Towards cancer treatment: synthesis and characterization of photoactive theranostic nanoclinics

This thesis describes the synthesis, the photophysical, photochemical and biological characterization of some organic aromatic heterocycle derivatives and their theranostical applications to cancer treatment. Although the topic thus falls within the general area of photomedicine, the approach adopted here takes advantage of the strong interdisciplinary character of a materials science PhD course to explore an original perspective in the molecular design of novel photosensitizers and fluorescent probes in the fight against cancer. In the initial part of this work, we explore the synthetic accessibility of squaraine dyes candidates as second generation sensitizers for Photodynamic Therapy (PDT). This well-established treatment involves the insurgence of citotoxic species in the cellular environment after irradiation of a dye with visible light. Typically, the sensitizer excited triplet state generated after irradiation can induce the formation of singlet oxygen, O2(1δg), the lowest excited state of molecular oxygen, and/or reactive oxygen species (ROS). It is generally accepted that production of sufficient quantities of O2(1δg) and/or ROS, can perturb cellular processes and ultimately cause cell death. Among the large number of known photosensitizers, squaraine dyes (1,3-dicondensation products of squaric acid and electron rich molecules) possess an intense one-photon absorption in the transparency window of biological tissues. Furthermore, squaraines possess an exceedingly strong two photon absorption enabling for their use at the wavelengths relevant for clinical applications (644 nm and 806 nm, respectively). We herein report multiple strategies aimed at the improvement of the reaction yield, of byproducts removal and of time sparing. Because of the many demanding requirements of modern PDT sensitizers, I felt important to investigate with greater effort the photophysical behavior of heterocycle-based π-extended polymethine dyes to get further insight into the possible oxygen-mediated mechanism leading to cellular phototoxicity. To this aim, we have measured singlet oxygen yields (Φ∆), fluorescence quantum yield (Φf), triplet yield (ΦT) and singlet oxygen rates (kq) in toluene and acetonitrile and cyclic voltammetry in dichloromethane. All of the data collected point out that these electronrich polymethine compounds have a strong affinity with molecular oxygen leading to the formation of a charge transfer encounter complex. This results in low singlet oxygen yields and high singlet oxygen rates for these dyes. To elucidate the photodamaging mechanism I designed simple photochemical experiments: firstly, I analyzed the photobleaching behavior of a benzothiazole-based squaraine by GC-MS and I observed the formation of two carbonyl compounds in accordance with a photooxygenation of the enaminic bond; secondly, I studied the product distribution of the reaction between light, squaraines and biologically relevant targets (i.e., limonene, cholesterol, and methyl linoleate) pointing out the presence of a radical chain of oxidative events. Consistently with the photophysical characterization, I established that the encounter complex between squaraine and molecular oxygen could evolve either by photooxygenation of the enaminic bond in the squaraine backbone or by ROS production leading to lipid peroxidation that culminate in cell death by a Type I mechanism. Indeed, the in-depth biological evaluation of two benzothiazole-based squaraine dyes showed that these dyes internalized in lipid vesicles in the cytoplasm and, although they are non-significantly cytotoxic in the dark, they promote a strong dose-dependent phototoxic effect in four different cancer cells after irradiation. In HeLa and MCF-7 cells 3.1c and 3.30, through their hydrocarbon chain substitutions, associate to the membranes and induce lipid peroxidation, as expected from the photophysical and photochemical study, causing cell death primarily by necrosis A challenge that occurs when dealing with photosensitizers is the way they are solubilized and specifically delivered to the biological target. In order to overcome thisp roblem, I have shown how the wide flexibility of the squaraine structure provides a powerful tool aimed at the improvement of bioavailability. In particular, we designed and synthesized a library of squaraine dyes functionalized with ionic groups (sulfonate), alkyl groups and biologically relevant groups (e.g. cholesterol), apt to be delivered in the free form, into liposomes or into low-density lipoproteins (LDL). Through photophysical and photochemical characterization, estimate of singlet oxygen generation efficiencies, subcellular localization and phototoxicity studies in cancer cell lines, we obtained encouraging results about the theranostical capabilities of some of these squaraine dyes, opening the way for their use in cancer-related PDT applications. To further validate the previous observations, I designed an experiment aim to activate the production of ROS in a family of cyanine dyes. I chose to engineer the progenitor Cy5 at the molecular level and increase its photooxidation capabilities by exploiting the effect plays by heteroaryl meso-substituents on the cyanine oxidation potential. By monitoring the degradation of 1,3-diphenylisobenzofuran (DPBF) after irradiation of a sensitizer in the presence of oxygen in solution I showed that it is possible to control the redox behavior, hence the ROS production, by modulating the extent of electron density pulled by the chromophoric side-group in the meso position and boost the photooxidative capability in the series of cyanine derivatives investigated. Moreover, I report on the synthesis, characterization and spectroscopic study both in solution and ex vivo of seven quadrupolar eteroaryl compounds which have been designed to be promising candidates as tumor-specific fluorescent molecular probes. Basic photophysical characteristics of these dyes, their subcellular localization in human umbilical vein endothelial cells (HUVEC), and their hydrophilicity based on logP values have provided in-depth insight into the processes that lead to accumulation in either mitochondria or lysosomes. I have also succeeded in recording its fluorescence spectrum ex vivo, obtaining further information about the interaction between this heteroaromatic dicationic dye and the biological environment.