on a FT-NMR Bruker Avance
DPX400 spectrometer at 400 MHz. Mass spectra were recorded on a Bruker micrOTOF-QII
Bruker Daltonics, MA, USA) equipped with an atmospheric pressure photoionization (APPI)
(
source. Absorption and fluorescence spectra were carried out in a Shimadzu UV-2401PC
spectrometer (Shimadzu Corporation, Tokyo, Japan) and on a Spex FluoroMax spectrofluorometer
(Horiba Jobin Yvon Inc, Edison, NJ, USA), respectively. Experiments of photooxidation of
substrates were carried out with a Cole-Parmer illuminator 41720-series (Cole-Parmer, Vernon
Hills, IL, USA) with a 150 W halogen lamp through a high intensity grating monochromator
(Photon Technology Instrument, Birmingham, NJ, USA). The visible light source used to irradiate
cell suspensions was a Novamat 130 AF (Braun Photo Technik, Nürnberg, Germany) slide
projector containing a 150 W lamp. A 2.5 cm glass cuvette filled with water was used to remove the
heat from the lamp. A wavelength range between 350 and 800 nm was selected by optical filters.
2
The fluence rate was determined as was 90 mW/cm (Radiometer Laser Mate-Q, Coherent, Santa
Clara, CA, USA).
All the chemicals from Aldrich (Milwaukee, WI, USA) were used as received. Sodium
bis(2-ethylhexyl)sulfosuccinate (AOT) from Sigma (St. Louis, MO, USA) was dried under vacuum.
Tetrasodium 2,2´-(anthracene-9,10-diyl)bis(methylmalonate) (ABMM) was synthetized as
previously described (13). Silica gel thin-layer chromatography (TLC) plates 250 microns from
Analtech (Newark, DE, USA) were used. Solvents (GR grade) from Merck (Darmstadt, Germany)
were distilled. Labconco (Kansas, MO, USA) equipment model 90901-01 was used to obtain
ultrapure water.
2
.2. Synthesis of fullerene C derivatives
60
N-Methyl-2-[4-(3-N,N-dimethylaminopropoxy)phenyl]fulleropyrrolidine (MPC ). A solution of C
60
6
0
(51 mg, 0.071 mmol), 4-(3-N,N-dimethylaminopropoxy)benzaldehyde (25 mg, 0.120 mmol) and N-
7

methylglycine (26 mg, 0.292 mmol) in 55 mL of dry toluene was stirred at reflux under an argon
atmosphere for 6 h. Then, the solvent was removed under vacuum. Flash column chromatography
(silica gel) using toluene/ethyl acetate (100:0 to 50:50 gradient, 1% triethylamine) as eluent
afforded 29 mg (43%) of MPC . TLC (sílica gel, toluene/ethyl acetate/triethylamine, 1:1:0.01) R =
60
f
-1
-1
1
0
.2. max (DMF) [nm] ( M cm ) 431 (3980). HNMR (CDCl , TMS)  (ppm) 1.97 (m, 2H, -CH -
3
2
), 2.28 (s, 6H, N-(CH ) ), 2.50 (t, 2H, N-CH -, J=7.0 Hz), 2.79 (s, 3H, N-CH pyrrolidine ring), 4.02
3 2 2 3
(
t, 2H, O-CH -, J=6.1 Hz), 4.24 (d, 1H, pyrrolidine ring, J=9.4 Hz), 4.88 (s, 1H, pyrrolidine ring);
2
4
.97 (d, 1H, pyrrolidine ring, J=9.4 Hz), 6.95 (d, 2H, Ar, J=8.0 Hz), 7.70 (d, 2H, Ar, J=8.0 Hz).
+
APPI-MS [m/z] 955.1810 [M+H] (954.1732 calculated for C H N O).
7
4
22
2
2+
N,N-Dimethyl-2-[4-(3-N,N,N-trimethylammoniopropoxy)phenyl]fulleropyrrolidinium (DPC60 ). A
mixture of MPC (10 mg, 0.010 mmol) and dimethyl sulfate (200 L, 2.11 mmol) in 2 mL of N,N-
60
dimethylformamide (DMF) was stirred for 40 h at 90 ºC under an argon atmosphere. The solvent
was removed under vacuum and the mixture was placed in Eppendorf tubes and ethyl ether was
added. The methylated product was precipitated. It was washed with 5% aqueous Na CO and then
2
3
2+
-1
-1
1
with water to obtain 96% of DPC60 . max (DMF) [nm] ( M cm ) 431 (3920). HNMR
+
(
CS /acetone-d , TMS)  [ppm]: 2.40 (m, 2H, -CH -), 3.28 (s, 9H, N -(CH ) ), 3.32 (t, 2H, N-CH -,
2
6
2
3 3
2
+
J=6.9 Hz), 3.88 (s, 6H, N -(CH ) pyrrolidine ring), 4.00 (t, 2H, O-CH -, J=6.0 Hz), 4.30 (d, 1H,
3
2
2
pyrrolidine ring, J=9.5 Hz), 4.94 (s, 1H, pyrrolidine ring); 5.02 (d, 1H, pyrrolidine ring, J=9.5 Hz),
+
6
.92 (d, 2H, Ar, J=8.0 Hz), 7.69 (broad signal, 2H, Ar). APPI-MS [m/z] 985.2280 [M+H]
(
984.2202 calculated for C H N O).
76
28
2
2
.3. Spectroscopic studies
Absorption and fluorescence spectra were performed in a quartz cell of 1 cm path length
using DMF, toluene and toluene/AOT (0.1 M)/water (W = 10) media at 25.00.5 C. Absorbances
0
<
0.05 were matched at the excitation wavelength (450 nm) and the areas of the emission spectra
were integrated in the range 600-800 nm. The fluorescence quantum yield ( ) of fullerenes were
F
8

calculated by comparison of the area below the corrected emission spectrum with that of C as a
6
0
-4
reference ( = 2.3x10 ), exciting at  = 450 nm in DMF [10]. AOT reverse micelles were
F
exc
prepared from a stock solution of 0.1 M AOT, which was prepared by weighing and dilution in
toluene. The amount of water dispersed in the system was reported as the molar ratio between water
and the AOT present in the reverse micelle (W = [H O]/[AOT]). In all experiments, W = 10 was
0
2
0
used and the mixtures were sonicated for about 10 s to obtain perfectly clear micellar system [14].
2
.4. Steady state photolysis
Photooxidation of 1,3-diphenylisobenzofuran (DPBF). Solutions of DPBF (20 M) and fullerene in
DMF were irradiated in 1 cm path length quartz cells (2 mL) with monochromatic light at  = 480
irr
2
nm (fullerene absorbance 0.1). The light fluence rate was determined as 0.60 mW/cm . The
photooxidation of DPBF was studied by following the decrease of the absorbance at max=415 nm.
After irradiation, the formation of products interfering in the absorption spectra was not detected
and the absorption due to fullerene derivatives was unchanged. Thus, the absorption changes can be
assigned to the photooxidation of DPBF mediated by fullerenes.
Photooxidation of 9,10-dimethylanthracene (DMA) and ABMM. Solutions of anthracene derivative
(35 M) and fullerene in different media (DMA in DMF or AOT micelles and ABMM in water or
DMF/water 1:1) were irradiated as described for DPBF using  = 450 nm (light fluence 0.45
irr
2
mW/cm ). Antracene photodecomposition was monitored following the decrease in absorbance at
max = 378 nm.
1
Quantum yields of O (  ) production ( ). The observed rate constants (k ) of DPBF or DMA
2
g

obs
photodecomposition were obtained by a linear least-squares fit of the semilogarithmic plot of ln
A /A) vs. time. Values of  in DMF were calculated comparing the k for the corresponding
(
0

obs
fullerene with that for C , which was used as a reference ( = 1) [15,16]. Measurements of the
60

sample and reference under the same conditions afforded  for photosensitizers by direct

comparison of the slopes in the linear region of the plots.
9

Photooxidaion of nitro blue tetrazolium (NBT). The NBT method was used to detect superoxide
.
-
anion radical (O ) formation in DMF [17,18]. The NBT method was carried out using 0.2 mM
2
NBT, 0.5 mM NADH and fullerene (10 M) in 2 mL of DMF. Control experiments were performed
in absence of NBT, NADH or fullerene. Samples were irradiated in 1 cm path length quartz cells
under aerobic condition with visible light filtered through a 2.5 cm glass cuvette filled with water
2
(
light fluence 44 mW/cm ). The progress of the reaction was monitored by following the increase of
the absorbance at =560 nm.
Photooxidation of L-tryptofan (Trp). Solutions of Trp (20 μM) and fullerene in DMF were treated
as described above for photodecomposition of DMA. Photooxidation of Trp were studied by
following the decrease of the fluorescence intensity at λ = 350 nm, exciting the samples at exc
=
2
90 nm. Control experiments showed that under these conditions the fluorescence intensity
correlates linearly with Trp concentration. The observed rate constants (k ) were obtained by a
obs
linear least-squares fit of semi-logarithmic plots of ln (F /F) vs. time.
0
2
.5. Bacterial strain and preparation of cultures
The Gram-positive strain S. aureus ATCC 25923 was used in this study. This bacterium was
grown on a rotator shaker (100 rpm) at 37 ºC in tryptic soy (TS, Britania, Buenos Aires, Argentina)
broth overnight. An aliquot (60 L) of this culture was aseptically transferred to 4 mL of fresh
medium (TS broth) and incubated at 37 ºC to mid logarithmic phase of growth (absorbance ~0.3 at
6
60 nm). After that, cells were harvested by centrifugation of broth cultures (3000 rpm for 15 min)
and re-suspended in 4 mL of 10 mM phosphate-buffered saline (PBS, pH=7.0). Then the cells were
6
8
diluted 1/1000 or 1/10 in PBS, corresponding to ~10 and ~10 colony forming units (CFU)/mL,
respectively.
2
.6. Photosensitized inactivation of bacteria cells
1
0

6
8
Cell suspensions of S. aureus (2 mL, ~10 CFU/mL or ~10 CFU/mL) in PBS were
incubated with 0.5 M fullerene in Pyrex culture tubes (13x100 mm) for 30 min in the dark at 37
ºC. Photosensitizers were added from a stock solution in DMF (0.5 mM). Then, two protocols were
6
followed: a) 2 mL of ~10 CFU/mL were directly irradiated in Pyrex culture tubes and b) 0.2 mL of
8
~
10 CFU/mL were placed in each well of 96-well microtiter plates (Deltalab, Barcelona, Spain). In
both cases, the cultures were exposed for different time intervals to visible light using the irradiation
system described in section 2.1. Serial dilutions of irradiated cell suspensions and controls were
performed with PBS and each sample was plated in triplicate on TS agar. The number of colonies
formed after ~24 h incubation at 37 ºC was counted.
2
.7. Controls and statistical analysis
Control experiments were performed in presence and absence of fullerene in the dark and in
the absence of photosensitizer with cells irradiated. The amount of DMF used in each experiment
was not toxic to S. aureus cells. Three values were obtained per each condition and each experiment
was repeated separately three times. The unpaired t-test was used to establish the significance of
differences between groups. Differences were considered statistically significant with a confidence
level of 95% (p < 0.05). Data were represented as the mean±standard deviation of each group.
3
. Results and discussion
3
.1. Synthesis of fullerene derivatives
The synthetic procedures to obtain fullerene derivatives are briefed in Scheme 1. First,
MPC60 was obtained by 1,3-dipolar cycloaddition reaction in dry toluene using 4-(3-N,N-
dimethylaminopropoxy)benzaldehyde, N-methylglycine and fullerene C , (1.7:4.1:1.0) molar
6
0
relationship, respectively. The solution was stirred for 6 h at reflux in an argon atmosphere. This
reaction mixture produced MPC , which was purified by flash chromatography (silica gel) giving
60
4
3 % yield. Thus, the synthesis of MPC60 was performed on the base of fulleropyrrolidine
1
1

formation developed by Prato et al. [19]. Similar fulleropyrrolidine bearing a flexible attachment
with an N-methylaniline end group was previously prepared [20]. The amine groups in the structure
2
+
2+
of MPC were the precursor of a dicationic DPC
by methylation. Thus, DPC60 was formed
60
60
treating MPC with an excess of dimethyl sulphate for 72 h at 80ºC. The exhaustive methylation
60
2+
produced DPC60 in high yields (96%).
2+
The structure of DPC60 is formed by a hydrophobic carbon sphere containing two cationic
groups that provide an amphiphilic character. The mainly difference between both fullerenes is the
2
+
presence of two intrinsic cationic charges in DPC60 in contrast with MPC , which is substituted
60
by an aliphatic amine group. This basis amine group in the periphery of MPC60 could acquire
positive charges, depending on the medium in which the fullerene is located. Moreover, in these
structures the cationic center is isolated from the fullerene by a propoxy bridge. Thus, this charge
has minimal influence on the electronic density of the sphere. This helps to retain the consistency of
the photophysical properties of the fullerene. Also, this chain provides a higher mobility of the
charge, which could facilitate the interaction with the membrane of the bacteria.
3
.2. Absorption and fluorescence spectroscopic studies
2
+
UV-visible absorption spectra of MPC and DPC
60
in DMF, toluene and toluene/AOT
6
0
(
0.1 M)/water (W =10) are shown in Scheme 1. Synthesis of fullerene derivatives
0
Figure 1. These fullerenes had moderately strong π-π* absorption bands in the UV region.
In the visible region, a broader range of absorption up to almost 710 nm was observed with a sharp
peak at 430 nm. Also, a very weak broader band characteristic of C derivatives was detected at
6
0
2
+
7
07 nm for MPC and at 708 nm for DPC
in DMF [21]. Monofunctionalization of the fullerene
60
60
core influences the electronic structure and leads to a change of the I -symmetry of pristine C ,
h
60
which adapts an effective C2´ symmetry [20]. On the other hand, similar shapes in the MPC60
absorption spectra were found in the organic solvents and in AOT micelles indicating a
solubilization mainly as monomer in these media. In contrast, a low intensity and a broadening of
2
+
the bands were observed in the spectrum of DPC60 in toluene due to aggregation of the dicationic
fullerene in this non-polar medium (Scheme 1. Synthesis of fullerene derivatives
2
+
Figure 1). However, the spectrum intensity of DPC60 increases in toluene/AOT/water,
indicating that this micellar system helps the solubilization of cationic fullerene as monomer.
2
+
The steady-state fluorescence emission spectra of MPC and DPC
were compared with
6
0
60
that of C in DMF exciting the samples at 450 nm (Figure 3). The spectra showed a band centered
60
1
2

2
+
at 717 nm for MPC60 and 716 nm for DPC60 , which are characteristic for similar
fulleropyrrolidines [10,22]. The fluorescence spectrum of N-methylfulleropyrrolidine is in
agreement with the mirror imaged absorption features [22]. This reflects the fact that the force
constants of vibrational levels in the first singlet excited state resemble those in the singlet ground
state. The good match of the longest-wavelength absorption and the shortest wavelength emission
and the fact that they exhibit the highest oscillator strengths are convincing evidence for an
assignment to 0*0 transition bands. From the intersection of the absorption and fluorescence
spectra, Stokes shifts of ~10 nm were calculated for these fullerenes. These Stokes shifts indicated
that in these molecules the spectroscopic energies are similar to the relaxed energies of the lowest
singlet excited state S , according to the rigid structure of the fullerenes. That suggests that only a
1
minor geometric relaxation occurs in the first excited state. Since the energy of the 0-0 electronic
transitions, the energy levels of the singlet excited stated (E ) were calculated giving in both cases a
s
value of 1.74 eV. This E value is similar to those previously reported for this kind of fullerene
s
derivatives [22]. Values of  for these photosensitizers were calculated by comparison with C as
F
60
-4
-4
a reference in the S state in DMF, giving (5.2±0.5)x10 and (2.8±0.3)x10 for MPC and
1
60
2+
DPC60 , respectively. In general, fullerene C derivatives present a low fluorescence emission and
60
these values agree with those previously reported by similar fullerenes [23,24].
3
3
.3. Photodynamic activity
.3.1. Photooxidation of DPBF
The photooxidation of DPBF induced by fullerene derivatives was studied in DMF. DPBF is
1
decomposed by O (  ) generated to produce 1,2-dibenzoylbenzene [25]. Thus, this substrate was
2
g
1
used to evaluate the ability of the fullerenes to produce O (  ). A time-dependent decrease in the
2
g
DPBF concentration was observed by following a decrease in its absorbance at 415 nm (Figure 4).
From first-order kinetic plots the values of the observed rate constant (k ) were calculate for
obs
DPBF. The results are shown in
1
3

Scheme and figures captions
Scheme 1. Synthesis of fullerene derivatives
Figure 1. Absorption spectra of (A) MPC and (B) DPC
60
2
+
in DMF (solid line), toluene (dashed
6
0
line) and toluene/AOT (0.1 M)/water (W =10) (dotted line).
0
2
+
Figure 3. Fluorescence emission spectra of MPC60 (solid line), DPC60 (dashed line) and C60
(
dotted line) in DMF,  =450 nm.
exc
Figure 4. First-order plots for the photooxidation of DPBF (20 M,  = 480 nm) photosensitized
irr
2+
by C (

), DPC () and DPC
() in DMF. Values represent mean ± standard deviation of three
6
0
60
60
separate experiments.
Figure 5. First-order plots for the photooxidation of DMA (35 M,  = 450 nm) photosensitized
irr
2+
by C (
), DPC60 () and DPC60
( ) in (A) DMF and (B) toluene/AOT (0.1 M)/water (W =10).

6
0
0
Values represent mean ± standard deviation of three separate experiments.
-
Figure 6. Time course of O2 generation detected by the NBT method as an increase in the
2
+
absorption at 560 nm, for NBT + β-NADH (
NADH+ MPC ( ) NBT + β-NADH + DPC60
.2 mM and [NADH] = 0.5 mM. Values represent mean ± standard deviation of three separate

); NBT + MPC (

); NBT + DPC60
(
); NBT + β-
6
0
2
+

0
(
) in DMF irradiated with visible light, [NBT] =
6
0
experiments.
Figure 7. First-order plots for the photooxidation of Trp (20 M,  =450 nm) photosensitized by
irr
2+
MPC60 () and DPC60
(
) in DMF. Values represent mean ± standard deviation of three separate
experiments.
6
Figure 8. Survival curves of S. aureus cells (2 mL, ~10 CFU/mL) incubated with 0.5 M of
2+
MPC (
) and DPC60
() for 30 min at 37 ºC in dark and exposed to visible light for different
6
0
irradiation times in culture tubes (13x100 mm). Control cultures: cells treated with 0.5 M MPC60
2+
(
) and DPC60
() in dark; cells untreated with the photosensitizer and irradiated (). Values
represent mean ± standard deviation of three separate experiments.
8
Figure 9. Survival curves of S. aureus cells (0.2 mL, ~10 CFU/mL) incubated with 0.5 M of
1
4

2+
MPC (
) and DPC60
() for 30 min at 37 ºC in dark and exposed to visible light for different
6
0
irradiation times in 96-well microtiter plates. Control cultures: cells treated with 0.5 M MPC (

0
)
6
2+
and DPC60
() kept in dark; cells untreated with the photosensitizer and irradiated (). Values
represent mean ± standard deviation of three separate experiments.
1
5

2
+
Table 1. Values of  were calculated comparing the slope for MPC and DPC
with the

60
60
corresponding slope obtained for the reference, C . MPC and C , photodecompose DPBF with
6
0
60
60
1
comparable rates, indicating that O (  ) was efficiently produced by MPC in this medium.
2
g
60
1
Similar results of O (  ) production were previously found a non-charged N-methyl-2-(4´-
2
g
acetamidophenyl)fulleropyrrolidine in DMF/water (10 %) [10]. Therefore, introduction of a
substituent on fullerene core produced a decrease in the photodynamic activity. On the other hand,
2
+
photodecomposition of DPBF photosensitized by DPC60 was lower than that of MPC . In
60
previous studies it was found a very low of  for N,N-Dimethyl-2-(4´-N,N,N-
trimethylaminophenyl)fulleropyrrolidinium due to an incomplete monomerization of the dicationic
2
+
1
fullerene in DMF/water (10 %) [10]. However, for DPC60 showed a O (  ) production about half
2
g
of that obtained for MPC .
60
3
.3.2. Photosensitized decomposition of DMA and ABMM
Photooxidation of anthracene derivatives induced by these photosensitizers was compared in
different media under aerobic conditions. Figure 5 shows characteristic semilogarithmic plots
DMA
describing the progress of the reaction for DMA. Values of kobs
were calculated from first-order
kinetic plots of the DMA absorption at 378 nm with irradiation time (
Scheme and figures captions
Scheme 1. Synthesis of fullerene derivatives
2
+
Figure 1. Absorption spectra of (A) MPC and (B) DPC
60
in DMF (solid line), toluene (dashed
6
0
line) and toluene/AOT (0.1 M)/water (W =10) (dotted line).
0
2
+
Figure 3. Fluorescence emission spectra of MPC60 (solid line), DPC60 (dashed line) and C60
(
dotted line) in DMF,  =450 nm.
exc
Figure 4. First-order plots for the photooxidation of DPBF (20 M,  = 480 nm) photosensitized
irr
2+
by C (

), DPC () and DPC
() in DMF. Values represent mean ± standard deviation of three
6
0
60
60
separate experiments.
1
6

Figure 5. First-order plots for the photooxidation of DMA (35 M,  = 450 nm) photosensitized
irr
2+
by C (
), DPC60 () and DPC60
( ) in (A) DMF and (B) toluene/AOT (0.1 M)/water (W =10).

6
0
0
Values represent mean ± standard deviation of three separate experiments.
-
Figure 6. Time course of O2 generation detected by the NBT method as an increase in the
2
+
absorption at 560 nm, for NBT + β-NADH (
NADH+ MPC ( ) NBT + β-NADH + DPC60
.2 mM and [NADH] = 0.5 mM. Values represent mean ± standard deviation of three separate

); NBT + MPC (

); NBT + DPC60
(
); NBT + β-
6
0
2
+

0
(
) in DMF irradiated with visible light, [NBT] =
6
0
experiments.
Figure 7. First-order plots for the photooxidation of Trp (20 M,  =450 nm) photosensitized by
irr
2+
MPC60 () and DPC60
(
) in DMF. Values represent mean ± standard deviation of three separate
experiments.
6
Figure 8. Survival curves of S. aureus cells (2 mL, ~10 CFU/mL) incubated with 0.5 M of
2+
MPC (
) and DPC60
() for 30 min at 37 ºC in dark and exposed to visible light for different
6
0
irradiation times in culture tubes (13x100 mm). Control cultures: cells treated with 0.5 M MPC60
2+
(
) and DPC60
() in dark; cells untreated with the photosensitizer and irradiated (). Values
represent mean ± standard deviation of three separate experiments.
8
Figure 9. Survival curves of S. aureus cells (0.2 mL, ~10 CFU/mL) incubated with 0.5 M of
2+
MPC (
) and DPC60
() for 30 min at 37 ºC in dark and exposed to visible light for different
6
0
irradiation times in 96-well microtiter plates. Control cultures: cells treated with 0.5 M MPC (

0
)
6
2+
and DPC60
() kept in dark; cells untreated with the photosensitizer and irradiated (). Values
represent mean ± standard deviation of three separate experiments.
1
7

Table 1). In DMF, MPC efficiently induced the decomposition of DMA, whereas lower
60
2+
reaction rate was detected using DPC60 as photosensitizer (Figure 5A). Moreover, the kinetic data
of DMA decomposition were used to estimate the  values considering that this substrate

1
quenches O (  ) by chemical reaction [25]. As can be observed in
2
g
Scheme and figures captions
Scheme 1. Synthesis of fullerene derivatives
2
+
Figure 1. Absorption spectra of (A) MPC and (B) DPC
60
in DMF (solid line), toluene (dashed
6
0
line) and toluene/AOT (0.1 M)/water (W =10) (dotted line).
0
2
+
Figure 3. Fluorescence emission spectra of MPC60 (solid line), DPC60 (dashed line) and C60
(
dotted line) in DMF,  =450 nm.
exc
Figure 4. First-order plots for the photooxidation of DPBF (20 M,  = 480 nm) photosensitized
irr
2+
by C (

), DPC () and DPC
() in DMF. Values represent mean ± standard deviation of three
6
0
60
60
separate experiments.
Figure 5. First-order plots for the photooxidation of DMA (35 M,  = 450 nm) photosensitized
irr
2+
by C (
), DPC60 () and DPC60
( ) in (A) DMF and (B) toluene/AOT (0.1 M)/water (W =10).

6
0
0
Values represent mean ± standard deviation of three separate experiments.
-
Figure 6. Time course of O2 generation detected by the NBT method as an increase in the
2
+
absorption at 560 nm, for NBT + β-NADH (
NADH+ MPC ( ) NBT + β-NADH + DPC60
.2 mM and [NADH] = 0.5 mM. Values represent mean ± standard deviation of three separate

); NBT + MPC (

); NBT + DPC60
(
); NBT + β-
6
0
2
+

0
(
) in DMF irradiated with visible light, [NBT] =
6
0
experiments.
Figure 7. First-order plots for the photooxidation of Trp (20 M,  =450 nm) photosensitized by
irr
2+
MPC60 () and DPC60
(
) in DMF. Values represent mean ± standard deviation of three separate
experiments.
6
Figure 8. Survival curves of S. aureus cells (2 mL, ~10 CFU/mL) incubated with 0.5 M of
2+
MPC (
) and DPC60
() for 30 min at 37 ºC in dark and exposed to visible light for different
6
0
irradiation times in culture tubes (13x100 mm). Control cultures: cells treated with 0.5 M MPC60
1
8

2+
(
) and DPC60
() in dark; cells untreated with the photosensitizer and irradiated (). Values
represent mean ± standard deviation of three separate experiments.
8
Figure 9. Survival curves of S. aureus cells (0.2 mL, ~10 CFU/mL) incubated with 0.5 M of
2+
MPC (
) and DPC60
() for 30 min at 37 ºC in dark and exposed to visible light for different
6
0
irradiation times in 96-well microtiter plates. Control cultures: cells treated with 0.5 M MPC (

0
)
6
2+
and DPC60
() kept in dark; cells untreated with the photosensitizer and irradiated (). Values
represent mean ± standard deviation of three separate experiments.
1
9

Table 1, comparable values of  were obtained for fullerenes using DMA or DPBF, which

are quite reasonable values for monosubstituted fullerenes dissolved as monomers [16].
1
ABMM was used as a molecular probe for the detection of O (  ) in aqueous solution [13].
2
g
Photooxidation of this substrate mediated by fullerenes was not detected in water after 10 min
irradiation. These fullerenes can form aggregates in this medium, producing a considerable decrease
1
in the formation of O (  ) [10]. Photodecomposition of ABMM was detected in DMF/water (1:1).
2
g
As shown in
Scheme and figures captions
Scheme 1. Synthesis of fullerene derivatives
2
+
Figure 1. Absorption spectra of (A) MPC and (B) DPC
60
in DMF (solid line), toluene (dashed
6
0
line) and toluene/AOT (0.1 M)/water (W =10) (dotted line).
0
2
+
Figure 3. Fluorescence emission spectra of MPC60 (solid line), DPC60 (dashed line) and C60
(
dotted line) in DMF,  =450 nm.
exc
Figure 4. First-order plots for the photooxidation of DPBF (20 M,  = 480 nm) photosensitized
irr
2+
by C (

), DPC () and DPC
() in DMF. Values represent mean ± standard deviation of three
6
0
60
60
separate experiments.
Figure 5. First-order plots for the photooxidation of DMA (35 M,  = 450 nm) photosensitized
irr
2+
by C (
), DPC60 () and DPC60
( ) in (A) DMF and (B) toluene/AOT (0.1 M)/water (W =10).

6
0
0
Values represent mean ± standard deviation of three separate experiments.
-
Figure 6. Time course of O2 generation detected by the NBT method as an increase in the
2
+
absorption at 560 nm, for NBT + β-NADH (
NADH+ MPC ( ) NBT + β-NADH + DPC60
.2 mM and [NADH] = 0.5 mM. Values represent mean ± standard deviation of three separate

); NBT + MPC (

); NBT + DPC60
(
); NBT + β-
6
0
2
+

0
(
) in DMF irradiated with visible light, [NBT] =
6
0
experiments.
Figure 7. First-order plots for the photooxidation of Trp (20 M,  =450 nm) photosensitized by
irr
2+
MPC60 () and DPC60
() in DMF. Values represent mean ± standard deviation of three separate
2
0

experiments.
Figure 8. Survival curves of S. aureus cells (2 mL, ~10 CFU/mL) incubated with 0.5 M of
6
2+
MPC (
) and DPC60
() for 30 min at 37 ºC in dark and exposed to visible light for different
6
0
irradiation times in culture tubes (13x100 mm). Control cultures: cells treated with 0.5 M MPC60
2+
(
) and DPC60
() in dark; cells untreated with the photosensitizer and irradiated (). Values
represent mean ± standard deviation of three separate experiments.
8
Figure 9. Survival curves of S. aureus cells (0.2 mL, ~10 CFU/mL) incubated with 0.5 M of
2+
MPC (
) and DPC60
() for 30 min at 37 ºC in dark and exposed to visible light for different
6
0
irradiation times in 96-well microtiter plates. Control cultures: cells treated with 0.5 M MPC (

0
)
6
2+
and DPC60
() kept in dark; cells untreated with the photosensitizer and irradiated (). Values
represent mean ± standard deviation of three separate experiments.
2
1

ABMM
2+
Table 1, a lower value of kobs
was found using DPC60 than MPC . A difficulty of ABMM
60
anionic trap is the interaction with cationic photosensitizers [13]. The interaction of ABMM with
2+
1
DPC60 can be interfering in the O (  ) generation.
2
g
Moreover, photooxidation of DMA mediated by fullerenes was performed in toluene/AOT
(
0.1 M)/water (W = 10) reverse micelles under aerobic conditions (Figure 5B). This
0
microheterogeneous system has been used as a simple biomimetic model of water pockets found in
bioaggregates [26]. In micellar systems, a solute can be located in the organic solvent, the dispersed
water pool or at the micellar interface [13]. Thus, compounds of different polarities can be
1
dissolved simultaneously in reverse micelles. Photodecomposition of DMA by O (  ) takes place
2
g
in the toluene pseudophase where this non-polar molecule is mainly solubilized [13]. As shown in
Scheme and figures captions
Scheme 1. Synthesis of fullerene derivatives
2
+
Figure 1. Absorption spectra of (A) MPC and (B) DPC
60
in DMF (solid line), toluene (dashed
6
0
line) and toluene/AOT (0.1 M)/water (W =10) (dotted line).
0
2
+
Figure 3. Fluorescence emission spectra of MPC60 (solid line), DPC60 (dashed line) and C60
(
dotted line) in DMF,  =450 nm.
exc
Figure 4. First-order plots for the photooxidation of DPBF (20 M,  = 480 nm) photosensitized
irr
2+
by C (

), DPC () and DPC
() in DMF. Values represent mean ± standard deviation of three
6
0
60
60
separate experiments.
Figure 5. First-order plots for the photooxidation of DMA (35 M,  = 450 nm) photosensitized
irr
2+
by C (
), DPC60 () and DPC60
( ) in (A) DMF and (B) toluene/AOT (0.1 M)/water (W =10).

6
0
0
Values represent mean ± standard deviation of three separate experiments.
-
Figure 6. Time course of O2 generation detected by the NBT method as an increase in the
2
+
absorption at 560 nm, for NBT + β-NADH (
NADH+ MPC ( ) NBT + β-NADH + DPC60
.2 mM and [NADH] = 0.5 mM. Values represent mean ± standard deviation of three separate

); NBT + MPC (

); NBT + DPC60
(
); NBT + β-
6
0
2
+

0
(
) in DMF irradiated with visible light, [NBT] =
6
0
experiments.
Figure 7. First-order plots for the photooxidation of Trp (20 M,  =450 nm) photosensitized by
irr
2+
MPC60 () and DPC60
() in DMF. Values represent mean ± standard deviation of three separate
2
2

experiments.
Figure 8. Survival curves of S. aureus cells (2 mL, ~10 CFU/mL) incubated with 0.5 M of
6
2+
MPC (
) and DPC60
() for 30 min at 37 ºC in dark and exposed to visible light for different
6
0
irradiation times in culture tubes (13x100 mm). Control cultures: cells treated with 0.5 M MPC60
2+
(
) and DPC60
() in dark; cells untreated with the photosensitizer and irradiated (). Values
represent mean ± standard deviation of three separate experiments.
8
Figure 9. Survival curves of S. aureus cells (0.2 mL, ~10 CFU/mL) incubated with 0.5 M of
2+
MPC (
) and DPC60
() for 30 min at 37 ºC in dark and exposed to visible light for different
6
0
irradiation times in 96-well microtiter plates. Control cultures: cells treated with 0.5 M MPC (

0
)
6
2+
and DPC60
() kept in dark; cells untreated with the photosensitizer and irradiated (). Values
represent mean ± standard deviation of three separate experiments.
2
3

1
2+
Table 1, a high O (  ) generation was found for MPC and DPC . These fullerenes can interact
2
g
60
60
with the AOT micelles through their substituents. Also, due to the high lipophilic character of the
fullerene sphere, it is expected that fullerene is located at the micellar interface with the moiety of
the C situated at the nonpolar solvent. Localization of C in the toluene pseudophase decrease the
6
0
60
1
formation of type I reaction, favoring the photosensitization of O (  ) from the fullerene triplet
2
g
state. On the other hand, the reaction rates of DMA sensitized by fullerenes in the AOT micellar
system were slower than those found in DMF (
Scheme and figures captions
Scheme 1. Synthesis of fullerene derivatives
2
+
Figure 1. Absorption spectra of (A) MPC and (B) DPC
60
in DMF (solid line), toluene (dashed
6
0
line) and toluene/AOT (0.1 M)/water (W =10) (dotted line).
0
2
+
Figure 3. Fluorescence emission spectra of MPC60 (solid line), DPC60 (dashed line) and C60
(
dotted line) in DMF,  =450 nm.
exc
Figure 4. First-order plots for the photooxidation of DPBF (20 M,  = 480 nm) photosensitized
irr
2+
by C (

), DPC () and DPC
() in DMF. Values represent mean ± standard deviation of three
6
0
60
60
separate experiments.
Figure 5. First-order plots for the photooxidation of DMA (35 M,  = 450 nm) photosensitized
irr
2+
by C (
), DPC60 () and DPC60
( ) in (A) DMF and (B) toluene/AOT (0.1 M)/water (W =10).

6
0
0
Values represent mean ± standard deviation of three separate experiments.
-
Figure 6. Time course of O2 generation detected by the NBT method as an increase in the
2
+
absorption at 560 nm, for NBT + β-NADH (
NADH+ MPC ( ) NBT + β-NADH + DPC60
.2 mM and [NADH] = 0.5 mM. Values represent mean ± standard deviation of three separate

); NBT + MPC (

); NBT + DPC60
(
); NBT + β-
6
0
2
+

0
(
) in DMF irradiated with visible light, [NBT] =
6
0
experiments.
Figure 7. First-order plots for the photooxidation of Trp (20 M,  =450 nm) photosensitized by
irr
2+
MPC60 () and DPC60
(
) in DMF. Values represent mean ± standard deviation of three separate
experiments.
6
Figure 8. Survival curves of S. aureus cells (2 mL, ~10 CFU/mL) incubated with 0.5 M of
2+
MPC (
) and DPC60
() for 30 min at 37 ºC in dark and exposed to visible light for different
6
0
2
4

irradiation times in culture tubes (13x100 mm). Control cultures: cells treated with 0.5 M MPC60
2+
(
) and DPC60
() in dark; cells untreated with the photosensitizer and irradiated (). Values
represent mean ± standard deviation of three separate experiments.
8
Figure 9. Survival curves of S. aureus cells (0.2 mL, ~10 CFU/mL) incubated with 0.5 M of
2+
MPC (
) and DPC60
() for 30 min at 37 ºC in dark and exposed to visible light for different
6
0
irradiation times in 96-well microtiter plates. Control cultures: cells treated with 0.5 M MPC (

0
)
6
2+
and DPC60
() kept in dark; cells untreated with the photosensitizer and irradiated (). Values
represent mean ± standard deviation of three separate experiments.
2
5

Table 1). Similar behavior was previously observed in AOT reverse micelles using porphyrins as
1
photosensitizers [13]. In the AOT micellar system, O (  ) is partitioned between the internal and
2
g
external pseudophases. Also, the reaction rate of DMA photooxidation can be lower in the toluene
pseudophase than in a more polar solvent, such as DMF [27].
3
.3.3. Photosensitized reduction of NBT
.
-
2
+
Generation of O by MPC and DPC
60
was observed using NBT reduction to diformazan
2
60
in presence of NADH, following the absorption at 560 nm in DMF (Figure 6). Photosensitized
decomposition of NBT ocurrs predominantly through a type I photoreaction process [28]. The
increase in diformazan absorption was investigated as a function of time after irradiation with
.
-
visible light. As can be observed in Figure 6, reduction of NBT by O2 was not found in the
photoirradiated samples without NADH. Decomposition of NBT significantly increases in presence
2+
of MPC or DPC
60
and NADH after irradiation with respect to solution without the fullerenes.
6
0
1
Therefore, even though O (  ) can be generated effectively by photoexcited triplet state of
2
g
.
-
fullerenes, it was observed that O can also be produced in the presence of NADH. However, the
2
biological microenvironment of the photosensitizer can produce important modifications in the
photophysics of the fullerene derivatives established in solution.
3
.3.4. Photooxidation of Trp
Photosensitized decomposition of Trp was investigated in DMF. Trp can be photooxidized
by both type I and type II reaction mechanisms and this amino acid can be a potential target of the
ROS generated by fullerenes in cells [29,30]. As shown in Figure 7, the photooxidation followed
first-order kinetics with respect to Trp concentration. From the plots in Figure 7, the values of the
Trp
kobs were determined for Trp decomposition.
Scheme and figures captions
Scheme 1. Synthesis of fullerene derivatives
2
+
Figure 1. Absorption spectra of (A) MPC and (B) DPC
60
in DMF (solid line), toluene (dashed
6
0
line) and toluene/AOT (0.1 M)/water (W =10) (dotted line).
0
2
6

2
+
Figure 3. Fluorescence emission spectra of MPC60 (solid line), DPC60 (dashed line) and C60
(
dotted line) in DMF,  =450 nm.
exc
Figure 4. First-order plots for the photooxidation of DPBF (20 M,  = 480 nm) photosensitized
irr
2+
by C (

), DPC () and DPC
() in DMF. Values represent mean ± standard deviation of three
6
0
60
60
separate experiments.
Figure 5. First-order plots for the photooxidation of DMA (35 M,  = 450 nm) photosensitized
irr
2+
by C (
), DPC60 () and DPC60
( ) in (A) DMF and (B) toluene/AOT (0.1 M)/water (W =10).

6
0
0
Values represent mean ± standard deviation of three separate experiments.
-
Figure 6. Time course of O2 generation detected by the NBT method as an increase in the
2
+
absorption at 560 nm, for NBT + β-NADH (
NADH+ MPC ( ) NBT + β-NADH + DPC60
.2 mM and [NADH] = 0.5 mM. Values represent mean ± standard deviation of three separate

); NBT + MPC (

); NBT + DPC60
(
); NBT + β-
6
0
2
+

0
(
) in DMF irradiated with visible light, [NBT] =
6
0
experiments.
Figure 7. First-order plots for the photooxidation of Trp (20 M,  =450 nm) photosensitized by
irr
2+
MPC60 () and DPC60
(
) in DMF. Values represent mean ± standard deviation of three separate
experiments.
6
Figure 8. Survival curves of S. aureus cells (2 mL, ~10 CFU/mL) incubated with 0.5 M of
2+
MPC (
) and DPC60
() for 30 min at 37 ºC in dark and exposed to visible light for different
6
0
irradiation times in culture tubes (13x100 mm). Control cultures: cells treated with 0.5 M MPC60
2+
(
) and DPC60
() in dark; cells untreated with the photosensitizer and irradiated (). Values
represent mean ± standard deviation of three separate experiments.
8
Figure 9. Survival curves of S. aureus cells (0.2 mL, ~10 CFU/mL) incubated with 0.5 M of
2+
MPC (
) and DPC60
() for 30 min at 37 ºC in dark and exposed to visible light for different
6
0
irradiation times in 96-well microtiter plates. Control cultures: cells treated with 0.5 M MPC (

0
)
6
2+
and DPC60
() kept in dark; cells untreated with the photosensitizer and irradiated (). Values
represent mean ± standard deviation of three separate experiments.
2
7

2
8

Table 1 shows that high photooxidation rates of Trp were obtained using these fullerenes as
photosensitizers. The deactivation of fullerenes by an energy transfer mechanism to Trp can be
ruled out on energetics grounds (E = 1.77 eV for fullerenes; E =3.51 eV for Trp [31]) and due to
s
s
the lack of appropriate spectral properties [32]. Possibly, interactions between these fullerenes and
Trp can be favoring an electron transfer process in the decomposition of Trp. Also, the
photosensitized oxidation of Trp was found to be more than two orders of magnitude more efficient
1
via electron transfer than via O (  ) [29]. Therefore photoinduced electron transfer can be involved
2
g
in the photooxidation. Comparing the kinetic results of Trp and DMA in DMF, it can be observed
Trp
DMA
2+
that the ratio kobs /kobs
takes values of 3.8 and 4.7 for MPC and DPC , respectively. These
60 60
results can be compared with similar studies using porphyrins as photosensitizers [33,34]. For non-
Trp
DMA
charged porphyrins, the photooxidation of Trp mainly follows a process type II with kobs /kobs
values of about 0.4. However, this ratio considerably increased when the Trp decomposition was
photosensitized by the fullerenes. Therefore, an electron transfer pathway may also be contributing,
together with type II photoprocess, to Trp decomposition in DMF.
3
.4. In vitro studies on S. aureus cells
2
+
The photodynamic activity of MPC and DPC
60
was investigated in vitro using a Gram-
6
0
6
8
positive bacterium, S. aureus. Photoinactivation studies were realized using ~10 and ~10 CFU/mL
cell densities. The cell toxicity produced by these fullerenes was evaluated in the absence of light at
6
different fullerene concentrations. No toxicity was found in 10 CFU/mL cultures treated with 2.5
2
+

M MPC60 for 30 min in dark. However, this concentration of DPC60 was cytotoxic without
irradiation. Similar bacteriostatic effects of cationic fullerene derivatives were previously observed
on Escherichia coli cells [10]. A regio isomer mixture of C -bis(N,N-dimethylpyrrolidinium
6
0
inhibited E. coli growth and oxygen uptake caused by cells. This result indicated that the
mechanism of the bacteriostatic effect of cationic fullerenes is the inhibition of energy metabolism
2
9