Synthesis, properties and photodynamic inactivation of Escherichia coli by novel cationic fullerene C60 derivatives

Article abstract of DOI:10.1016/j.ejmech.2007.06.014

A novel N,N-dimethyl-2-(4′-N,N,N-trimethylaminophenyl)fulleropyrrolidinium iodide (DTC₆₀²⁺) has been synthesized by 1,3-dipolar cycloaddition using 4-(N,N-dimethylamino) benzaldehyde, N-methylglycine and fullerene C₆₀. This approach produced an N-methyl-2-(4′-N,N-dimethylaminophenyl)fulleropyrrolidine with 38% yield. Exhaustive methylation of this fullerene derivative with methyl iodide yielded 95% of dicationic DTC₆₀²⁺. The spectroscopic and photodynamic properties of the DTC₆₀²⁺ were compared with a non-charged N-methyl-2-(4′-acetamidophenyl)fulleropyrrolidine (MAC₆₀) and a monocationic N,N-dimethyl-2-(4′-acetamidophenyl)fulleropyrrolidinium iodide (DAC₆₀⁺). The dicationic DTC₆₀²⁺ is essentially aggregated in solution of different solvents and it is partially dissolved as monomer in benzene/benzyl-n-hexadecyldimethyl ammonium chloride (BHDC) 0.1 M/water (W₀ = 10) reverse micelles. The singlet molecular oxygen, O₂ (¹Δ_g), production was evaluated using 1,3-diphenylisobenzofuran. The photodynamic effect was strongly dependent on the medium, diminishes when the sensitizer is aggregated and increases in an appropriately surrounded microenvironment. The photodynamic inactivation produced by these fullerene derivatives was investigated in vitro on a typical Gram-negative bacterium, Escherichia coli. Photosensitized inactivation of E. coli cellular suspensions by DTC₆₀²⁺ exhibits a ～3.5 log decrease of cell survival (99.97% of cellular inactivation), when the cultures are treated with 1 μM of sensitizer and irradiated for 30 min. This photosensitized inactivation remains high even after one washing step. Also, the photodynamic activity was confirmed by growth delay of E. coli cultures. The growth was arrested when E. coli was exposed to 2 μM of cationic fullerene and irradiated, whereas a negligible effect was found for the non-charged MAC₆₀. These studies indicate that dicationic DTC₆₀²⁺ is an interesting agent with potential applications in photodynamic inactivation of bacteria.

Full text of DOI:10.1016/j.ejmech.2007.06.014

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European Journal of Medicinal Chemistry 43 (2008) 853e861
http://www.elsevier.com/locate/ejmech
Original article
Synthesis, properties and photodynamic inactivation of Escherichia
coli by novel cationic fullerene C60 derivatives
*
Mariana B. Spesia, M. Elisa Milanesio, Edgardo N. Durantini
Departamento de Qu´ımica, Universidad Nacional de R´ıo Cuarto, Agencia Postal Nro 3, X5804BYA R´ıo Cuarto, Cordoba, Argentina
Received 29 March 2007; received in revised form 19 June 2007; accepted 21 June 2007
Available online 10 July 2007
Abstract
A novel N,N-dimethyl-2-(40-N,N,N-trimethylaminophenyl)fulleropyrrolidinium iodide (DTC620þ) has been synthesized by 1,3-dipolar cycload-
dition using 4-(N,N-dimethylamino) benzaldehyde, N-methylglycine and fullerene C60. This approach produced an N-methyl-2-(40-N,N-dimethy-
laminophenyl)fulleropyrrolidine with 38% yield. Exhaustive methylation of this fullerene derivative with methyl iodide yielded 95% of dicationic
2þ
DTC260þ. The spectroscopic and photodynamic properties of the DTC were compared with a non-charged N-methyl-2-(40-acetamidophenyl)ful-
60
leropyrrolidine (MAC60) and a monocationic N,N-dimethyl-2-(40-acetamidophenyl)fulleropyrrolidinium iodide (DACþ60). The dicationic DTC260þ
is essentially aggregated in solution of different solvents and it is partially dissolved as monomer in benzene/benzyl-n-hexadecyldimethyl ammo-
nium chloride (BHDC) 0.1 M/water (W0 ¼ 10) reverse micelles. The singlet molecular oxygen, O2 (1Dg), production was evaluated using 1,3-
diphenylisobenzofuran. The photodynamic effect was strongly dependent on the medium, diminishes when the sensitizer is aggregated and
increases in an appropriately surrounded microenvironment. The photodynamic inactivation produced by these fullerene derivatives was inves-
2þ
tigated in vitro on a typical Gram-negative bacterium, Escherichia coli. Photosensitized inactivation of E. coli cellular suspensions by DTC
exhibits a w3.5 log decrease of cell survival (99.97% of cellular inactivation), when the cultures are treated with 1 mM of sensitizer and irradiated
for 30 min. This photosensitized inactivation remains high even after one washing step. Also, the photodynamic activity was confirmed by growth
60
delay of E. coli cultures. The growth was arrested when E. coli was exposed to 2 mM of cationic fullerene and irradiated, whereas a negligible
2þ
effect was found for the non-charged MAC60. These studies indicate that dicationic DTC is an interesting agent with potential applications
60
in photodynamic inactivation of bacteria.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Fullerene; Photosensitizer; Photodynamic inactivation; Bacteria; Escherichia coli
1. Introduction
candidates as electron acceptors [3,4]. Also, fullerene
compounds have avid reactivity with free radicals. Potential
biological activities of fullerenes have been investigated with
the aim of using it in the field of medicine [5e7]. An impor-
tant inconvenience for this application is the low solubility of
fullerenes in polar solvents and the consequent formation of
aggregates in aqueous solutions [1]. However, the develop-
ment of covalent chemistry of C60 has opened the possibility
to attach this spherical structure with several groups, which al-
lows increment in the biological activity [1,3,8].
Chemical and physical features of fullerene C60, together
with its spherical shape, have aroused the hope of successful
use in many different fields either in biological or material
chemistry [1,2]. The condensed aromatic rings present in the
compound lead to an extended p conjugation of molecular or-
bitals causing significant absorption of visible light. The facile
electron acceptability of up to six electrons makes them good
The emergence of antibiotic resistance amongst pathogenic
bacteria has led to a major research effort to find alternative an-
tibacterial therapies [9]. Bacterial photodynamic inactivation
* Corresponding author. Tel.: þ54 358 4676157; fax: þ54 358 4676233.
E-mail address: edurantini@exa.unrc.edu.ar (E.N. Durantini).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.06.014

854
M.B. Spesia et al. / European Journal of Medicinal Chemistry 43 (2008) 853e861
(PDI) is based on the administration of a photosensitizer, which
is preferentially accumulated in the microbial cells. The subse-
quent irradiation with visible light, in the presence of oxygen,
specifically produces cell damages that inactivate the microor-
ganisms [10,11]. Different oxidative mechanisms can occur af-
ter photoactivation of the photosensitizer. In the type I
photochemical reaction, the photosensitizer interacts with
a biomolecule to produce free radicals, while in the type II
mechanism, singlet molecular oxygen, O2(1Dg), is produced
as the main species responsible for cell inactivation [12,13].
Photoexcited fullerenes react with various electron donors to
give the C60 radical anion (Cꢀ6ꢁ0 ) via type I electron-transfer
pathway. Also, reduced active oxygen species, such as superoxide
(Oꢀ2ꢁ) can be generated by electron transfer from C6ꢀ ꢁ0 to molec-
ular oxygen [14]. A variety of photosensitizers have shown to
be effective to inactivate Gram-positive bacteria. However,
Gram-negative bacteria exhibit a remarkable resistance to neg-
atively charged or neutral agents [15,16]. This resistance has
been ascribed to the presence of highly organized outer mem-
brane, which hinders the interaction of the photosensitizer with
the cytoplasmic membrane and intercepts the photogenerated
reactive species [17e20]. In general, photosensitizers contain-
ing cationic groups produce direct photoinactivation of Gram-
negative bacteria even in the absence of additives [21e27].
Fullerenes have a long lifetime of triplet excited state to
produce efficiently O2(1Dg) [28]. Therefore, they are interest-
ing candidates for use in photosensitization processes, espe-
cially in situations where absorption in the red part of the
spectra is not required. According to its photochemical prop-
erties, fullerene has been proposed for PDI of viruses in bio-
logical fluids [29]. Recently, cationic fullerene derivatives
have shown interesting applications against the HIV strains
[30]. Furthermore, cationic fullerenes bearing quaternary pyr-
rolidinium groups have been evaluated as effective antimicro-
bial photosensitizers [31].
2. Materials and methods
2.1. General
Absorption and fluorescence spectra were recorded on
a Shimadzu UV-2401PC spectrometer and on a Spex Fluoro-
Max fluorometer, respectively. Proton nuclear magnetic reso-
nance (1H NMR) spectra were recorded on an FT-NMR
Bruker Avance DPX400 multinuclear spectrometer at
400 MHz. FAB mass spectra were taken with a ZAB-SEQ
Micromass equipment. Silica gel thin-layer chromatography
(TLC) plates of 250 mm from Aldrich (Milwaukee, WI,
USA) were used. All the chemicals from Aldrich were used
without further purification. Benzyl-n-hexadecyldimethylam-
monium chloride (BHDC) from Sigma was recrystallized
twice from ethyl acetate and dried under vacuum over P2O5.
Solvents (GR grade) from Merck were distilled. Ultrapure
water was obtained from Labconco (Kansas, MO, USA)
equipment model 90901-01. Semiempirical molecular orbital
calculations (AM1) were carried out using HyperChem
software.
2.1.1. Sensitizers
Fulleropyrrolidines were synthesized according to
modifications of established procedures [36].
2.1.1.1. N,N-Dimethyl-2-(40-N,N,N-trimethylaminophenyl)ful-
leropyrrolidinium iodide (DTC260þ). A solution of C60
(50 mg, 0.069 mmol), 4-N,N-dimethylaminobenzaldehyde
(10 mg,
0.067 mmol)
and
N-methylglycine
(6 mg,
0.069 mmol) in 52 mL of dry toluene was stirred at reflux in
atmosphere of argon for 6 h. Then, the solvent was removed
under vacuum. Flash column chromatography (silica gel) us-
ing toluene/heptane (50:50 to 80:20 gradient) as eluent
afforded 22 mg (38%) of N-methyl-2-(40-N,N-dimethylamino-
phenyl)fulleropyrrolidine (DC60). TLC (silica gel, toluene/
heptane, 1:1) analysis Rf 0.10. MS [m/z] 896 (Mþ)
(896.1313 calculated for C71H16N2). Anal. calcd. C 95.08, H
1.80, N 3.12; found C 95.12, H 1.85, N 3.07. 1H NMR
(CDCl3, TMS) d [ppm] 2.88 (s, 3H), 3.10 (s, 6H), 4.34 (d,
1H, J ¼ 9.5 Hz), 5.01 (s, 1H), 5.09 (d, 1H, J ¼ 9.5 Hz), 6.99
(d, 2H, J ¼ 9.0 Hz), 7.52 (d, 2H, J ¼ 9.0 Hz). Then, a mixture
of this fullerene derivative (18 mg, 0.020 mmol) and 2 mL of
methyl iodide in 2 mL of N,N-dimethylformamide (DMF) was
stirred for 72 h at 70 ꢂC. The solvents were removed under
vacuum. The solid was re-suspended in heptane/toluene
(1:1) and filtered to yield 22 mg (95%) of DTC260þ. MS [m/z]
926 (Mþ ꢁ 2I) (926.1783 calculated for C73H22N2). Anal.
calcd. C 74.25, H 1.88, N 2.37; found C 74.33, H 1.84, N
2.42. lmax (DMF) [nm] (3, Mꢁ1 cmꢁ1) 429 (2100).
In previous works, we have investigated the photodynamic
activity of porphyrins with different number of cationic
charges in vitro as agents to eradicate Gram-negative bacteria
[32,33]. Amphiphilic porphyrin derivatives showed to be ac-
tive photosensitizers to inactivate Escherichia coli cells.
Also, we have recently used for the first time a porphyrine
fullerene dyad for the photodynamic inactivation of tumoral
cells [34,35]. The results showed that molecular dyads, which
can form photoinduced charge-separated state, offer a promis-
ing molecular architecture for photosensitizing agents that can
produce cellular inactivation still under anoxic condition.
In the present work, we have synthesized a novel N,
N-dimethyl-2-(40-N,N,N-trimethylaminophenyl)fulleropyrro-
lidinium iodide (DTC260þ) bearing two cationic groups. The
2þ
spectroscopic and photodynamic properties of DTC were
60
compared with a non-charged N-methyl-2-(40-acetamidophe-
nyl)fulleropyrrolidine (MAC60) and a monocationic N,
N-dimethylþ-2-(40-acetamidophenyl)fulleropyrrolidinium io-
2.1.1.2.
N,N-Dimethyl-2-(40-acetamidophenyl)fulleropyrro-
lidinium iodide (DACþ60). A mixture of C60 (30 mg,
0.042 mmol), 4-acetamidobenzaldehyde (14 mg, 0.086
mmol) and N-methylglycine (5 mg, 0.056 mmol) in 5 mL of
dry toluene was stirred at reflux in atmosphere of argon for
7 h. The solvent was evaporated under vacuum and flash
dide (DAC60) in different media and in a typical Gram-neg-
2þ
ative bacterium, E. coli. These studies show that DTC is an
60
interesting photosensitizer with potential applications in PDI
of bacteria.

M.B. Spesia et al. / European Journal of Medicinal Chemistry 43 (2008) 853e861
855
column chromatography (silica gel) using toluene/ethyl ace-
tate (100:0 to 80:10 gradient) as eluent to yield 32 mg
(41%) of N-methyl-2-(40-acetamidophenyl)fulleropyrrolidine
(MAC60). TLC (silica gel, toluene/ethyl acetate, 1:1) analysis
Rf 0.25. MS [m/z] 910 (Mþ) (910.1106 calculated for
C71H14N2O). Anal. calcd. C 93.62, H 1.55, N 3.08; found C
93.55, H 1.61, N 3.02. 1H NMR (CDCl3, TMS) d [ppm]
2.37 (s, 3H), 2.86 (s, 3H), 4.31 (d, 1H, J ¼ 9.5 Hz), 4.97 (s,
1H), 5.07 (d, 1H, J ¼ 9.5 Hz), 7.03 (d, 2H, J ¼ 8.5 Hz), 7.54
(d, 2H, J ¼ 8.5 Hz), 7.71 (br s, 1H). lmax (DMF) [nm] (3,
rate constants (kobs) were obtained by a linear least-squares
fit of the semilogarithmic plot of ln A0/A vs time. Photooxi-
dation of DPBF was used to determine singlet molecular ox-
ygen, O2(1Dg), production by the photosensitizers. Fullerene
C60 was used as the standard (FD ¼ 1) [39,40]. Measure-
ments of the sample and reference under the same condi-
tions afforded FD for sensitizers by direct comparison of
the slopes in the linear region of the plots [41]. All the ex-
periment were performed at 25.0 ꢃ 0.5 ꢂC. The pooled stan-
dard deviation of the kinetic data, using different prepared
samples, was less than 5%.
M
ꢁ1 cmꢁ1) 430 (4000). Then, a mixture of MAC60 (17 mg,
0.020 mmol) and 2 mL of methyl iodide in 2 mL of N,N-dime-
thylformamide (DMF) was stirred for 72 h at 70 ꢂC. The
solvents were removed under vacuum. The solid was re-
suspended in heptane and filtered to yield 18 mg (96%) of
DACþ60. MS [m/z] 925 (Mþ ꢁ I) (925.1341 calculated for
C72H17N2O). Anal. calcd. C 82.20, H 1.70, N 2.73; found C
82.14, H 1.63, N 2.66. lmax (DMF) [nm] (3, Mꢁ1 cmꢁ1) 429
(3500).
2.4. Bacterial strain and preparation of cultures
E. coli strain (EC7) recovered from clinical urogenital
material was previously characterized and identified
[32,33]. E. coli strain was grown aerobically at 37 ꢂC in
30% w/v tryptic soy (TS) broth overnight. Aliquots
(w40 mL) of this culture were aseptically transferred to
4 mL of fresh medium (30% w/v TS broth) and incubated
at 37 ꢂC to mid logarithmic phase (absorbance w0.6 at
660 nm). Cells in the logarithmic phase of growth were har-
vested by centrifugation of broth cultures (3000 rpm for
15 min) and re-suspended in 4 mL of 10 mM phosphate-buff-
ered saline (PBS, pH ¼ 7.0). Then the cells were diluted
1/1000 in PBS, corresponding to w106 colony forming units
(CFU)/mL. In all the experiments, 2 mL of the cell suspen-
sions in Pyrex brand culture tubes (13 ꢄ 100 mm) were
used and the sensitizer was added from a stock solution of
sensitizer (4.5 ꢄ 10ꢁ4 M) in DMF. Viable bacteria were
monitored and their number was calculated by counting the
number of colony forming units after appropriate dilution
on TS agar plates [32]. Bacterial cultures grown under the
same conditions with and without photosensitizers kept in
the dark as well as illuminated cultures without sensitizer
served as controls.
2.2. Spectroscopic studies
Spectra were recorded using 1-cm path length quartz cells
at 25.0 ꢃ 0.5 ꢂC. The fluorescence quantum yield (fF) of ful-
lerenes was calculated by comparison of the area below the
corrected emission spectrum with that of C60 as a fluores-
cence standard, excited at lexc ¼ 450 nm [37]. A value of
fF ¼ (2.3 ꢃ 0.1) ꢄ 10ꢁ4 for C60 in DMF was calculated by
comparison with the fluorescence spectrum in toluene using
fF ¼ 2.2 ꢄ 10ꢁ4 and taking into account the refractive index
of the solvents [28,37]. Studies in reverse micelles were per-
formed using a stock solution of BHDC 0.1 M, which was
prepared by weighing and dilution in benzene. The addition
of water to the corresponding solution was performed using
a calibrated microsyringe. The amount of water present in
the system was expressed as the molar ratio between water
and the BHDC present in the reverse micelle (W0¼[H2O]/
[BHDC]). In all experiments, W0 ¼ 10 was used. The
mixtures were sonicated to obtain perfectly clear micellar
system [38].
2.5. Photosensitized inactivation of bacteria cells
2.3. Steady-state photolysis
Cell suspensions of E. coli (2 mL, w106 CFU/mL) in PBS
were incubated with 1 mM of sensitizer for 30 min in the dark
at 37 ꢂC. After that, two protocols were followed: (a) irradia-
tion of cultures without cell washing step and (b) washing the
cells once with PBS prior to irradiation. In both cases, the cul-
tures were exposed for different time intervals to visible light.
The light source used was a Novamat 130 AF slide projector
equipped with a 150 W lamp. The light was filtered through
a 2.5-cm glass cuvette filled with water to absorb heat. A
wavelength range between 350 and 800 nm was selected by
optical filters [42]. The light intensity at the treatment site
was 90 mW/cm2 (Radiometer Laser Mate-Q, Coherent). Con-
trol and irradiated cell suspensions were serially diluted with
PBS, each solution was plated in triplicate on TS agar and
the number of colonies formed after 18e24 h incubation at
37 ꢂC was counted.
Solutions of 1,3-diphenylisobenzofuran (DPBF, 20 mM)
and photosensitizer in different media were irradiated in
1-cm path length quartz cells (2 mL) with monochromatic
light at lirr ¼ 470 nm (sensitizer absorbance 0.1) from
a 75 W high-pressure Xe lamp through a high intensity grat-
ing monochromator, Photon Technology Instrument [38].
The light intensity was determined as 0.55 mW/cm2 (Radio-
meter Laser Mate-Q, Coherent). The kinetics of photooxi-
dation were studied by following the decrease in the
absorbance (A) at lmax ¼ 415 nm for DPBF. Under these
conditions, absorption due to fullerene derivatives at
415 nm is unchanged and therefore, the absorption changes
observed in the presence of DPBF sensitized by fullerenes
can be assigned to the oxidation of DPBF. The observed

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M.B. Spesia et al. / European Journal of Medicinal Chemistry 43 (2008) 853e861
2.6. Growth delay of E. coli cultures
a
reverse micellar system formed by benzene/BHDC
2þ
(0.1 M)/water (W0 ¼ 10). Representative results for DTC
60
Cultures of E. coli cells were grown overnight as described
above. A portion (60 mL) of this culture was transfer to 20 mL
of fresh TS broth (10%) medium. The suspension was homoge-
nized and aliquots of 2 mL were incubated with 2 mM ofsensitizer
at 37 ꢂC. The culture grown was measured by turbidity at 660 nm
using a Tuner SP-830 spectrophotometer [27,32]. Then the flasks
were irradiated with visible light at 37 ꢂC, as described above.
In all cases, control experiments were carried out without
illumination in the absence and in the presence of sensitizer.
Each experiment was repeated separately three times.
are shown in Fig. 1. The spectra of these fullerene derivatives
are typical of most C60 monoadducts [3]. Similarly to C60, the
electronic spectra of the pyrrolidine derivatives are dominated
by strong absorptions mainly in the UV region. In the visible
region, these fullerenes show a broader range of absorption up
to almost 710 nm, with a characteristic sharp peak at 430 nm
and broader band at around 700 nm. The absorption spectrum
of MAC60 shows similar shape in DMF and in micelles indi-
cating a moderate solubility as monomer in both media. On
the other hand, a low intensity and a broadening of the bands
2þ
are observed in the spectrum of DTC in DMF, probably due
60
to aggregation of dicationic fullerene in this medium (Fig. 1).
2þ
3. Results and discussion
Similar behavior was also found for DTC in toluene and
60
2þ
PBS. However, the spectrum intensity of DTC increases in
60
3.1. Synthesis of fullerene derivatives
Fulleropyrrolidine derivatives were synthesized by 1,3-dipolar
cycloaddition of azomethine ylides to C60 (Schemes 1 and 2) [3].
benzene/BHDC/water, indicating that this micellar system
helps the solubilization of cationic fullerene as monomer.
The steady-state fluorescence emission spectra of fullerenes
in DMF are shown in Fig. 2. The spectra show a band centered
at w712 nm, which are characteristic for similar fulleropyrro-
lidines [43]. Taking into account the absorption spectrum, this
band has been assigned to 0* / 0 transition band [28]. By
2þ
A two-step method was used to synthesize DTC . First, the
60
reaction of C60 with N,N-dimethylaminobenzaldehyde and
N-methylglycine ([1:1:1] molar relation) in refluxing toluene
afforded N-methyl-2-(40-N,N-dimethylaminophenyl)fulleropyr-
rolidine with 38% yield after purification by flash chromatogra-
phy. This fullerene amino derivative was treated with an excess
of methyl iodide at 70 ꢂC for 72 h in DMF (Scheme 1). The ex-
haustive methylation produces DTC with 95% yield.
60
comparison with C60 as
fF ¼ (3.0 ꢃ 0.3) ꢄ 10ꢁ4
a
reference, the values
,
(2.0 ꢃ 0.2) ꢄ 10ꢁþ4 and (5.4 ꢃ
2þ
0.3) ꢄ 10ꢁ4 were obtained for MAC60, DAC60 and DTC , re-
60
2þ
spectively. According to the photophysical properties of fuller-
opyrrolidines, low values of fF are expected [3,28,43]. In all
cases, a small Stokes shift (w6 nm) was observed indicating
that the spectroscopic energy is nearly identical to the relaxed
energy of the singlet state. Taking into account the energy of
0 / 0 electronic transitions, the energy levels of the singlet
excited state (Es) were calculated giving in the three cases
a value of 1.77 eV. These results are in agreement with those
previously reported for this family of photosensitizers [3,43].
A similar procedure of cycloaddition was used to obtain
MAC60 from a mixture of C60, 4-acetamidobenzaldehyde
and N-methylglycine with 41%þyield (Scheme 2). Methylation
of MAC60 yields 96% of DAC60.
2þ
The compound DTC bears a hydrophobic carbon sphere
60
substituted by two cationic groups forming one amphiphilic
monoadduct. To evaluate the effect produced by the last
groups upon the intramolecular polarity, the dipole moment
of fullerene derivatives was estimated. Semiempirical method
for molecular modeling (AM1) was used in structure geometry
3.3. Photodynamic activity
optimization and calculatþions giving values of 3.4, 17.3 and
2þ
46.0 D for MAC60, DAC60 and DTC , respectively. As ex-
60
The photooxidation of 1,3-diphenylisobenzofurane
(DBPF) sensitized by fullerene derivatives was investigated
in homogenic DMF/water (10% v/v) medium and reverse
micellar system formed by benzene/BHDC (0.1 M)/water
(W0 ¼ 10). It is generally assumed that DBPF is decomposed
only by O2(1Dg) generated by type II photoprocess to pro-
duce 1,2-dibenzoylbenzene. Moreover, it is known that
DPBF quenches almost exclusively by chemical reaction,
physical quenching being negligible [44,45]. Therefore, it
pected, the presence of a dicationic moiety in the periphery
of fullerene derivative considerably enhances the dipole mo-
ment with respect to the non-charged structures.
3.2. Spectroscopic studies
The UVevisible absorption spectra of MAC60, DACþ60 and
2þ
DTC
were performed in homogeneous media and in
60
I-
+N
I-
1. toluene
+
reflux, 6 h, Ar
38%
N
O
H
OH
N
H
C60
+
+
N
O
2. CH3I, DMF
reflux, 72 h, Ar
95%
2+
DTC60
Scheme 1.

M.B. Spesia et al. / European Journal of Medicinal Chemistry 43 (2008) 853e861
857
+N
I-
1. toluene
reflux, 6 h, Ar
MAC60, 41%
NH
O
H
OH
N
H
C60
+
+
NH
O
O
O
2. CH3I, DMF
reflux, 72 h, Ar
96%
+
DAC60
Scheme 2.
was used in this work to evaluate the ability of the sensitizers
to produce O2(1Dg). A time-dependent decrease in the DBPF
concentration was observed by following a decrease in its
absorbance at 415 nm (Fig. 3). From first-order kinetic plots
the values of the observed rate constant (kobs) were calcu-
lated for DBPF. The results are gathered in Table 1. In this
condition, the quantum yield of O2(1Dg) production (FD)
was calculated comparing the slope for fulleropyrrolidines
with the corresponding slope obtained for the reference,
C60. In DMF/water, non-charged MAC60 and C60, photode-
composed DBPF with comparable rates, indicating that
O2(1Dg) is efficiently produced by MAC60 in this medium.
Values of FD ¼ 1 and 0.72 were calculated for C60 and
MC60, respectively. Similar results of singlet oxygen produc-
tion were previously found for C60 and bis(ethoxycarbo-
nyl)C61 measuring 1268 nm emissions in CS2 [40].
Introduction of substituents on fullerene core produces a de-
crease in the photodynamic activity and it appears that this
Although, fullerene in micelles was not used to perform the
biological experiments, these microheterogeneous systems are
frequently used as an interesting model to mimic the water
pockets that are often found in various bioaggregates such as
proteins, enzymes and membranes [46]. Thus, water-soluble
and water-insoluble compounds can be dissolved simulta-
neously in reverse micelles, which simulate a biomimetic micro-
environment [47]. The rates of DBPF decomposition sensitized
by fullerene derivatives in benzene/BHDC/water micelles are
given in Table 1. As can be observed, in micellar system the
value of kobs sensitized by MAC60 is w4 times higher than
that obtained with DTC26þ0 , while in the homogenic medium
this ratio reaches a value of w15 times. This means that in the
microheterogeneous media the photosensitizing activity of
2þ
DTC is more similar to that of the non-charged fullerene.
60
This enhancement in reverse micellar system is probably facil-
itated for a better solubilization as monomer of DTC260þ, like it
was previously shown by absorption spectroscopic studies.
Thus, it can be noted that O2(1Dg) production is very dependent
on the medium where the sensitizer is localized and consider-
ably diminishes when the photosensitizer is aggregated.
effect is not significantly dependent on the kind of addend.
2þ
On the other hand, DBPF reaction sensitized by DTC
was one order of magnitude lower than those of non-charged
60
2þ
fullerene derivatives. This low O2(1Dg) production of DTC
60
is probably due to an incomplete monomerization of the cat-
ionic fullerene in the DMF/water medium.
3.4. Studies in vitro on E. coli cells
2þ
Fullerene derivatives, MAC60, DACþ60 and DTC , were
60
evaluated as photodynamic agents in vitro using a typical
1.0
0.8
0.6
1.0
0.8
0.6
0.4
0.2
0.0
600
700
800
0.4
0.2
0.0
400
600
800
wavelength (nm)
600
700
800
2þ
Fig. 1. Absorption spectra of DTC in different media, DMF (dashed line),
60
wavelength (nm)
PBS (dotted line), toluene (dashed dotted line) and benzene/BHDC (0.1 M)/
2þ
2þ
60
W0 ¼ 10 (solid line). Inset: DTC spectrum enlargement in benzene/BHDC
60
Fig. 2. Fluorescence emission spectra of MAC60 (solid line) and DTC
(dashed line) in DMF, lexc ¼ 450 nm.
(0.1 M)/W0 ¼ 10.

858
M.B. Spesia et al. / European Journal of Medicinal Chemistry 43 (2008) 853e861
toxicity was detected for MAC60. However, compound
2þ
A
DTC was completely dark toxic to E. coli and formation of
1.0
0.8
0.6
0.4
0.2
0.0
60
colonies was not detected. When 2.5 mM of sensitizer was
2þ
used, compound DTC showed only minor dark toxicity to-
60
ward E. coli producing 23% of cellular inactivation. In previous
studies, two isomers of C60-bis(N,N-dimethylpyrrolidinium) io-
dide were used to study the bacteriostatic effects of fullerene
derivatives on E. coli cells [48]. The inactivation of E. coli
growth was attributed to the inhibition of energy metabolism
by two opposite dose-dependent mechanisms. At low fullerene
concentration the oxygen uptake is decreased, on the contrary at
high concentration the oxygen uptake increases and oxygen is
converted to H2O2. Besides, these fullerene concentrations in-
hibit the respiratory chain activity without photoirradiation. Fi-
nally in our case, no dark toxicity was found when cell cultures
2þ
were treated with 1 mM of DTC and therefore it was selected
60
for photodynamic in vitro studies.
0
100
200
300
time (s)
400
500
The capacity of these photosensitizers to bind to bacterial
cells was analyzed by fluorescence emission spectra according
to the procedure previously described in the literature [32,33].
The E. coli cultures were incubated with 1 mM of fullerene for
different times (5, 10 and 30 min) at 37 ꢂC in the dark.
However, the emission of fullerene in SDS (2%) solution
was negligible due to the low fluorescence intensity of these
compounds.
Suspensions of E. coli cells in PBS were treated with 1 mM
of sensitizer for 30 min at 37 ꢂC in dark and then the cultures
were irradiated with visible light. Fig. 4 shows the survival of
bacterial cells after different irradiation times. Control exper-
iments showed that the viability of E. coli was unaffected by
illumination alone or by dark incubation with 1 mM of the
1.5
1.2
0.9
0.6
0.3
0.0
B
0
100
200
time (s)
300
400
6
5
4
3
2
Fig. 3. First-order plots for the photooxidation of DPFB (20 mM) photosensi-
tized by MAC60 ) and C60 (C) in (A) DMF/wa-
), DACþ60 (-), DTC2þ
(
(
:
;
60
ter (10%) and (B) benzene/BHDC (0.1 M)/W0 ¼ 10, lirr ¼ 470 nm. Values
represent mean ꢃ standard deviation of three separate experiments.
Gram-negative bacterium, E. coli. First, the cell toxicity in-
duced by these fullerenes was analyzed in the absence of light
at different photosensitizer concentrations. When the cultures
were treated with 5 mM of sensitizer for 30 min in dark, no
Table 1
Kinetic parameters (kobs) and quantum yield of O2 (1Dg) production (FD) of
fullerene derivatives in different media
a
0
10
irradiation time (min)
20
30
Fullerenes
kobs (sꢁ1) DMF/watera
FD
kobs (sꢁ1) micellesc
C60
MAC60
(2.97 ꢃ 0.08) ꢄ 10ꢁ3
(2.14 ꢃ 0.07) ꢄ 10ꢁ3
(2.05 ꢃ 0.06) ꢄ 10ꢁ3
(0.20 ꢃ 0.02) ꢄ 10ꢁ3
1b
(3.24 ꢃ 0.09) ꢄ 10ꢁ3
(1.60 ꢃ 0.08) ꢄ 10ꢁ3
(3.40 ꢃ 0.09) ꢄ 10ꢁ3
(0.80 ꢃ 0.05) ꢄ 10ꢁ3
0.72 ꢃ 0.05
0.69 ꢃ 0.05
0.07 ꢃ 0.01
Fig. 4. Survival curves of E. coli cells (w106 CFU/mL) incubated with 1 mM
of MAC60
), DACþ60 (-) and DTC2þ ) for 30 min at 37 ꢂC in dark and
DACþ60
(
(
:
;
60
2þ
DTC
60
exposed to visible light for difþferent irradiation times and washing once the
a
2þ
DMF/water (10%).
Ref. [28].
Benzene/BHDC (0.1 M)/water (W0 ¼ 10).
cells before illumination DAC60 (,) and DTC
(
6
). Control culture un-
60
b
c
treated irradiated (C). Values represent mean ꢃ standard deviation of three
separate experiments.

M.B. Spesia et al. / European Journal of Medicinal Chemistry 43 (2008) 853e861
859
2þ
photosensitizer for 30 min, indicating that the cell mortality
obtained after irradiation of the cultures treated with the fuller-
ene is due to the photosensitization effect of the agent pro-
duced by visible light.
higher concentration than that used with DTC [27]. Also,
60
in previous studies cationic fullerenes bearing two quaternary
pyrrolidinium groups on sphere were evaluated to eradicate
microbes. After incubation, the bis-cationic fullerene was
highly active in killing E. coli cells producing a 4 log of kill-
ing [31].
On the other hand, after one washing step the photoinacti-
vation of E. coli cells follows the tendency shown in Fig. 4.
Under this condition, the photodynamic effect is mainly asso-
ciated with porphyrins that are tightly bound to cells. As can
be observed, the photoinactivation activity of dicationic fuller-
ene remains similar to those found for unwashed cells. Thus,
the enhanced photodynamic activity imputed to the interac-
tions of positive charges on the E. coli cellular surface is stable
enough to survive the washing.
Photosensitized growth delay of E. coli cultures by fuller-
enes was carried out in medium (10% w/v TS broth). This
experiment was performed to ensure that PDI of cells is still
possible when the cultures were not under starvation condi-
tions or the potential damaging effects of phosphate buffer
washing [18,27,33,49]. The cultures of E. coli in lag phase
were treated with 2 mM of sensitizer and the flasks were ir-
radiated with visible light at 37 ꢂC. As can be observed in
The viability of E. coli cells after irradiation was dependent
upon both the fullerene derivatives used in the treatment and
the light exposure level. As expected for non-charged photo-
sensitizers, no inactivation effect was found for cultures
treated with 1 mM of MAC60 still after 30 min of irradiation.
Similar behavior was also found using 5 mM of MAC60.
This result is in agreement with that reported before for
non-charged fullerene derivatives, indicating that these non-
cationic sensitizers are unsuccessful sensitizers for Gram-
negative bacteria under these conditions [31].
On the other hand, as it can be observed in Fig. 4 the E. coli
cells are rapidly photoinactivated when the unwashed cultures
2þ
treated with dicationic DTC are exposed to visible light. In
60
particular, the dicationic fullerene exhibits a photosensitizing
activity causing a w3.5 log decrease of cell survival, when
the cultures are irradiated for 30 min. These results represent
a value greater than 99.97% of cellular inactivation.
2þ
The photodynamic activity sensitized by DTC can be di-
60
rectly compared with those of other different family of cat-
ionic sensitizers under similar irradiation conditions by using
the same E. coli strain. Thus, when E. coli cultures were
treated with 1 mM of a cis dicationic porphyrin, 5,10-bis(4-
methylphenyl)-15,20-bis(N,N,N-trimethylammoniumphenyl)-
porphyrin iodide, 70% of cellular inactivation was found after
Fig. 5, growth was arrested when E. coli cultures were
2þ
treated with DTC
and illuminated. After irradiation in
60
the presence of dicationic fullerene, the cells no longer ap-
peared to be growing as measured by turbidity at 660 nm.
Furthermore, a minor effect in the growth delay was found
for cells treated with MAC60. On the other hand, E. coli
cells exposed to sensitizers in the dark or not treated with
sensitizer and illuminated showed no growth delay
5 min of irradiation with visible light, whereas under compa-
2þ
rable condition, DTC
inactivates 86% of bacterial cells
60
[23]. When the E. coli cultures were incubated with 5 mM of
cis dicationic porphyrin and irradiated for 30 min, the PDI ef-
fect produces a w3.2 log decrease of cell survival. This value
is comparable with that obtained using 1 mM of DTC
2þ
0.6
0.5
0.4
0.3
0.2
0.1
0.0
60
(Fig. 4) [32]. Also, the behavior of dicationic fullerene deriv-
ative can be compared with that of a trans dicationic porphyrin,
5,15-bis[(4-3-N,N,N-trimethylammoniumpropoxy)phenyl]-10,
20-bis(4-trifluoromethylphenyl)porphyrin iodide [33]. Irradia-
tion of E. coli cultures treated with 1 mM of this trans
dicationic porphyrin photoinactivates w87% of the cells
even after 20 min of irradiation. Therefore these comparisons
2þ
indicate that 1 mM of DTC produces a similar photoinacti-
60
vation of E. coli cells than those of dicationic porphyrin after
2þ
5 min of irradiation; though DTC causes a higher decrease
60
of cell survival after 30 min of irradiation. On the other hand,
porphyrin derivatives bearing three of four cationic charges
produce a higher photoinactivation than that found for
DTC26þ0 , probably due to a tighter electrostatic interaction
with negatively charged sites at the outer surface of E. coli
2þ
cells [23,32,33]. Also, DTC can be compared with cationic
60
0
100
200
300
400
phthalocyanine derivatives. Per example, 1 mM of dicationic
fullerene is more effective than 10 mM of a cationic Zn(II)
tetramethyltetrapyridinoporphyrazinium salt, which produces
w2 log decrease of cell survival after 30 min of irradiation
[26]. The photoinactivation sensitized by 10 mM of a cationic
Zn(II) N-methylpyridyloxyphthalocyanine causes a w4.5 log
decrease of E. coli cell survival, even so it required 10 times
time (min)
Fig. 5. Photosensitized growth delay curves of E. coli cells incubated with
2 mM of MAC60
(
) and DTC2þ
(
) and exposed to different irradiation
:
;
60
times with visible light in 10% w/v TS broth at 37 ꢂC. Control culture un-
treated irradiated (C), untreated in dark (B), treated with 2 mM of MAC60
(
7
) and DTC2þ
of three separate experiments.
(
) in dark. Values represent mean ꢃ standard deviation
6
60

860
M.B. Spesia et al. / European Journal of Medicinal Chemistry 43 (2008) 853e861
compared with controls. Therefore, the data illustrate that
the observed growth delay is due to the photoinactivation
effect of the sensitizers on the cells [32]. Similar behavior
was previously observed for E. coli cultures in the presence
of 10 mM of dicationic porphyrin derivatives under analo-
gous experimental conditions [32,33].
CET. M.B.S. and M.E.M. thank CONICET for a doctoral
fellowship.
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