A cyclodextrin-based nanoassembly with bimodal photodynamic action

Article abstract of DOI:10.1002/chem.201101635

We have developed a supramolecular nanoassembly capable of inducing remarkable levels of cancer cell mortality through a bimodal action based on the simultaneous photogeneration of nitric oxide (NO) and singlet oxygen ( ¹O₂). This was achieved through the appropriate incorporation of an anionic porphyrin (as ¹O₂ photosensitizer) and of a tailored NO photodonor in different compartments of biocompatible nanoparticles based on cationic amphiphilic cyclodextrins. The combination of steady-state and time-resolved spectroscopic techniques showed the absence of significant intra- and interchromophoric interaction between the two photoactive centers embedded in the nanoparticles, with consequent preservation of their photodynamic properties. Photodelivery of NO and ¹O₂ from the nanoassembly on visible light excitation was unambiguously demonstrated by direct and real-time monitoring of these transient species through amperometric and time-resolved infrared luminescence measurements, respectively. The typical red fluorescence of the porphyrin units was essentially unaffected in the bichromophoric nanoassembly, allowing its localization in living cells. The convergence of the dual therapeutic action and the imaging capacities in one single structure makes this supramolecular architecture an appealing, multifunctional candidate for applications in biomedical research. Copyright

Full text of DOI:10.1002/chem.201101635

DOI: 10.1002/chem.201101635
A Cyclodextrin-Based Nanoassembly with Bimodal Photodynamic Action
Noufal Kandoth,[a] Elisa Vittorino,[a] Maria Teresa Sciortino,[b] Tiziana Parisi,[b]
Ivana Colao,[b] Antonino Mazzaglia,*[c] and Salvatore Sortino*[a]
Abstract: We have developed a supra-
molecular nanoassembly capable of in-
ducing remarkable levels of cancer cell
showed the absence of significant intra-
and interchromophoric interaction be-
tween the two photoactive centers em-
bedded in the nanoparticles, with con-
sequent preservation of their photody-
namic properties. Photodelivery of NO
and 1O2 from the nanoassembly on
visible light excitation was unambigu-
ously demonstrated by direct and real-
time monitoring of these transient spe-
cies through amperometric and time-
resolved infrared luminescence meas-
urements, respectively. The typical red
fluorescence of the porphyrin units was
essentially unaffected in the bichromo-
phoric nanoassembly, allowing its local-
ization in living cells. The convergence
of the dual therapeutic action and the
imaging capacities in one single struc-
ture makes this supramolecular archi-
tecture an appealing, multifunctional
candidate for applications in biomedi-
cal research.
mortality through
a bimodal action
based on the simultaneous photogener-
ation of nitric oxide (NO) and singlet
oxygen (1O2). This was achieved
through the appropriate incorporation
1
of an anionic porphyrin (as O2 photo-
sensitizer) and of a tailored NO photo-
donor in different compartments of
biocompatible nanoparticles based on
cationic amphiphilic cyclodextrins. The
combination of steady-state and time-
Keywords: cyclodextrins
·
drug
delivery · imaging agents · nitric
oxide · phototherapeutic agents ·
singlet oxygen
resolved
spectroscopic
techniques
Introduction
make phototherapeutic agents a powerful arsenal for treat-
ing cancer diseases in a noninvasive way,[3] avoiding the pos-
sible complications of surgery.[4]
Bimodal therapies aim to exploit either additive or synergis-
tic effects arising from the generation of two active species
in the same region of space, with the final goal of maximiz-
ing the therapeutic efficacy. The site of action, timing, and
dosage of the delivered species play key roles in determin-
ing the therapeutic outcome.[1] Light is a highly orthogonal
trigger for the rapid introduction of therapeutic agents in a
desired bio-environment with the potential for precise con-
trol over all the above factors and the additional advantage
of not affecting important physiological parameters such as
temperature, pH, and ionic strength.[2] These properties
Nitric oxide (NO) is one of the most appealing and in-
tensely studied molecules in the fascinating realm of the bio-
medical sciences.[5] Besides its pivotal role in the mainte-
nance and bioregulation of vital functions,[6] NO has recently
stimulated an upsurge of interest because of its promising
anticancer activity.[7] This exciting discovery has made the
development of new strategies and methods for NO photo-
delivery a hot topic with the intriguing prospect of tackling
cancer diseases.[8] In this regard, the use of NO in conjunc-
tion with other anticancer species is highly desirable for ef-
fective therapeutic action.[9]
Singlet oxygen (1O2) is the best-known phototherapeutic
agent and its role in photodynamic therapy (PDT) is well es-
1
tablished.[10] In PDT, the cytotoxic O2 is generated by pho-
[a] N. Kandoth, Dr. E. Vittorino, Prof. S. Sortino
Laboratory of Photochemistry
toinduced energy transfer between the lowest excited triplet
state of a suitable photosensitizer (e.g., a porphyrin or
phthalocyanine) and nearby molecular oxygen.[11] Unlike
other reactive oxygen species (that is, hydrogen peroxide
and superoxide radical), 1O2 is not consumed by enzymes
such as catalase and superoxide dismutase produced by
cancer cells.[12] Furthermore, PDT is not affected by multiple
drug resistance, overcoming the major problems faced in
chemo- and radiotherapy.[13] The combination of 1O2 with
NO therefore in principle represents an ideal strategy for bi-
modal treatments. On the basis of these considerations the
objective of this work was to develop a multifunctional mo-
lecular ensemble capable of simultaneous photogeneration
Department of Drug Sciences, Viale Andrea Doria 6
95125 Catania (Italy)
Fax : (+39)095580138
E-mail: ssortino@unict.it
[b] M. T. Sciortino, T. Parisi, I. Colao
Dipartimento di Scienze della Vita
Sezione di Scienze Microbiologiche Genetiche e Molecolari
Universitꢀ di Messina, Salita Sperone, 98166 Messina (Italy)
[c] Dr. A. Mazzaglia
Istituto per lo Studio dei Materiali Nanostrutturati, ISMN-CNR
c/o Dipartimento di Chimica Inorganica Analitica e Chimica Fisica
Universitꢀ di Messina, Salita Sperone, 98166 Messina (Italy)
Fax : (+39)090393756
E-mail: antonino.mazzaglia@ismn.cnr.it
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Chem. Eur. J. 2012, 18, 1684 – 1690

FULL PAPER
of 1O2 and NO and offering imaging capacities in living
cells.
Results and Discussion
Nanotechnology offers enormous opportunities for the
construction of effective delivery vehicles.[14] In the case of
photoactivated compounds, three of the most important cri-
teria that a carrier system would meet are: 1) biocompatibil-
ity, 2) cell-penetrating properties, and 3) preservation of the
photodynamic activity of the photosensitizer.
When CD 1 and the anionic porphyrin 2 are mixed together
at a molar ratio of approximately 50:1, spontaneous forma-
tion of amphiphilic NPs approximately 300 nm in diameter
is observed, according to our previous results.[18] Under
these experimental conditions, the porphyrin is mainly en-
tangled as the monomeric form, which is responsible for the
1O2 generation (a and b in Figure 1).
Cyclodextrins (CDs) are water-soluble oligosaccharides
well-known for the formation of host–guest inclusion com-
plexes with a range of substrates.[15] Appropriate functionali-
zation of the primary, secondary, or both sides of CDs leads
to intriguing derivatives with amphiphilic character, able to
self-organize in a variety of assemblies such as micelles, vesi-
cles, and nanoparticles[16] with great potential in drug deliv-
ery.[17] Previously, we prepared and characterized heterotop-
ic colloidal nanoparticles (NPs) based on the cationic am-
phiphilic CD 1 (Scheme 1), which is capable of trapping the
oppositely charged porphyrin 2, mainly through electrostatic
interactions. The NPs have hydrodynamic radii ranging from
ꢀ140 to ꢀ1000 nm, depending on the porphyrin loading.[18]
We also showed that such NPs are promising vehicles for
photodynamic therapy, because they combine low immuno-
genic activity, high capability to convey photosensitizers into
tumor cells, and excellent levels of cell mortality upon light
irradiation, due to effective photogeneration of 1O2.[19] In ad-
dition, we demonstrated that the porphyrin units are not
hosted in the interiors or vicinities of the CD cavities, offer-
ing the opportunity to exploit the empty cages in the CD-
based NPs for the accommodation of additional guests.[20] In
the wake of these promising results and motivated by our
ongoing interest in developing NO photoreleasing sys-
tems,[21] here we report a fluorescent supramolecular nano-
assembly capable of photo-deactivating cancer cells through
Figure 1. Absorption spectra (phosphate buffer, 10 mm, pH 7.4) of 2 a) in
the absence and b) in the presence of CD 1, c) of 3 in the presence of
CD 1, d) of the model compound 4 , and e) of 2+3 in the presence of
CD 1. [1]=40 mm, [2]=0.8 mm, [3]=10 mm, T=258C, cell path=1 cm.
The inset shows the size distribution of the bichromophoric nanoassem-
bly obtained by dynamic light scattering measurements.
1
a bimodal action due to the simultaneous generation of O2
To introduce an appropriate NO photoreleaser within
and NO under visible light excitation conditions (Scheme 1).
these NPs, we designed and synthesized compound
3
Scheme 1. Idealized view of the photoactive CD-based bichromophoric nanoassembly.
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1685

S. Sortino, A. Mazzaglia et al.
(Scheme 1), in which a commercial nitroaniline derivative
and an adamantane appendage are joined together through
an alkyl spacer. We have discovered the nitroaniline deriva-
tive to be a suitable NO photodonor[22] because it satisfies
several prerequisites for bio-applications. In this compound,
the twisted conformation of the nitro group with respect to
the aromatic plane is crucial for the NO photorelease. How-
ever, it has been shown that incorporation of this chromo-
phore within constrained environments, such as b-CD cavi-
ties[23] or densely packed vesicles,[24] can lead to partial pla-
narization of the nitro group with consequent dramatic
modification of the NO photoreleasing properties. With this
in mind, the rationale behind the design of compound 3 was
i) that the adamantane, a well-known and effective guest for
b-CD,[25] would be expected to occupy the empty cavities in
NPs based on CD 1,[26] and ii) that use of an alkyl spacer of
appropriate length should further encourage the binding of
3 with the NPs through cooperative hydrophobic interchain
interactions involving the alkyl branches of CD 1. It was en-
visaged that these effects should leave the nitroaniline chro-
mophore mainly exposed to an aqueous environment, pre-
serving the out-of-plane geometry and consequently the NO
photoreleasing capacity. The validity of our design is demon-
strated by the absorption spectra shown in Figure 1. Com-
pound 3 is insoluble in aqueous solution. On the other hand,
it becomes fairly soluble in the presence of the NPs based
on CD 1, as shown by the appearance of the characteristic
absorption of the nitroaniline chromophore in the visible
region (spectrum c). These absorption features are basically
the same as those exhibited by the water-soluble model
compound 4 in the absence of NPs (spectrum d). Because
the energy of the visible absorption band is very sensitive
both to microenvironment polarity and to changes in the ge-
ometry of the nitro group,[24] the absence of any significant
spectral shift is highly consistent with the photoactive core
being mainly exposed to an aqueous environment.
nature of compound 3, which does not influence the charge
balance between the anionic 2 and the cationic CD 1. Such
a balance has been demonstrated to be critical in driving
porphyrin reorganization and promoting the fusion of the
amphiphilic NPs into large aggregates of several hundreds
of nanometers.[18]
The entangling of the monomeric porphyrin within the
CD-based NPs results in a significant redshift of its typical
dual band fluorescence emission with negligible changes in
the fluorescence quantum yield (Figure 2A).[27] Interestingly,
these emission properties were only slightly affected in the
case of the bichromophoric nanoassembly, ruling out any in-
tramolecular quenching (i.e., photoinduced electron-trans-
fer) of the excited porphyrin by the NO photodonor. It is
also worth noting that the positions of the two emission
bands were independent of the excitation energy (data not
shown), suggesting the presence in the nanoassembly mainly
The bichromophoric nanoassemblies were prepared by
loading the CD-based NPs with 2 and 3 at the appropriate
molar ratio. The spectral characteristics observed (e in
Figure 1) match the profile obtained by summing the spectra
of the NPs loaded with the individual chromophores 2 or 3
(b and c in Figure 1) fairly well, with an experimental uncer-
tainty of approximately 15% (in absorbance values). This
confirms their concurrent presence within the NPs and the
absence of relevant interactions with one another in the
ground state. Moreover, the unaltered position of the maxi-
mum of the Soret band of 2 indicates that the presence of
compound 3 does not induce any rearrangement (i.e., dis-
placement/aggregation) of the porphyrins. This might be the
consequence of different affinities of the two components
for different binding sites in the NPs, consistently with the
idealized view illustrated in Scheme 1.
Figure 2. A) Fluorescence emission spectra (lexc =440 nm) of 2 a) in the
absence and b) in the presence of CD 1, and c) of 2+3 in the presence of
CD 1; the inset shows a representative microscopy image of HeLa cancer
cells incubated with the bichromophoric nanoassembly for 1 h at 378C.
B) Transient absorption spectra observed upon laser excitation (532 nm)
Dynamic light scattering measurements showed the mean
diameter of the bichromophoric nanoassemblies to be ap-
proximately 300 nm (Figure 1, inset), a value similar to that
already reported for the NPs loaded with 2 alone at the
same molar ratio.[18] This is consistent with the uncharged
&
of Ar-saturated CD 1 solution loaded with 2 ( ) and 2+3 (*), recorded
0.1 ms after the laser pulse. Each point was obtained by signal averaging
of 10 traces. The inset shows the decay trace monitored at 450 nm. E532
ꢀ12 mJ per pulse. Phosphate buffer (10 mm, pH 7.4), [1]=40 mm, [2]=
0.8 mm, [3]=10 mm, T=258C.
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A Cyclodextrin-Based Nanoassembly with Bimodal Photodynamic Action
FULL PAPER
of a single population of fluorophores (i.e., the monomeric
form). The preservation of the fluorescence properties of
the porphyrins under these experimental conditions repre-
sents a great advantage for mapping the nanoassembly in
living cells. The inset of Figure 2A shows a representative
optical image recorded in fluorescence mode after incuba-
tion of the bichromophoric NPs with HeLa tumor cells. This
clearly represents the internalization of the supramolecular
system in the cell compartment and shows that the localiza-
tion takes place mainly at the cytoplasmatic level.
The excited triplet state of the porphyrin is the key inter-
1
mediate for the photosensitization of O2 and its effective
generation is thus crucial for the photodynamic action.[11]
Figure 2B shows the characteristic triplet absorption of 2
when entangled within the NPs in the absence and in the
presence of the NO photodonor 3. Because the two samples
were optically matched at the excitation wavelength, the in-
tensity of the transient absorption is directly related to the
triplet quantum yield. The comparable values obtained for
the two samples indicate that the triplet state of the porphy-
rin is still populated efficiently in the bichromophoric nano-
assembly. Furthermore, the monoexponential decay of the
triplet with a lifetime of approximately 1500 ms (inset, Fig-
ure 2B), which is the same as that observed in the absence
of 3,[19] indicates that no quenching by the NO photodonor
occurs. Note that the observed lifetime is much longer than
that of the free porphyrin in aqueous medium in the ab-
sence of NPs (ca. 200 ms),[19] ruling out any exit dynamics of
Figure 3. A) NO released upon light irradiation (400 nm) of the bichro-
mophoric CD-based nanoassembly, and B) representative kinetic trace of
1O2 generated upon laser excitation (532 nm). Phosphate buffer (10 mm,
pH 7.4), [1]=40 mm, [2]=0.8 mm, [3]=10 mm, T=258C.
the porphyrin triplet from the nanoassembly on this time-
scale.
The most convenient method for testing the suitability of
1
the nanoassembly for photogeneration of NO and O2 is the
real-time monitoring of these transient species. To this end,
an ultrasensitive NO electrode was used to detect NO con-
centrations by an amperometric technique, whereas time-re-
solved infrared luminescence was employed to monitor the
typical phosphorescence of 1O2 at 1270 nm.[28] Figure 3
shows unambiguous evidence of the light-controlled genera-
1
tion of NO and O2. Indeed, we observed the linear photo-
generation of NO, which promptly stopped when the light
was turned off and restarted as the light was turned on
again. It is also noteworthy that the rate of NO release in
the nanoassembly is comparable to that obtained for the
NO photodonor within the NPs in the absence of por
ACHTUNGTNERpNUNG hy-
ACHTUNGTRENNUNGrins, indicating that the porphyrin units do not quench the
photoexcited NO donor. In addition, we observed the char-
acteristic infrared luminescence of 1O2, decaying by first-
order kinetics with a lifetime of approximately 4 ms.
To validate the feasibility of using the bichromophoric
nanoassembly for dual-function phototherapeutic activity,
cancer cells were incubated for 1 h under different experi-
mental conditions and either kept in the dark or irradiated
with visible light for 30 min. The results illustrated in
Figure 4 show that only irradiation of the photoactive com-
ponents in the presence of the CD-based NPs leads to suc-
cessful photomortality of the cells, thus confirming the pho-
todynamic effects.[29] No significant cell death was detected
Figure 4. Cell viabilities of HeLa cells incubated with the NPs based on
CD 1 (40 mm): a) without the photoactive components and loaded with
b) 2 (0.8 mm), c) 3 (10 mm), and d) 2+3.
in the cells that were incubated in the dark or in the absence
of the photoactive compounds, indicating a good biocompat-
ibility of the CD-based NPs. The almost complete photody-
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S. Sortino, A. Mazzaglia et al.
(3.92 mL, 22.96 mmol) was added. The reaction mixture was stirred over-
night at room temperature and afterwards the excess of NaH was
quenched by addition of water. The mixture was concentrated under re-
duced pressure and the aqueous phase was extracted with Et2O (3ꢂ
10 mL). The organic phases were then collected, washed, dried with
Na2SO4, filtered, concentrated under reduced pressure, and purified by
column chromatography (dichloromethane/cyclohexane 30:70) to give 3a
(yield 90%). 1H NMR (CDCl3, 500 MHz): d=3.56 (d, J=6.50 Hz, 2H),
3.26 (t, J=6.68 Hz, 2H), 1.90–1.84 (m, 3H), 1.72–1.35 ppm (m, 24H).
namic inactivation induced by the bichromophoric nanoas-
sembly—approximately 98%—in relation to the value ob-
served with the NPs loaded with the single components 2 or
3 provides clear-cut evidence of the involvement of a
double-action photoinactivation mechanism in the cell
1
death, in which NO and O2 are believed to play a key role.
[7-(Adamantan-1-yloxy)heptyl]-[4-nitro-3-(trifluoromethyl)phenyl]amine
(3): A mixture of 3a (1 g, 3.03 mmol) and 4-nitro-3-(trifluoromethyl)ani-
line (200 mg, 1.01 mmoli) was heated at reflux in acetonitrile for 5 days.
The organic mixture was dried under vacuum and purified by column
chromatography (dichloromethane/cyclohexane 70:30) to afford com-
Conclusion
We have developed a multifunctional photoactive nanoas-
sembly that exploits the different affinities of a O2 photo-
1
pound
3 as a
yellowish powder (yield 60%). 1H NMR (CDCl3,
sensitizer and a tailor-made NO photodonor towards differ-
ent compartments in CD-based NPs. Both guests constitute
independent photoactive centers, as demonstrated by the
preservation of their photophysical and photochemical prop-
erties after confinement within the NPs network. We would
like to stress that this finding, in contrast with the case of
non-photoresponsive compounds, is not obvious. In most
cases, in fact, the photoresponse of single or multiple photo-
active units located in a confined space can be considerably
affected by the occurrence of competitive photoprocesses
(e.g., photoinduced energy and/or electron transfer, hydro-
gen abstraction, nonradiative deactivation, etc.),[30] which
preclude the final goal.
500 MHz): d=7.95 (d, J=9.2 Hz, 1H), 6.80 (d, J=2.4 Hz, 1H), 6.56 (dd,
J1 =9.2 Hz, J2 =2.4 Hz, 1H), 4.47 (broad, 1H), 3.34 (dd, J1 =9.5 Hz, J2 =
6.5 Hz, 2H), 3.15 (dd, J1 =9.8 Hz, J2 =6.9 Hz, 2H), 1.83–1.77 (m, 3H),
1.63–1.57 (m, 6H) 1.44–1.18 ppm (m, 16H).
Instrumentation: UV/Vis absorption and fluorescence spectra were re-
corded with a Jasco V-560 spectrophotometer and a Fluorolog-2 (mod. F-
111) spectrofluorimeter, respectively. Nanoparticle sizes were measured
with a dynamic light scattering Horiba LS 550 apparatus fitted with a
diode laser (wavelength 650 nm). Fluorescence images were taken with a
Biomed fluorescence microscope (Leitz, Wetzlar, Germany).
Sample preparation: NPs based on CD 1 were prepared from stock solu-
tions (180 mm) in CHCl3, which were allowed to evaporate slowly to form
thin films. The films were hydrated, sonicated for 20 min at 508C, and al-
lowed to equilibrate overnight. An aqueous solution of the porphyrin 2
was then added and each sample was adjusted to a final volume of 2 mL
with phosphate buffer. Compound 3 was dissolved in acetonitrile and al-
lowed to evaporate slowly to form a thin film. This film was then hydrat-
ed with the colloidal solutions of CD 1 either with or without the porphy-
rin 2. All the final solutions were allowed to equilibrate overnight at 48C,
sonicated for 15 min, and allowed to equilibrate at room temperature for
20 min.
We have demonstrated that the fluorescence emission of
the porphyrin units allows the localization of the nanoas-
sembly in living cells and provides the bichromophoric
1
system with the ability to generate O2 and NO effectively
and concurrently, resulting in an amplified level of cancer
cell mortality. To the best of our knowledge this is the first
report in which cancer cellular death due to the combined
action of these two transient species has been shown. The
uniting of the dual photodynamic action and the imaging ca-
pacities in one single nanostructure, together with its bio-
compatibility, make this supramolecular architecture an ap-
pealing candidate for applications in biomedical research.
We finally envision that the extension of our results to a va-
Laser flash photolysis: All of the samples were excited with the second
harmonic of a Nd-YAG Continuum Surelite II-10 laser (532 nm, 6 ns
FWHM), in quartz cells with a path length of 1.0 cm. The excited solu-
tions were analyzed with a Luzchem Research mLFP-111 apparatus with
an orthogonal pump/probe configuration. The probe source was a ceram-
ic xenon lamp coupled to quartz fiber-optic cables. The laser pulse and
the mLFP-111 system were synchronized with a Tektronix TDS 3032 digi-
tizer, operating in pre-trigger mode. The signals from a compact Hama-
matsu photomultiplier were initially captured by the digitizer and then
transferred to a personal computer, controlled by Luzchem Research
software operating in the National Instruments LabView 5.1 environ-
ment. The solutions were deoxygenated by bubbling with a vigorous and
constant flux of pure argon (previously saturated with solvent). In all of
these experiments, the solutions were renewed after each laser shot (in a
flow cell of 1 cm optical path), to prevent probable autooxidation pro-
cesses. The sample temperature was 295ꢁ2 K. The energy of the laser
pulse was measured at each shot with a SPHD25 Scientech pyroelectric
meter.
1
riety of O2 and NO photodispensers chosen ad hoc might
open fascinating possibilities for novel classes of light-acti-
vated, nanoscaled systems in the emerging field of nanome-
dicine, for multimodal therapy.
Experimental Section
1
Singlet oxygen detection: Photogeneration of O2 upon laser excitation of
Materials: CD 1 and the model compound 4 were synthesized by the pre-
viously reported procedures.[16b,31] The tetraanionic porphyrin 2 was pur-
chased from Sigma–Aldrich and used as received. All other reagents
were of the highest commercial grade available and used without further
purification. All solvents used (Carlo Erba) were analytical grade.
the photosensitizer was monitored by luminescence measurements in
oxygen-saturated solutions. The near-IR luminescence of singlet oxygen
!
3
ꢂ
at 1.27 mm (resulting from the forbidden transition Sg
1Dg) was probed
orthogonally to the exciting beam with a pre-amplified (low impedance)
Ge-photodiode (Hamamatsu EI-P, 300 ns resolution) maintained at
ꢂ1968C and coupled to a long-pass silicon filter (>1.1 mm) and an inter-
Syntheses: The synthesis of [7-(adamantan-1-yloxy)heptyl]-[4-nitro-3-(tri-
fluoromethyl)phenyl]amine (3) was carried out in two steps. Syntheses
were carried out at low light intensity levels.
1
ference filter (1.27 mm). The pure signal of O2 was obtained as the differ-
ence between signals in air- and Ar- saturated solutions. The temporal
profile of the luminescence was fitted to a single-exponential decay func-
tion with exclusion of the initial portion of the plot, which was affected
by scattered excitation light, fluorescence, and the formation profile of
singlet oxygen itself.
1-(7-Bromoheptyloxy)adamantane (3a): A THF solution of adamantanol
(1 g, 6.56 mmol) was added at 08C under argon to a suspension of
sodium hydride (377 mg, 9.84 mmol) in dry THF (10 mL). After the mix-
ture had been left at room temperature for 2 h, 1,7-dibromoheptane
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A Cyclodextrin-Based Nanoassembly with Bimodal Photodynamic Action
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ized with a four-channel recording system and transferred to a PC. The
sensor was accurately calibrated by mixing standard solutions of NaNO2
with H2SO4 (0.1m) and KI (0.1m) according to Equation (1):
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ꢂ
4 Hþ þ 2 Iꢂ þ 2 NO2 ! 2 H2O þ 2 NO þ I2
ð1Þ
Irradiation was performed in a thermostatted quartz cell (1 cm path
length, 3 mL capacity) with gentle stirring and use of monochromatic ra-
diation (400 nm) from the fluorimeter described above (mod. F-111) as
light sources. NO measurements were carried out with the electrode posi-
tioned outside the light path to avoid false NO signals due to photoelec-
tric interference on the ISO-NO electrode.
Experiments with cells: HeLa cells were obtained from the American
Type Culture Collection and propagated at 1:6 ratio with Dulbeccoꢃs
modification of Eagleꢃs Minimal Essential Medium supplemented with
FBS (Fetal Bovine Serum, 10%). Samples of cells treated with the differ-
ent samples of NPs based on CD 1 were placed separately in a cuvette
and irradiated with a halogen lamp (Osram) for 30 min. The irradiating
beam was filtered through a UV filter (Hoya glass type UV-34, cut-off:
340 nm) to cut the UV component and through a 1 cm cell filled with
water to remove the IR component. Before and after irradiation, 8ꢂ104
cells were placed in 96-well plates with RPMI-1640 medium (100 mL) and
incubated in the presence of tetrazolium compound [3-(4,5-dimethylthia-
zol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt; MTS, Promega] and an electron coupling reagent phenazine
methosulfate (PMS) dye (MTS 20 ml per well). After further incubation
(1 h), the absorbance was read at 490 nm in a microplate reader (Labsys-
tems Multiskan Bichromatic). The cell viability (%) was calculated with
the aid of Equation (2):
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cell viability ð%Þ ¼
½A ðbefore lampÞꢂA ðafter lampÞ=A ðbefore lampÞꢃ ꢄ 100
ð2Þ
in which A (sample before lamp) represents measurements from the
wells treated with samples before the exposure to the halogen lamp and
A (sample after lamp) represents measurements from the wells treated
with samples after the exposure to the halogen lamp.
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Acknowledgements
We thank the Marie Curie Program 237962 CYCLON (FP7-PEOPLE-
ITN-2008) for financial support to MC fellow N.K. and funding of the re-
search. Financial support from MIUR (PRIN 2008) is also gratefully ac-
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excitation wavelength.
Received: May 27, 2011
Revised: September 7, 2011
Published online: December 30, 2011
1690
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ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 1684 – 1690