A host-guest supramolecular complex with photoregulated delivery of nitric oxide and fluorescence imaging capacity in cancer cells

Article abstract of DOI:10.1002/asia.201200640

Herein we report the design, preparation, and properties of a supramolecular system based on a tailored nitric oxide (NO) photodonor and a rhodamine-labeled β-cyclodextrin conjugate. The combination of spectroscopic and photochemical experiments shows the absence of significant interchromophoric interactions between the host and the guest in the excited states. As a result, the complex is able to release NO under the exclusive control of visible light, as unambiguously demonstrated by direct detection of this transient species through an amperometric technique, and exhibits the typical red fluorescence of the rhodamine appendage. The supramolecular complex effectively internalizes in HeLa cancer cells as proven by fluorescence microscopy, shows a satisfactory biocompatibility in the dark, and induces about 50 % of cell mortality upon irradiation with visible light. The convergence of all these properties in one single complex makes the present host-guest ensemble an appealing candidate for further delevopment of photoactivatable nanoscaled systems addressed to photostimulated NO-based therapy. NO delivery today: A supramolecular host-guest complex, based on a rhodamine-labeled β-cyclodextrin conjugate and a tailored NO photodonor, effectively internalizes in cancer cells, can be easily mapped intracellularly due to its satisfactory red fluorescence emission, and induces about 50 % of cellular death under the exclusive control of visible light stimuli. Copyright

Full text of DOI:10.1002/asia.201200640

DOI: 10.1002/asia.201200640
A Host–Guest Supramolecular Complex with Photoregulated Delivery of
Nitric Oxide and Fluorescence Imaging Capacity in Cancer Cells
Noufal Kandoth,[a] Milo Malanga,[b] Aurore Fraix,[a] Lꢀszlꢁ Jicsinszky,[b] ꢂva Fenyvesi,*[b]
Tiziana Parisi,[c] Ivana Colao,[c] Maria Teresa Sciortino,[c] and Salvatore Sortino*[a]
Abstract: Herein we report the design,
preparation, and properties of a supra-
molecular system based on a tailored
nitric oxide (NO) photodonor and
of visible light, as unambiguously dem-
onstrated by direct detection of this
transient species through an ampero-
metric technique, and exhibits the typi-
cal red fluorescence of the rhodamine
appendage. The supramolecular com-
plex effectively internalizes in HeLa
cancer cells as proven by fluorescence
microscopy, shows a satisfactory bio-
compatibility in the dark, and induces
about 50% of cell mortality upon irra-
diation with visible light. The conver-
gence of all these properties in one
single complex makes the present
host–guest ensemble an appealing can-
didate for further delevopment of pho-
toactivatable nanoscaled systems ad-
dressed to photostimulated NO-based
therapy.
a
rhodamine-labeled b-cyclodextrin
conjugate. The combination of spectro-
scopic and photochemical experiments
shows the absence of significant inter-
chromophoric interactions between the
host and the guest in the excited states.
As a result, the complex is able to re-
lease NO under the exclusive control
Keywords: cyclodextrins · fluores-
cence · nitric oxide · phototherapy ·
supramolecular chemistry
Introduction
donors much more appealing than those based on spontane-
ous thermolysis for potential use in nanomedicine.[9] Light is
in fact the most elegant and finely tunable external input for
the non-invasive introduction of therapeutic agents in a de-
sired biological environment, mimicking an “optical syringe”
with an exquisite spatiotemporal control.[10] In addition to
providing fast reaction rates, light triggering represents a “bi-
ofriendly” and easily manipulated reactant, and offers the
great benefit of not affecting physiological parameters such
as temperature, pH, and ionic strength, a fundamental requi-
site for biomedical applications.[10]
Due to its half-life of approximately 5 seconds in tissues,
its very small size, its lack of charge, and its lipophilic char-
acter, NO is capable to diffuse some micrometers (40–
200 mm) in the cellular environment. Therefore, this radical
species offers the advantage to confine its reactivity in the
restricted region of space where it has been photogenerated.
Furthermore, the NO radical is able to attack biological sub-
strates of different nature like the plasma membrane,[11] the
mitochondria,[12] and the cell nucleus,[13] thus representing
a multitarget cytotoxic agent and avoiding problems of mul-
tidrug resistance encountered with several conventional an-
ticancer drugs. Finally, since the NO release from NO pho-
todonors is independent from O2 availability, the NO-photo-
stimulated therapy can potentially very well complement
the photodynamic therapy, in which the phototoxicity mech-
anism is entirely dependent on the presence of oxygen,[14] at
the onset of hypoxic conditions.
Nitric oxide (NO) is one of the most appealing and studied
molecules in the fascinating realm of the biomedical scien-
ces.[1] Besides its pivotal role in the maintenance and biore-
gulation of vital functions including neurotransmission, hor-
mone secretion, and vasodilatation in living bodies,[2] this
ephemeral free radical has recently stimulated an upsurge of
interest because of its promising antioxidant, antibacterial,
and anticancer activity.[3–5] These exciting discoveries have
made the development of new strategies and methods for
NO delivery a hot topic[6] with the intriguing prospect to
tackle important diseases, especially cancer.[7] The biological
effects of NO have been shown to be highly site-, concentra-
tion-, and dosage-dependent, creating a complex role for
this molecule in opposing beneficial and deleterious
events.[8] This dichotomy has made the light-controlled NO
[a] N. Kandoth, Dr. A. Fraix, Prof. S. Sortino
Laboratory of Photochemistry, Department of Drug Sciences
University of Catania
Viale Andrea Doria 6, I-95125 Catania (Italy)
Fax : (+39)095-580138
E-mail: ssortino@unict.it
[b] M. Malanga, Dr. L. Jicsinszky, Dr. ꢀ. Fenyvesi
CycloLab Ltd.
Illatos fflt 7, H-1097 Budapest (Hungary)
E-mail: fenyvesi.e@cyclolab.hu
[c] T. Parisi, I. Colao, Dr. M. T. Sciortino
Dipartimento di Scienze della Vita
The cell-penetrating property of the NO photodonors is
another indispensable prerequisite to be fulfilled in NO-
based therapy. This issue has inspired an intense research
activity devoted to develop biocompatibile vehicles.[6a,9a] Cy-
Sezione di Scienze Microbiologiche Genetiche e Molecolari
Università di Messina
Salita Sperone, I-98166 Messina (Italy)
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clodextrins (CDs) comprise a family of biocompatible cyclic
oligosaccharide nanocages, made of a-d-glucopyranose units
joined in a circular way to form a ring with a hydrophilic ex-
terior and a hydrophobic cavity, that are able to host
a range of substrates.[15] Although the chemistry of CDs and
their implementation as carriers of “conventional” drugs
have been greatly developed during the last thirty years,[16]
the suitability of CDs as carriers for photoactivatable thera-
peutic compounds has been only recently object of atten-
tion.[17] Due to their hydrophobic cavity, natural CDs can
host a variety of photosensitive agents by supramolecular in-
teractions.[18] However, in most cases the low binding con-
stants between unmodified CDs and guest molecules are
a major limitation of these systems as bio-carriers. This
drawback makes the modification of the CD structure strict-
ly necessary in view of actual applications.[19] CDs can be se-
lectively mono- or perfacially modified with a plethora of
substituents which can 1) elongate the cavity or make it flex-
ible, thus modifying its binding capacity, 2) alter the hydro-
philic characteristics, 3) introduce charged groups that will
enable cell penetration, and 4) enable them to form conju-
gates with other functional mol-
lar ensemble consisting of the photoresponsive components
1 and 2 (Scheme 1).
The photoactive guest 1 integrates a commercial nitroani-
line derivative and an adamantane appendage, joined to-
gether by an alkyl spacer, within the same molecular skele-
ton. We have demonstrated that such a nitroaniline deriva-
tive is a suitable NO photodonor as it satisfies several pre-
requisites for biological applications.[21a,24] In this chromo-
phoric unit, the twisted conformation of the nitro group
with respect to the aromatic plane is crucial for the NO pho-
torelease. Similarly to other nitroaromatics having the same
conformation, the mechanism of NO release involves an ini-
tial nitro-to-nitrite photorearrangement followed by cleav-
age of the O-NO bond.[25] However, it has been shown that
incorporation of this chromophore within constrained
media, such as either a b-CD cavity[26] or densely packed
vesicles,[27] can lead to a partial planarization of the nitro
group with consequent drastic modification of the NO pho-
toreleasing properties. This finding spurred us to devise
compound 1 in which the adamantane moiety, a very effec-
tive guest for b-CD,[28] is expected to have a much more pro-
ecules.[20]
In the last years we have ach-
ieved a number of NO-photore-
leasing nanoassemblies,[9a] in-
cluding nanoparticles,[21] thin
films[22], and molecular conju-
gates.[23] In the framework of
this ongoing interest, we report
herein the design, preparation,
and properties of a multifunc-
tional, water-soluble host–guest
supramolecular complex based
on a tailor-made NO photodo-
nor and a novel fluorescent b-
CD conjugate. We demonstrate
that such a complex internalizes
in living cells, can be easily lo-
calized therein by fluorescence
microscopy, exhibits a good bio-
compatibility in the dark, re-
leases NO under the exclusive
control of visible-light stimuli
and, as result, induces an appre-
ciable level of photomortality
in tumor cell lines.
Results and Discussion
In search of a viable strategy to
construct a stable, non-covalent
photoactivable inclusion com-
plex able to display the conver-
gence of all the above proper-
Scheme 1. Schematic of the photoresponsive host–guest supramolecular complex and molecular structures of
ties, we have designed a molecu- the compounds used in this work.
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A Multifunctional Host–Guest Supramolecular Complex
nounced preference for the CD cavity of the host than the
nitroaniline chromophore.[29] As a consequence, the NO
photoreleasing unit should locate outside the CD cage, thus
preserving the suitable conformation for the NO photodeliv-
ery (see below).
that containing 2 alone (inset in Figure 1, curve e). These
absorption features are very similar to those exhibited by
the model compound 3, which is completely water soluble
(inset in Figure 1, curve f). Our previous work demonstrated
that the energy of the visible absorption band of the nitroa-
niline unit is very sensitive to both microenvironment polar-
ity and changes in the geometry of the nitro group.[27] In this
view, the absence of any shift in the absorption maximum of
1 with respect to 3 suggests a binding mode in which the
NO photodonor is mainly exposed to a water pool while the
adamantane unit occupies the CD cavity, as illustrated in
Scheme 1. Based on the absorbance values of the NO photo-
donor at 403 nm and of the rhodamine label at 558 nm, and
the related extinction coefficients at the same wavelengths
(ca. 10000mꢀ1 cmꢀ1 and 100000mꢀ1 cmꢀ1, respectively), an
nearly complete complexation between 1 and 2 with a 1:1
stoichiometry can be estimated. In view of the low concen-
trations of the components (ca. 10 mm), this finding accounts
for a high binding constant (Ka >105 mꢀ1). Taking into ac-
count that the Ka values for adamantane and unfunctional-
ized b-CD are on the order of 104 mꢀ1,[28] we believe that the
effective binding process might stem from cooperative intra-
cavity interactions involving the adamantane moiety and hy-
drophobic interchain interactions involving the alkyl spacer
of 1 and the isopropyl termini of 2. Such an efficient com-
plexation of the active guest is a fundamental requisite for
its intracellular delivery (see below).
The photoactive host 2 consists of a b-CD scaffold which
has been ad hoc modified at both the upper and the lower
rim. The rationale behind the design of compound 2 was
that 1) the peripheral amino groups, which are expected to
be protonated at physiological pH, should assist in cell pene-
tration, owing to their positive charges, and ensure an excel-
lent solubility in aqueous media, 2) the isopropyl termini
should further promote the binding with the guest through
cooperative hydrophobic interchain interactions involving
the alkyl spacer of 1, and 3) the rhodamine fluorogenic unit
is expected to facilitate the localization of the supramolec-
ular system in living cells by fluorescence microscopy, with-
out affecting the NO photoreleasing properties of the guest.
The absorption and fluorescence spectra of 2 in aqueous
solution are dominated by the rhodamine chromophore and
exhibit maxima at 558 and 586 nm, respectively (Figure 1,
Interestingly, the emissive properties of the host 2 were
only little influenced by the presence of the guest 1 in the
complex (Figure 1, curves b and d), thereby offering a great
advantage for its easy mapping in living cells by fluores-
cence microscopy (see below). This result is in line with the
binding mode suggested in Scheme 1, which implies a dis-
tance between the two chromophores long enough to avoid
any potential intramolecular quenching by photoinduced
electron transfer.[31]
The most convenient methodology to demonstrate the
generation of NO from the supramolecular complex is the
direct and real-time monitoring of this radical species. To
this end, we have used an ultrasensitive NO electrode,
which directly detects NO, with a sensitivity in the nanomo-
lar concentration range, by an amperometric technique.[32]
The results illustrated in Figure 2A provide unambiguous
evidence that the complex is stable in the dark but supplies
NO upon illumination with lexc =420 nm. The release pro-
cess is strictly dependent on the external light input, as con-
firmed by the linear NO photodelivery that promptly stops
when the light is turned off and restarts when the illumina-
tion is turned on again.
Figure 1. Absorption and fluorescence emission spectra of 2 (10.8 mm,
lexc =525 nm, isosbestic point) in 10 mm phosphate buffer (pH 7.4) in the
absence (curves a and b) and in the presence (curves c and d) of
1 (10 mm). The inset shows the difference absorption spectrum between
sample c and a (curve e) and absorption spectrum of the model com-
pound 3 (curve f). T=258C, cell path=1 cm.
curves a,b). The fluorescence quantum yield for this com-
pound was Ff =0.21. This value is close to that reported for
rhodamine B under identical experimental conditions (Ff =
0.31),[30] thereby accounting for a CD conjugate with satis-
factory emission properties.
Compound 1 is completely insoluble in aqueous media.
However, it becomes soluble in an aqueous solution of 2 as
a consequence of the formation of a supramolecular com-
plex. This is proven by the appearance of an additional ab-
sorption contribution in the region below 500 nm, which is
due to the nitroaniline chromophore (Figure 1, curve c). The
presence of this unit is better visible in the difference spec-
trum between the sample containing both components and
Figure 2B shows the changes in the electronic absorption
spectrum of the complex observed upon light irradiation. It
can be noted that bleaching occurs in the corresponding ab-
sorption region of the NO photodonor (around 400 nm),
whereas only negligible changes are observed in the band of
the rhodamine chromophore (around 558 nm). This photo-
bleaching accounts for a photochemical reaction involving
exclusively the guest 1 and is in excellent agreement with
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To demonstrate the suitability of the supramolecular con-
struct as a biocompatible vehicle with imaging and photo-
therapeutic capacities, we performed cell culture experi-
ments with HeLa cancer cells. The cells were treated with
the complex for three hours, harvested, and then analyzed
by fluorescence microscopy. The presence of the CD deriva-
tive inside the cells can be easily revealed by the red fluores-
cence emission typical of the rhodamine label, while the lo-
cation of the nuclear compartment is accomplished by stain-
ing the cells with the fluorescent DNA-binding dye Hoechst
33342. Figure 3A shows a well detectable level of intracellu-
Figure 3. Fluorescence microscopy analysis of HeLa cells treated with the
complex of 1 with 2 (ca. 10 mm) and stained with Hoechst 33342 dye in
10 mm phosphate buffer (pH 7.4). The cells were analyzed by fluores-
cence microscopy (40”) with
a rhodamine emission filter (A) or
a Hoechst emission filter (B). C) Merge of images shown in panels A and
B.
Figure 2. A) NO release profile observed upon irradiation with mono-
chromatic light at l=420 nm of the complex of 1 with 2 (ca. 10 mm) in
10 mm phosphate buffer (pH 7.4) at 258C. B) Absorption spectral
changes observed upon light irradiation at l>400 nm, from 0 min (curve
a) to 40 min (curve e), of the complex under the same conditions as in
panel A. Fluorescence emission spectra of the supramolecular complex
before (curve f) and after (curve g) photolysis (lexc =525 nm). The inset
shows the kinetic profiles, monitored at l=400 nm, and the related linear
lar red fluorescence within HeLa cells, confirming the ability
of the CD conjugate to penetrate the cells. Figure 3B evi-
dences nuclear staining of the same cells with Hoechst
33342, as revealed by the blue fluorescence emission of this
dye. Figure 3C shows the corresponding merged image ob-
tained by double fluorescence analysis. A close inspection of
this image excludes the confinement of the CD derivative in
the nuclear compartment of the HeLa cells, accounting for
a localization mainly in the cytoplasm or a possible colocali-
zation with mitochondria, in agreement with typical obser-
vations for rhodamine derivatives.[33]
To evaluate the effectiveness of this system to induce anti-
tumor effects upon light irradiation, the cell cultures were
treated for 3 hours with the complex and, for comparison,
with the host 2 alone, and the samples were either kept in
the dark or irradiated with visible light for 30 minutes. The
results reported in Figure 4 show that both 2 and its com-
plex with 1 exhibit a low level of cytotoxicity in the dark. By
contrast, considerable cell mortality is observed under light
excitation. It can be seen that the extent of cellular death
observed for the complex is greater than that noted in the
case of the host alone. This result provides clear-cut evi-
dence for the capability of 2 to deliver 1 to the cell compart-
ment, although we cannot exclude the possibility that the
complex dissociates after cell internalization. The photoin-
duced cell mortality induced by 2 alone can be reasonably
*
fittings, observed in the case of the complex of 1 with either 2 ( ) or
&
with the model compound 4 ( ).
the photochemical pathway leading to NO release previous-
ly proposed in the case of the single NO photodonor
unit.[21a] The notion that the rhodamine fluorophore is not
involved in any photochemical process was also confirmed
by the almost complete preservation of the fluorescence
spectrum observed after the photolysis experiments (Fig-
ure 2B, curves f and g).
To investigate whether the rhodamine unit influences the
photoconversion rate of 1, we carried out comparative ex-
periments with a supramolecular complex between 1 and
the model compound 4 (see Scheme 1). In this case, the b-
CD scaffold bears both the amino and isopropyl appendages
but, in contrast to 2, lacks of the rhodamine fluorophore. As
shown in the inset in Figure 2, the kinetic profiles for the
photobleaching exhibit basically the same slopes, thereby
excluding any relevant effect of the rhodamine fluorophore
on the photoreactivity of the NO photodonor.
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A Multifunctional Host–Guest Supramolecular Complex
The described experiments demonstrate the validity of
our logical design and justify further research in this direc-
tion with the intriguing prospect to achieve a novel class of
photoactivatable nanoscaled systems based on similar com-
ponents that exhibit an improved photoaction. In this re-
spect, we are currently working on the synthesis of rhoda-
mine-labeled CD-based oligomers. The presence of multiple
CD compartments should lead, in principle, to nanostruc-
tures able to accommodate a larger amount of NO photodis-
pensers, with a consequential increase in both the light-har-
vesting properties and the reservoir of NO available. The re-
sults of this investigation will be reported in due course.
Experimental Section
Figure 4. Cell mortality of HeLa cells in the dark or upon photoinduction
in the absence of photoactive components (a), in the presence of 2
(10 mm) (b), and in the presence of the complex of 1 with 2 (c).
Materials
All cyclodextrin intermediates were synthesized by CycloLab (Budapest,
Hungary). All other reagents were of the highest commercial grade avail-
able and used without further purification. All solvents used (from Carlo
Erba) were analytical grade, and dried by conventional methods and dis-
tilled immediately prior to use. Slide-A-Lyzer Dialysis Casettes G2, with
a cut-off MW of 2000 (Thermo Scientific), were used for dialyses. Thin
layer chromatography (TLC) was performed on aluminum sheets pre-
coated with silica gel 60 F254 (Merck, Art. No.: 1.05554). Plates were de-
veloped in a saturated chamber of 1,4-dioxane/ammonium hydroxide
(25%, 10:7 (v/v)), visualized by UV light at 254 nm and 366 nm, and
charring with a solution of EtOH (96%)/H2SO4 (96%, 9:1) followed by
heating at 105–1108C. Phosphate buffer (10 mm, pH 7.4) was prepared
with biological grade reagents and all solutions were prepared with nano-
pure water (18 MW).
ascribed to the generation of reactive oxygen species (ROS)
by the rhodamine dye, which has been reported to act as
a photosensitizer although with low efficiency.[34]
Conclusions
We have presented here a novel multifunctional, photores-
ponsive supramolecular “Lego system” based on the effec-
tive formation of an inclusion complex between a tailor-
made NO photodonor and an ad hoc devised CD conjugate
carrying a rhodamine fluorophore. Both the guest and the
host behave as independent photoactive centers in the
supramolecular complex, as proven by the excellent preser-
vation of their photochemical and photophysical properties.
As a result, this nanoassembly exhibits the convergence of
photoregulated release of NO and fluorescence imaging in
one single nanostructure. We would like to highlight that, in
contrast to non-photoresponsive compounds, the preserva-
tion of the photobehavior of independent components after
their assembly is not a “trivial result”. In most cases, the re-
sponse to light of single or multiple photoactive units locat-
ed in a confined space can be, in fact, considerably influ-
enced, in both nature and efficiency, by the occurrence of
competitive photoprocesses (i.e., photoinduced energy and/
or electron transfer, hydrogen abstraction, nonradiative de-
activation, etc.),[18,35] which preclude the final goal.
The host–guest complex internalizes in cancer cells, prob-
ably assisted by its cationic nature at physiological pH, can
be easily mapped therein in view of its satisfactory fluores-
cence emission, and is able to induce about 50% cell photo-
mortality. In this regard, it appears that the extent of the
cellular death is not exclusively due to the light-triggered
generation of NO but also to the involvement of ROS pho-
togenerated by the rhodamine unit. Studies addressed to
clarify this point deserve certainly attention.
Synthesis
Compound 1 and the model compound 3 were synthesized according to
previously reported procedures.[21b,23a] 6-Tetradeoxy-6-triamino-6-rhoda-
minylthioureido-tri-O-(2-hydroxypropyl)cyclomaltoheptaose (2) was syn-
thesized in five steps (see Scheme 2A). The key intermediate 6-tetrazido-
6-tetradeoxycyclomaltoheptaose was synthesized by following the meth-
ods reported in the literature.[36a,b] The hydroxypropylation and the reduc-
tion steps were performed as reported by Malanga et al.[37]
The rhodaminylation was performed as follows: Rhodamine B isothio-
cyanate (RBITC, 0.37 g, 0.69 mmol) was dissolved in DMF (5 mL) and
then added dropwise to a stirred solution of 6-tetramino-6-tetradeoxy-tri-
O-(2-hydroxypropyl)cyclomaltoheptaose (0.91 g, 0.69 mmol) and N,N-dii-
sopropylethylamine (DIPEA, 99%, 0.6 mL, 3.4 mmol) in DMF (30 mL).
The solution was stirred at 90–1008C for 24 h, cooled to 808C, and then
the solvent was removed under reduced pressure (T=808C). The crude
product was dissolved in water (20 mL) and extracted with dichlorome-
thane (3”100 mL). Subsequently, water was removed under reduced
pressure (T=608C) and the resulting material was dialyzed against de-
ionized water. Freeze-drying yielded the target compound 2 as a violet
powder (0.66 g, 52%); m.p.: 205–2128C (dec.); IR (KBr): n˜ =3349, 2973,
2928, 1670, 1591, 1466, 1340, 1153, 1083, 1039, 946, 754, 607, 580 cmꢀ1
;
1H NMR (D2O): d=1.13–1.14 (m, 9H, hydroxypropyl-methyl-H), 1.29–
1.36 (m, 12H, RBITC-methyl-H), 3.33–4.13 (m, 59H, H2, H3, H4, H5,
H6, hydroxypropyl-methylene-H, hydroxypropyl-methyne-H, RBITC-
methylene-H), 5.09–5.25 (d, 7H, H1) 6.76–7.39 (bs, 6H, aromatic-H),
8.00–8.04 (d, 1H, aromatic-H), 8.11–8–17 (d, 1H, aromatic-H), 8.45 ppm
(m, 1H, aromatic-H); ESI-MS (MeOH, HP=2-hydroxypropyl fragment):
[M0+Na]+ =1826.77; [M1+Na]+ =1768.73; M1 =M0ꢀHP; [M2+Na]+
1710.73; M2 =M1ꢀHP; [M3+Na]+ =1884.82; M3 =M0+HP; [M4+Na]+
1942.91; M4 =M3+HP.
=
=
The DS (degree of substitution) for HP and rhodamine was determined
from the H NMR data.
1
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photochemical reactor equipped with
8 RPR lamps, with an emission in the
380–480 nm range with a maximum at
420 nm in the presence of a 400 nm
cut-off filter. The incident photon flux
on quartz cuvettes was approximately
0.8”1015 quanta sꢀ1
.
Sample Preparation
Compound 1 was dissolved in metha-
nol and slowly evaporated to form
a thin film. This film was then hydrat-
ed with an aqueous solution of either
2 or the model compound 4 in phos-
phate buffer at pH 7.4. The mixtures
were stirred for 1 h at 508C, and then
the final solutions were left to equili-
brate at room temperature.
NO Detection
NO release was measured with
a
World Precision Instrument, ISO-
NO meter, equipped with a data ac-
quisition system, and based on direct
amperometric detection of NO with
short response time (<5 s) and a sensi-
tivity range of 1 nm–20 mm. The analog
signal was digitalized with a four-chan-
nel recording system and transferred
to a PC. The sensor was accurately
calibrated by mixing standard solu-
tions of NaNO2 with 0.1m H2SO4 and
0.1m KI according to the reaction:
Scheme 2. Synthetic route for the preparation of the CD conjugate 2 (A) and the model compound 4 (B).
6-Monodeoxy-6-monoamino-tri-O-(2-hydroxypropyl)cyclomaltoheptaose
(4) was synthesized in three steps (see Scheme 2B). The hydroxypropyla-
ꢀ
4 Hþ þ 2 Iꢀ þ 2 NO2 ! 2 H2O þ 2 NO þ I2
tion and the reduction steps were performed as reported by Malanga
et al.[36] 6-Monodeoxy-6-monoazido-tri-O-(2-hydroxypropyl)cyclomalto-
heptaose (60 g, 0.044 mol) was dissolved in a water/ethanol solution
(400 mL, 1:1) at room temperature. Palladium on charcoal (6 g, 10% Pd
content) was suspended in water (60 mL) at room temperature and
added to the solution; the catalyst was washed-in with water (60 mL),
then hydrazine carbonate (20 mL) was added, and the reaction mixture
was heated to reflux. After 20 min, the reaction mixture was cooled to
room temperature, the solids were filtered, and the volatiles were re-
moved from the filtrate. Traces of hydrazine were removed with dry, acti-
vated H+ ion-exchanger (30 g) in methanol. Subsequently, the ion-ex-
changer was removed by filtration, the filtrate was filtered through
a membrane, and the solvent was removed to yield 4 as a white solid
(49 g, 82%); m.p.: 202–2058C (dec.); IR (KBr): n˜ =3403, 2969, 2929,
1646, 1457, 1411, 1374, 1336, 1299, 1155, 1084, 1034, 948, 853, 757,
Irradiation was performed in a thermostated quartz cell (1 cm path
length, 3 mL capacity) under gentle stirring by using the monochromatic
radiation at 420 nm of the Fluorolog-2 as the light source. NO measure-
ments were carried out with the electrode positioned outside the light
path in order to avoid false-positive NO signals due to photoelectric in-
terference on the ISO-NO electrode.
Cell Culture Experiments
HeLa cells were obtained from the American Type Culture Collection
and propagated at 1:6 ratio using Dulbeccoꢂs modification of Eagleꢂs
Minimal Essential Medium supplemented with 10% FBS (Fetal Bovine
Serum). The samples of cells treated with the different samples of either
2 or its complex with 1 were trypsinized, placed separately in spectropho-
tometric cuvettes, and either kept in the dark or irradiated with visible
light in the photoreactor as described above. Before and after irradiation,
8”104 cells were placed in 96-well plates with 100 mL of phosphate buffer
and incubated in the presence of a tetrazolium compound [3-(4,5-dime-
thylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra-
zolium, inner salt; MTS, Promega] and an electron coupling reagent,
phenazine methosulfate (PMS dye, MTS, 20 ml/well). After further incu-
bation for 1 h, the absorbance was read at 490 nm in a Labsystems Mul-
tiskan Bichromatic plate reader.The cell viability (%) was calculated ac-
cording to the following equation:
580 cmꢀ1 1H NMR (D2O): d=1.13–1.14 (m, 9H, hydroxypropyl-methyl-
;
H), 3.33–4.13 (m, 51H, H2, H3, H4, H5, H6, hydroxypropyl-methylene-
H, hydroxypropyl-methyne-H), 5.09–5.25 ppm (d, 7H, H1).
1
The DS for HP was determined from the H NMR data.
Instrumentation
UV/Vis absorption and fluorescence spectra were recorded with a Jasco
V-560 spectrophotometer and
a Fluorolog-2 (model F-111) spectro-
fluorimeter, respectively. Fluorescence images were taken with a Biomed
fluorescence microscope (Leitz, Wetzlar, Germany). The ESI-MS experi-
cell viability ð%Þ ¼
ðA ðbefore LampÞ-ðA ðafter LampÞ=-A ðbefore LampÞÞ ꢁ 100
ments were performed using
a Waters Q-TOF Premier instrument
(Waters Corporation, MA, USA). Melting points were determined on
a Büchi OP545 melting point apparatus and are uncorrected. 1H- and
13C NMR spectra were recorded on a Varian VXR-600 spectrometer at
400 or 600 MHz. IR spectra were recorded in KBr disks on a Nicolet 205
FTIR spectrometer. Photolysis experiments were performed in a thermo-
stated quartz cell (1 cm path length, 3 mL capacity) by using a Rayonet
where A (before Lamp) denotes the absorbance of treated samples
before light exposure and A (after Lamp) denotes the absorbance of
treated samples after light exposure. The cell mortality (%) is the com-
plement of cell viability.
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A Multifunctional Host–Guest Supramolecular Complex
128; g) F. L. Callari, A. Mazzaglia, L. Monsœ Scolaro, L. Valli, S.
Sortino, J. Mater. Chem. 2008, 18, 802.
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We thank the Marie Curie Program # 237962 CYCLON (FP7-PEOPLE-
ITN-2008) for financial support to MC fellows N. Kandoth, M. Malanga
and A. Fraix, and funding of the research. Financial support from MIUR
(PRIN 2008), Rome Italy, is also acknowledged. We thank one of the
anonymous referees for his/her constructive comments.
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Published online: && &&, 0000
Chem. Asian J. 2012, 00, 0 – 0
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
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FULL PAPER
Supramolecular Chemistry
NO delivery today: A supramolecular
host–guest complex, based on a rhoda-
mine-labeled b-cyclodextrin conjugate
and a tailored NO photodonor, effec-
tively internalizes in cancer cells, can
be easily mapped intracellularly due to
its satisfactory red fluorescence emis-
sion, and induces about 50% of cellu-
lar death under the exclusive control
of visible light stimuli.
Noufal Kandoth, Milo Malanga,
Aurore Fraix, Lꢀszlꢁ Jicsinszky,
ꢂva Fenyvesi,* Tiziana Parisi,
Ivana Colao, Maria Teresa Sciortino,
Salvatore Sortino*
&&&& — &&&&
A Host–Guest Supramolecular
Complex with Photoregulated
Delivery of Nitric Oxide and Fluores-
cence Imaging Capacity in Cancer
Cells
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ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 0000, 00, 0 – 0
ÝÝ These are not the final page numbers!