Elisa Vittorino

A Cyclodextrin‐Based Nanoassembly with Bimodal Photodynamic Action

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 ((1)O(2)). This was achieved through the appropriate incorporation of an anionic porphyrin (as (1)O(2) 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 (1)O(2) 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.

Bichromophoric multilayer films for the light-controlled generation of nitric oxide and singlet oxygen

In this contribution we report the design, preparation and characterization of bichromophoric Langmuir–Schafer multilayer films incorporating a tailored nitric oxide (NO) photodonor (1) and an ethylene-bridged zinc porphyrin dimer (2). They are achieved by exploiting the coordination of a pyridine appendage of the NO photodonor to the metal centers of the porphyrin units. Quite uniform floating films at the water–air interface are obtained spreading a mixture of 1:2 in the molar ratio 20:1 as confirmed by Brewster angle microscopy. The floating films are successfully transferred onto quartz slides by horizontal lifting deposition and the resulting hybrid multilayers are characterized by steady-state UV-Vis absorption, fluorescence and laser flash photolysis. The high ratio between the two chromogenic centers prevents the porphyrin aggregation, leading to photoresponsive bichromophoric films able to generate NO and singlet oxygen, 1O2 (1Δg), under the exclusive control of visible light stimuli.

Photofunctional multilayer films by assembling naked silver nanoparticles and a tailored nitric oxide photodispenser at water/air interface.

This contribution reports the design, preparation, and characterization of nanostructured hybrid films of silver nanoparticles (AgNPs) and a tailored nitric oxide (NO) photodonor. They were achieved by exploiting effective interfacial interactions between an amino-terminated NO photodonor spread onto water surface and naked AgNPs dissolved in the water subphase. The morphology, the spectroscopic features, and the interaction between the two components in the floating films at the air/water interface were inspected by Brewster Angle Microscopy, UV-Vis reflection, and polarization-modulation infrared reflection-absorption spectroscopy. AgNPs and the NO photodonor were successfully transferred onto hydrophobized quartz substrates by horizontal lifting deposition and the resulting multilayer films were characterized by UV-Vis absorption spectroscopy and transmission electron microscopy, respectively. The results obtained showed the presence of both isolated AgNPs and assemblies of AgNPs having nanodimensional character in the films. The photochemical properties of the NO photodonor were well preserved in the hybrid multilayers. In fact, they were able to release NO under visible light excitation, as unambiguously demonstrated by the direct and in real-time monitoring of this transient species using an ultrasensitive electrode, and the transfer of the released NO to a protein such as myoglobin.

Light-Activated Release of Nitric Oxide with Fluorescence Reporting in Living Cells

The rapid delivery of biologically relevant species with precise spatiotemporal control is a hot topic in life science. Light is the most elegant on/off trigger to fulfill these criteria. In fact, the fast response of the photochemical reactions and their instantaneous initiation/stopping depending on the presence or absence of the illumination, represent a powerful tool for the introduction of given amounts of “photocaged” chemical entities in biological systems in a noninvasive way. In addition, the use of photons as external input offers the additional advantage of not affecting important physiological parameters, such as pH, temperature and ionic strength, which is a critical condition for bio-applications. Quantification of the delivery upon uncaging of the chemical entity is another important issue to be faced. One possibility to address this task is to use a fluorescent reporter. This elegant strategy relies on the simultaneous photorelease of the desired caged bioactive species and a fluorescent component from the same nonfluorescent caged precursor. In this way, the uncaging process can be quantified by monitoring the fluorescence emission of the reporter and, furthermore, the spatial distribution of the released species in a biological environment can be followed in real time by fluorescence microscopy. Nitric oxide (NO) is one of the most appealing and well-studied molecules in biomedical sciences. This inorganic radical is a biological mediator in many physiological processes, including neurotransmission, hormone secretion and vasodilatation. Furthermore, NO has proven to reduce radical-mediated oxidative processes, exert cytotoxic effects against infective microorganisms, and inhibit tumor growth. This multifaceted role has stimulated a massive interest in developing NO-releasing systems with the prospect to tackle important diseases. However, the biological effects of this radical have been shown to be highly site, concentration and dosage dependent, creating a complex role for the molecule in opposing beneficial and deleterious events. This dichotomy has made NO-photoreleasing compounds much more desirable than those based on spontaneous thermolysis as demonstrated by the increasing number of papers on NO photodonors with improved photophysical and photochemical characteristics appearing in recent years. Conversely, NO uncaging quantification based on fluorescence reporting has attracted very little attention. As a part of our ongoing interest in developing functional light-controlled NO delivering systems, we recently reported the design and synthesis of molecular conjugate 1 (Scheme 1). It integrates two chromogenic centers within the same covalent skeleton: an anthracene moiety (unit a) and a nitroaniline derivative that we have recently discovered to be a suitable NO photodonor (unit b). This compound was devised in such a way to amplify the NO photorelease from unit b through an effective photoinduced energy transfer a*! b. As a result, the typical emission of the anthracene fluorophore is completely suppressed, making conjugate 1 intrinsically nonfluorescent. Herein, we report novel and intriguing properties of compound 1. In particular, we demonstrate that NO photo-uncaging: 1) leads to the formation of a strongly fluorescent coproduct that can be used as an optical reporter for NO delivery; 2) induces a remarkable level of mortality in cancer cell. The absorption spectrum of 1 shows the distinctive spectral features of the anthracene chromophore below 400 nm and an intense absorption at longer wavelengths extending beyond 450 nm, due to the contribution of the nitroaniline moiety (a in Figure 1 a). These spectral features allow the NO photodonor unit to be selectively excited at lexc>400 nm. Changes in the electronic absorption spectrum observed upon irradiation of a solution of 1 at lexc = 420 nm are strictly related to the photorelease of NO from the unit b (Figure 1 a). A typical NO-release profile, obtained by the direct amperometric detection of this species alternating cycles of light/dark is shown in the inset of Figure 1 a, for the sake of clarity. The photobleaching observed is in line with the photochemical pathway leading to NO release previously proposed in the case of the single NO photodonor unit b. This mechanism involves a nitro-to-nitrite photo-rearrangement followed by the simultaneous release of NO and a phenoxy radical. Eventually, this intermediate generates a phenol derivative as the main stable photoproduct that, in our case, does not absorb in the visible region. Phenol derivatives as the main stable photoproducts have also been reported in the case of other nitroaromatics, for which a NO release mechanism similar to the chromophoric center b applies. Electrospray ionization (ESI) mass spectrometric analysis of the crude reaction mixture, performed immediately after the photolysis experiments (~20 % transformation), validates this proposal ; a main peak with an m/z value of 425.2 corresponding to [M + 1] of the conjugate 2 (see [a] Dr. E. Vittorino, Prof. S. Sortino Laboratory of Photochemistry, Department of Drug Sciences University of Catania, Viale Andrea Doria 6, 95125 Catania (Italy) Fax: (+ 39) 095-580-138 E-mail : ssortino@unict.it [b] Dr. M. T. Sciortino, Dr. G. Siracusano Dipartimento di Scienze della Vita, Sezione di Scienze MicrobiologicheGenetiche e Molecolari, Universit di Messina Salita Sperone, 98166 Messina (Italy)

Gold nanoparticles decorated with a photoactivable nitric oxide donor/cyclodextrin host/guest complex

Hybrid gold nanoclusters exhibiting photoregulated release of nitric oxide are obtained by appropriate self-assembling of a suitable host, a photoactivable guest and Au nanoparticles. The excitation of the core, the shell or both components of the nanoclusters with visible light, together with their dark stability and water solubility, make these nanoarchitectures intriguing candidates to be tested in biomedical research studies.

Reversible molecular motion of a bis-calix[5]arene host driven by a photoresponsive guest.

Modulation and control of the molecular features of a given receptor/substrate, and hence their supramolecular properties, by means of external, nondestructive, stimuli is still a topic of current and increasing interest, not only from a fundamental perspective but also from that of future nanodevices. Light is the most appealing on/off trigger. The combination of its ready availability and easy manipulation, together with the fast rate of photochemical reactions, makes photoregulated systems particularly suited for multifaceted applications in different fields, which range from molecular optoelectronics and energy conversion, to the environment and biomedicine. Control over host–guest complexation events is one of the fundamental areas in which photoresponsive centers play a key role. In this respect, the reversible trans–cis photoisomerization of azobenzene moieties has proved to be one of the most convenient tools for inducing conformational modifications that may influence functional properties at the nanoscale level. Incorporation of an azobenzene moiety either into a receptor framework or into a substrate has provided orthogonal triggers for the selective capture of target species and/or controlled release of bound substrates. Very recently, the use of azobenzene cores has allowed control over the reversible assembly/disassembly of capsular complexes. As part of our ongoing interest in calix[5]arenes as selective receptors for linear a,w-alkanediyldiammonium guests, we have demonstrated that receptor 1 (Figure 1), composed of two calix[5]arene units covalently linked to each other through their upper rims, forms either capsular or noncapsular 1:1 complexes according to the molecular length of the alkanediyldiammonium guests present in solution. Bis-calix[5]arene 1 is a heterotetratopic receptor capable of simultaneously and synergically binding, with remarkable efficiency, linear alkanediyldiammonium dications along with their counterions by means of the ureido moieties located in the phenylene bridging unit. Depending on the length of the alkyl chain of the diammoniun dication, receptor 1 adopts in solution (CDCl3/CD3OD, 4:1) either an open “S-shaped” conformation, in which the two p-rich cavities arrange themselves in an antiperiplanar fashion with respect to the phenylene spacer, or an opposite closed “C-shaped” synperiplanar conformation, to allow either one or both terminal ammonium moieties of the substrate to nest inside the bis-calixarene cavityACHTUNGTRENNUNG(ies) (Figure 1 a). Intrigued by this behavior and motivated by the above considerations, herein, we have explored the possibility of reversibly controlling the openQclosed conformational interconversion of host 1 without changing the nature of the guest, using instead photons alone as external triggers. Based on an inspection of the solid-state structure of 1 (see below), diammonium salt 2·2HCl (incorporating a photochromic azobenzene moiety in its skeleton) was designed and synthesized as a potential guest. The shape/length of this photoactive guest can be modulated by the trans–cis isomerization of its azo group. When it adopts the trans configuration, the guest has the correct shape (antiparallel ditopic guest with a nitrogen-to-nitrogen distance of 16.6 ; Spartan’10) to encourage the formation of the closed “Cshaped” capsular complex through a positive self-assembly cooperativity. On the other hand, the more compact [a] Dr. G. Brancatelli, Prof. S. Geremia Centro di Eccellenza in Biocristallografia Dipartimento di Scienze Chimiche e Farmaceutiche Universit di Trieste Via L. Giorgieri 1, 34127 Trieste (Italy) [b] Dr. C. Capici, Dr. G. Gattuso, Dr. A. Notti, Prof. M. F. Parisi Dipartimento di Chimica Organica e Biologica Universit di Messina Viale F. Stagno d’Alcontres 31, 98166 Messina (Italy) E-mail : mparisi@unime.it [c] Prof. S. Pappalardo Dipartimento di Scienze Chimiche Universit di Catania, Viale A. Doria 6, 95125 Catania (Italy) E-mail : spappalardo@unict.it [d] Prof. S. Sortino, Dr. E. Vittorino Laboratory of Photochemistry Department of Drug Sciences University of Catania Viale A. Doria 6, 95125 Catania (Italy) E-mail : ssortino@unict.it Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201100738.

Synthesis and photophysics of a fullerene-triquinoxaline ensemble

In this contribution, we report the synthesis and photophysical characterization of a molecular conjugate, in which C60 is covalently attached to a triquinoxaline-based cavitand through a pyrrolidine ring. Comparative experiments performed by using suitable model compounds demonstrate negligible interactions between the quinoxaline chromophores and the fullerene centre in the ground state. On the other hand, fluorescence emission and excitation spectra provide evidence for the occurrence of efficient photoinduced singlet–singlet energy transfer from the quinoxaline moiety to the C60 core. Laser flash photolysis experiments show that such an intramolecular process precludes the population of the lowest triplet state of the quinoxaline. By way of contrast, the lowest triplet state of the fullerene is effectively populated and is capable of sensitizing the formation of singlet oxygen in high yield, as unambiguously demonstrated by its typical infrared phosphorescence, detected by using time-resolved luminescence apparatus. The fullerene-quinoxaline conjugate exhibits photoinduced DNA-cleaving activity, as confirmed by preliminary photocleavage experiments carried out with a pBR322 supercoiled plasmid.