Thèses sur le sujet « Pdt photodynamic therapy imaging cancer »

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.

This thesis presents the development and in-vivo applications of wearable and portable devices for the investigation of light interaction with tissue involved in Photodynamic therapy (PDT) and during contraction of muscles. A hand-held device and a clinical method were developed for time course in-vivo imaging of the fluorescence of the photosensitizer Protoporphyrin IX (PpIX) in healthy and diseased skin with the aim to guide improvement of PDT protocols. The device was used in a small clinical study on 11 healthy volunteers and 13 patients diagnosed with non-melanoma skin cancer (NMSC). Two types of PpIX precursors were administered: Ameluz gel and Metvix® cream. The fluorescence was imaged with a 10 minute time step over three hours which was the recommended metabolism time before commencing PDT treatment at Ninewells Hospital, Dundee. The fluorescence time course was calculated by integrating the areas with the highest intensity. The fluorescence continued to grow in all subjects during the three hours. The time course varied between individuals. There was no statistical significance between either healthy volunteers or patients in Ameluz vs Metvix® groups; nor was there statistical difference between the three lesions groups (Actinic keratosis (AK) Ameluz vs AK Metvix® vs Basal cell carcinoma (BCC) Metvix®). The p-value was larger than 0.05 in a two sample t-test with unequal variances for all the groups. However, there was strong body site dependence between the head & neck compared to the lower leg & feet, or the trunk & hands body site groups (p-value < 0.01). One of the possible explanations for this was temperature and vasculature variation in skin at different body sites: the temperature is higher and the vasculature structure is denser at the head and the neck compared to the lower leg or the trunk. The temperature was not measured during the study. So in order to support this hypothesis, typical skin temperatures at the lesion sites were taken from the IR thermal images of healthy skin available in literature. PpIX fluorescence had a positive correlation to temperature. If this hypothesis is true, it will be highly important to PDT treatment. Increasing the temperature could speed up the metabolism and reduce the waiting time before starting the treatment; ambient temperature should be taken into account for daylight PDT; cooling air as pain management should be administered with caution. Potential improvements for wearable PDT light sources were investigated by modelling light transport in skin for the current LED-based Ambulight PDT device, a commercial OLED for future devices and a directional OLED developed in the group. The optical models were implemented in commercial optical software (with intrinsic Monte Carlo ray tracing and Henyey-Greenstein scattering approximation) which was validated on diffuse reflectance and transmittance measurements using in-house made tissue phantoms. The modelling was applied to investigate the benefits from diffusive and forward scattering properties of skin on light transmission in treatment light sources. 1 mm thick skin can only compensate approximately 10% of non-uniform irradiance. It means that uniform illumination is crucial for the treatment light sources. Forward scattering in skin showed a 10% improved light transmission from a collimated emission compared to a wide angle Lambertian emission. However, depth-dependent transmission measurements of directional vs Lambertian emission from organic light emitting films (a nano-imprinted grating was fabricated to provide directional emission in one of the films), collimated vs diffused HeNe laser light through fresh porcine skin did not show the expected improvement. This could be explained by skin roughness which was previously found to change the optical properties and may also affect light coupling. The modelling was applied to guide an optical design of another wearable device – a muscle contraction sensor. Muscle is fibrous and because of that scatters light differently in different directions. The sensor detects the change in backscattered light in parallel and perpendicular directions with respect to muscle fibres. The sensor was implemented on a wearable bandage on fully flexible substrate with flexible OLED and organic photodiodes. The major advantages of organic optoelectronic sensing compared to conventional electromyography (EMG) sensors are the ability to distinguish two types of contractions (isotonic and isometric), insensitivity to electromagnetic interference and the absence of an immune response due to non-invasive electrode-free sensing. Optical modelling was performed to understand the operation of the sensor. A 3D anisotropic optical model of scattering in muscle was created by geometrical manipulations with the standard Henyey-Greenstein scattering volumes. The penetration depth from the Super Yellow OLED was found to be 20-25 mm; the optimal separation between the source and the detector was found to be 20 mm. This distance provided a still detectable signal along with the best discrimination between the two backscatterings. When a 2 mm thick layer of skin and a 2 mm thick layer of adipose tissue were added to the model, the signal was hugely diffused. The discrimination between the two backscatterings decreased by three orders of magnitude, the penetration depth in muscle was reduced, and the intensity of the signal dropped down but was still detectable. With 5 mm thick adipose tissue and 2 mm thick skin the signal was too diffused and interacted with very shallow layers of muscle which approached the limits of the optical sensing of muscle activity.

Photodynamic therapy (PDT) has shown to be a promising technique to treat various forms of malignant neoplasia. The photodynamic eradication of the tumor cells is achieved by applying a photosensitizer either locally or systemically and following local activation through irradiation of the tumor mass with light of a specific wavelength after a certain time of incubation. Due to preferential accumulation of the photosensitizer in tumor cells, this procedure allows a selective inactivation of the malignant tumor while sparing the surrounding tissue to the greatest extent. These features and requirements make the PDT an attractive therapeutic option for the treatment of retinoblastoma, especially when surgical enucleation is a curative option. This extreme solution is still in use in case of tumours that are resistant to conventional chemotherapy or handled too late due to poor access to medical care in less advanced country. In this study we initially conducted in-vitro investigations of the new cationic water-soluble photo sensitizer tetrahydroporphyrin-tetratosylat (THPTS) regarding its photodynamic effect on human Rb-1 and Y79 retinoblastoma cells. We were able to show, that neither the incubation with THPTS without following illumination, nor the sole illumination showed a considerable effect on the proliferation of the retinoblastoma cells, whereas the incubation with THPTS combined with following illumination led to a maximal cytotoxic effect on the tumor cells. Moreover the phototoxicity was lower in normal primary cells from retinal pigmented epithelium demonstrating a higher phototoxic effect of THPTS in cancer cells than in this normal retinal cell type. The results at hand form an encouraging foundation for further in-vivo studies on the therapeutic potential of this promising photosensitizer for the eyeball and vision preserving as well as potentially curative therapy of retinoblastoma.

Photodynamic therapy (PDT) and Magnetic resonance imaging (MRI) are two techniques used in therapeutic and diagnostic purpose respectively. PDT can selectively kill the cancer cells by utilizing light and photosensitizer, while MRI provides invasive imaging on our interior bodies. If these two techniques combine, the probe can act as both PDT and MRI agent at the same time. This theranostic agents can bring great efficiency in the cancer treatment. In this project, a porphyrin-cyclen gadolinium based dual functions bio-probe, PZnGdL, is designed for diagnostic and photodynamic therapeutic functions. PZnGdL demonstrated a great T1 signal enhancement for MRI, in which its T1 relaxivity is 15.06 mM-1s-1 (at 1.4T, 37oC). The T1 relaxivity is five-fold higher than the clinically approved MRI contrasting agent Gd-DOTA, (2.92 at 1.4T, 37oC). Furthermore, PZnGdL exhibits low dark toxicity and high photocytotoxicity. Therefore, its photodynamic therapeutic index (PDI) in HeLa cells is as high as 1348 upon 1 J/cm2 light irradiation. Results from the present study show that PZnGdL is an effective photodynamic therapy agent as well as a safe and promising MRI contrasting agent.

La thérapie photodynamique (PDT) est une méthode de lutte contre le cancer basée sur l’utilisation de la lumière et d’un composé sensible à la lumière, appelé photosensibilisateur (PS). Le PS absorbe la lumière et, en présence d’oxygène, engendre la production des dérivés réactifs de l'oxygène (DRO), lesquels sont toxiques et provoquent la régression de la tumeur. La limitation principale des PSs utilisés dans les tests cliniques est leur faible sélectivité envers les tissus cancéreux. Le but principal de ce projet est de créer des agents multifonctionnels combinant sur une même molécule l’activité PDT, la vectorisation et l’imagerie optique proche infrarouge. Dans cette région du spectre optique, les cellules possèdent une faible autofluorescence, et la lumière proche infrarouge pénètre plus profondément dans les tissus biologiques que la lumière visible. Nous proposons ici de modifier une structure dendrimérique de type poly(amidoamine) de génération 3, en tant que plateforme polyvalente. En effet, ce dernier possède trente-deux groupes terminaux qui peuvent être facilement substitués par des PSs. De plus, cette macromolécule peut complexer dans ses cavités jusqu’à 8 cations lanthanides émettant dans le proche infrarouge. Quatre nouveaux ligands dendrimère ont été synthétisés avec différents PSs tels que des dérivés de naphtalimide, d’anthraquinone et de tétraphénylporphyrine. De plus, le naphtalimide a été couplé avec des groupes dérivés de l’acide folique pour assurer la vectorisation envers les tissus cancéreux. Les complexes de lanthanide émettant dans le proche infrarouge ont été préparés pour chaque dendrimère. La caractérisation des performances des différents complexes a été réalisée. La production de DRO et la présence de complexes d’Yb(III) a été démontrée dans les cellules HL60. Les dendrimères modifiés par les groupes anthraquinone et tétraphénylporphyrine en tant que PS, ont montré, dans les cellules vivantes, une émission proche infrarouge lorsqu’ils sont sous la forme de complexe d’Yb(III). Les résultats obtenus montrent que les complexes de lanthanides formés avec des ligands dendrimères peuvent servir comme des agents de PDT et des rapporteurs luminescents proche infrarouge in cellulo
PDT is a cancer treatment that uses the combination of a nontoxic photoactivated molecule (photosensitizer), an appropriate source of light excitation and molecular oxygen to generate reactive oxygen species (ROS) leading to the decrease of size or to the destruction of tumors. However, the PDT efficiency of currently used drugs is limited by the selectivity for the cancer tissue. The main goal of this work is to develop a multifunctional agent which combines a PDT activity, a tumor targeting and near-infrared (NIR) optical imaging. The use of reporters that absorb at low energy is justified by low tissue autofluorescence and high tissue penetration depth in the NIR spectrum window. For this purpose, we have chosen the generation-3 poly(amidoamine) dendrimers as a versatile platform. Such macromolecules can incorporate eight NIR emitting lanthanide ions inside their branches forming species with thirty-two end groups at the periphery that can be substituted by suitable photosensitizers. Four new dendrimer ligands were synthesized with different photosensitizers, such as derivatives of naphthalimide, anthraquinone, and porphyrin. In addition the naphthalimide photosensitizer was functionalized with a targeting molecule, based on folic acid, to induce selectivity of the molecule towards cancer tissues. The corresponding NIR emitting lanthanide complexes were prepared for each dendrimer. Four Yb(III)-dendrimer complexes were characterized for their photophysical and ROS production properties. All complexes demonstrated a ROS production. The dendrimer functionalized with anthraquinone and tetraphenylporphyrin photosensitizers show strong NIR emission in living cells. These new multifunctional Yb(III)-dendrimer complexes have been designed to broaden the current scope of PDT agents and of NIR optical imaging agents

Le cancer colorectal (CCR) est l’un des cancers les plus diagnostiqués dans le monde mais surtout le 2ème cancerle plus mortel. Malgré les progrès de la recherche médicale dans les traitements anticancéreux, de nombreux effetssecondaires subsistent chez les patients ainsi que l’apparition de résistances aux traitements conventionnels. Ledéveloppement de nouvelles stratégies thérapeutiques anticancéreuses est donc nécessaire afin d’améliorer la priseen charge de ces patients. La thérapie photodynamique (PDT) utilisant des photosensibilisateurs (PS) se présentecomme une stratégie thérapeutique innovante limitant fortement ces effets secondaires indésirables. La PDT a étéapprouvée pour le traitement de certains cancers grâce à la génération d’espèces réactives de l’oxygènecytotoxiques uniquement après photoactivation des PS. Cependant, une faible solubilité et un manque de sélectivitédes PS vis à vis des sites tumoraux sont les principales limites en clinique. En effet, l’administration ciblée demédicaments est un point essentiel dans la thérapie anticancéreuse. La nanomédecine par l’utilisation denanoparticules permet d’améliorer le ciblage tumoral car elles sont capables de s’accumuler spontanément dansles tumeurs solides grâce à l’effet de perméabilité et de rétention accrue. L’objectif de cette étude a été dedémontrer l’intérêt de la vectorisation de la 5-(4-hydroxyphényl)-10,15,20-triphénylporphyrine-xylane (TPPOHX)sur des nanoparticules de silice (SNPs) afin d’augmenter l’efficacité anticancéreuse par un meilleur ciblagetumoral du traitement. Il a été démontré une augmentation significative de l’efficacité anticancéreuse des TPPOHXSNPs-PDT grâce à l’amélioration de l’internalisation cellulaire par rapport à la TPPOH libre-PDT sur 3 lignéescellulaires de CCR humain. De plus, il a été caractérisé que la mort cellulaire induite par les TPPOH-X SNPs-PDTest dépendante de la voie apoptotique et que l’autophagie joue un rôle de résistance à la mort cellulaire. Par ailleurs,in vivo et en l’absence de toxicité, les TPPOH-X SNPs-PDT induisent une augmentation de l’efficacitéanticancéreuse grâce à un meilleur ciblage tumoral par rapport à la TPPOH libre-PDT. Cette étude a donc permisde démontrer l’intérêt de la combinaison de la PDT et de la nanomédecine afin d’améliorer les futurs traitementsanticancéreux
Colorectal cancer (CRC) is one of the most common cancer globally but above all the second leading cause ofdeath for oncological reasons. Despite medical research advances in anti-cancer treatments, many side effectspersist in patients as well as development of resistances to conventional treatments. The development of new anticancertherapeutic strategies is necessary in order to improve care of patients. Photodynamic therapy (PDT) usingphotosensitizers (PS) comes as an innovative therapeutic strategy severely restricting these undesirable sideeffects. PDT has been approved for treatment of some cancers due to the generation of cytotoxic reactive oxygenspecies only with photoactivated PS. However, low physiological solubility and lack of selectivity towards tumorsites are the main limitations of their clinical use. Indeed, targeted drug delivery is a crucial point in cancer therapy.Nanomedicine through the use of nanoparticles improves tumor-targeting because they are able to spontaneouslyaccumulate in solid tumors through an enhanced permeability and retention effect. The purpose of this study wasto prove added value of 5-(4-hydroxyphenyl)-10,15,20-triphenylporphyrin-xylan (TPPOH-X) vectorization bysilica nanoparticles (SNPs) in order to enhance anti-cancer efficacy through better tumor-targeting. It has beendemonstrated significant anti-cancer efficacy increase of TPPOH-X SNPs-PDT thanks to cellular uptakeimprovement relative to free TPPOH-PDT in 3 human CRC cell lines. Moreover, it has been characterized thatcell death induced by TPPOH-X SNPs-PDT is conducted via apoptosis and autophagy acts as a resistance pathwayto cell death. Furthermore, in vivo and without toxicity, TPPOH-X SNPs-PDT induce an elevated anti-cancerefficacy through improvement of tumor-targeting compared to free TPPOH-PDT. This study therefore highlightedthe added value of PDT and nanomedicine combination in order to improve future cancer treatments

A terapia fotodinâmica (Photodynamic Therapy - PDT) é um método de tratar neoplasias baseado na interação entre luz, oxigênio molecular e um agente fotossensibilizador. Após a administração do agente, o tumor é iluminado com luz visível, ativando-o e produzindo espécies reativas de oxigênio, altamente citotóxicas, que provocam morte celular e destruição tecidual. Com a destruição do tumor há ativação do sistema imune inato e o subsequente processo inflamatório determina a apresentação de antígenos tumorais aos linfócitos, promovendo uma resposta imunológica adaptativa contra o tecido tumoral. O presente trabalho visou estudar a PDT associando um laser de diodo como fonte de luz e o fotossensibilizante Azul de Metileno (AM) a 1%, avaliando a sua efetividade no tratamento do Tumor de Ehrlich (TE) em sua forma sólida e a resposta imunológica nos animais tratados. Em um primeiro estudo, avaliou-se macro e microscopicamente tumores tratados, determinando a capacidade do protocolo em induzir inflamação e destruição do tecido tumoral. No segundo estudo, a resposta imune foi estudada em camundongos desafiados com um segundo implante de células do tumor de Ehrlich. O primeiro implante tumoral foi tratado com a PDT ou a excisão cirúrgica, comparando-se com um grupo controle sem tratamento. Os parâmetros avaliados após 17 dias foram o crescimento tumoral (p>0,05), peso relativo dos órgãos linfóides [Baço (p<0,05) e Linfonodo poplíteo (p>0,05)], tamanho relativo do linfonodo (p<0,05), presença de metástase em linfonodo poplíteo (p>0,05), contagem de leucócitos sanguíneos (p>0,05) e análise morfométrica quantitativa do tumor secundário [determinação da fração volumétrica de células tumorais (p<0,05), infiltrado inflamatório (p<0,05), necrose (p>0,06) e porcentagem da área tumoral em necrose (p<0,05)]. A PDT com o AM foi capaz de induzir necrose do TE e inflamação, havendo diferenças da resposta imune sistêmica quando comparado aos animais tratados por meio de excisão cirúrgica do tumor de Ehrlich.
Photodynamic therapy (PDT) is a method of treating neoplasms based on the interaction between light, molecular oxygen and a photosensitizing agent. After administration of the photosensitizer, the tumor is illuminated with visible light, activating the agent and producing reactive oxygen species (ROS). This highly cytotoxic ROS cause cell death and tissue destruction. The activation of the innate immune system and the subsequent inflammation induces tumor antigen presentation to lymphocytes, promoting an adaptive immune response against the tumor cells. This work aimed to study the PDT using a diode laser as light source and Methylene Blue (MB) 1% as photosensitizer. It was accessed its effectiveness in treating Ehrlich Solid tumor (ET) and the immune response produced in treated animals. First the treated tumors were evaluated macroscopically and microscopically, determining the ability of the protocol to induce inflammation and tumor tissue destruction. In a second study, the immune response was studied in mice challenged with a second tumor cell implant. The primary tumor was treated with PDT or surgical excision, comparing with a control group without treatment. The parameters evaluated after 17 days were tumor growth (p> 0.05), relative weight of lymphoid organs [spleen (p <0.05) and popliteal lymph node (p> 0.05)], the relative size of the lymph node (p <0, 05), metastasis at lymph node (p>0,05), blood leukocyte count (p> 0.05) and quantitative morphometric analysis of secondary tumor [determining the volume fraction of tumor cells (p <0.05), inflammatory infiltrate (p <0.05), necrosis (p> 0.06) and tumor necrosis area (p <0.05)]. PDT with MB was able to induce necrosis of the ET and inflammation, with differences in the immune response when compared to animals treated surgically to remove the Ehrlich tumor in its solid form.

La thérapie photodynamique (PDT) est une technique utilisée cliniquement pour traiter certaines maladies de la peau, la dégénérescence maculaire liée à l’âge et certains types de cancer. Elle fait intervenir trois composants : une molécule photosensible ou photosensibilisateur (PS), la lumière et l’oxygène. Après administration du PS, celui-ci va se localiser plus ou moins sélectivement dans les zones tumorales où il est alors activé par irradiation lumineuse à une longueur d’onde et une puissance données. Ceci engendre la formation d’espèces réactives de l’oxygène (ROS) très réactives, dont l'oxygène singulet1 O2, qui entraînent la destruction des tissus tumoraux par nécrose ou apoptose. Afin d’améliorer la sélectivité du traitement, différentes pistes sont actuellement exploitées dont l’élaboration de « photodynamic molecular beacons » (PMB). Dans un PMB, le photosensibilisateur (PS) est associé via un peptide à un inhibiteur 1O2, appelé quencher. Ce quencher inhibe la formation d’1O2 tant que le composé n’a pas atteint sa cible. Une fois la zone cancéreuse atteinte, des enzymes spécifiques clivent le peptide, libérant ainsi le PS qui retrouve alors sa capacité à former de l’1O2. Trouver un couple PS/quencher adéquat reste un challenge en PDT. Les propriétés photophysiques particulières des caroténoïdes et leur aptitude à inhiber la production d’1O2 font de ces derniers des quenchers potentiellement utilisables pour l’élaboration de PMBs. Chez les plantes, les caroténoïdes (carotènes et xanthophylles) sont des pigments associés à la photosynthèse, qui ont deux rôles principaux : un rôle de collecteur de lumière et un rôle photoprotecteur en protégeant le(s) système(s) photosynthétique(s) contre les dommages photooxydatifs liés à une exposition trop intense à la lumière. Ceci s’opère, entre autre, via le cycle des xanthophylles. Cette aptitude à capter de l’énergie présente un intérêt potentiel à ne pas négliger dans la perspective de la conception de PMB utilisables en thérapie photodynamique. Dans le cadre de cette thèse en co-tutelle avec la Faculté des Sciences de Bizerte nous avons ciblé les caroténoïdes présents dans trois fruits produits en Tunisie à savoir les kakis (Diospyros kaki L.), les abricots (Prunus armeniaca L.) et les pêches (Prunus persica L.) connus pour leur richesse globale en ces pigments. Divers procédés d’extractions ont été étudiés : (i) L’extraction de type Soxhlet par solvants organiques à pression atmosphérique, utilisée comme référence, (ii) l'extraction accélérée par solvant organique (ASE : Accelerated solvent Extraction) effectuée sous pression, enfin (iii) l'extraction par CO2 supercritique avec l’éthanol comme cosolvant. Pour ces deux derniers procédés, une approche par plan d’expériences (surfaces de réponses) a été utilisée pour identifier les facteurs clé et les conditions optimales d’extractions de divers caroténoïdes (pression, température, débit, % de cosolvant, temps, nombre de cycles). L'analyse par chromatographie liquide à haute performance couplée à la détection UV-Visible et à la spectrométrie de masse a ensuite permis l'identification et la quantification des caroténoïdes présents dans les extraits obtenus, permettant ainsi de comparer les profils caroténoïdiques propres à chaque fruit et les performances de chaque procédé d’extraction. Cette étude ayant révélé un profil caroténoïdique particulièrement intéressant chez le kaki par rapport aux autres fruits, une extraction et une purification des caroténoïdes de ce fruit par chromatographie liquide haute pression préparative a ensuite été effectuée afin de disposer d’une quantité suffisante de chaque caroténoïde, et parfois de leurs isomères conformationnels, en vue de l’étude de leurs propriétés photophysiques (absorption, émission de fluorescence, inhibition d’1O2) et de l’évaluation de leur intérêt potentiel en tant que quencher d’1O2 dans un édifice de type PMB
Photodynamic therapy (PDT) is a clinically used technique for treating skin diseases, age-relatedmacular degeneration but mainly some types of cancer. PDT involves three components: a photosensitive molecule named photosensitizer (PS), light and oxygen. After administration of the PS, this one will be located more or less selectively in tumoral regions where it is activated by light irradiation at appropriate wavelength and power. This leads to the formation of highly reactive and cytotoxic reactive oxygen species (ROS), especially singlet oxygen, resulting in the destruction of the tumor by necrosis or apoptosis. To improve the treatment selectivity, different strategies are being exploited, one of which is the development of "photodynamic molecular beacons" (PMB). In PMB the photosensitizer is linked via a peptide to an inhibitor of 1O2 (quencher). This quencher inhibits the formation of 1O2 as long as the compound has not reached its target, namely cancer cells. In order to inhibit the toxicity of the PS in non-target cells and restore toxicity only close to the biological target, it is necessary to find an adequate PS/quencher couple. This remains a challenge for PDT. Carotenoids are interesting candidates due to their specific photophysical properties and ability to inhibit 1O2, which makes them potential quenchers for building PMBs. In plants, carotenoids (carotenes and xanthophylls) are pigments involved in the photosynthesis, in which they play two main roles: a light collecting role and a protecting role by preserving the photosynthetic systems against photoxydative damages induced by a too intense light exposure. This protection can for instance occur via the well-known xanthophylls cycle. This capacity to catch energy presents a potential interest that should not be neglected in the framework of the design of PMBs usable in photodynamic therapy. Within the framework as part of this PhD thesis in Cotutelle with the Faculty of Sciences of Bizerte, we focused on carotenoids from three fruits produced in Tunisia: persimmon (Diospyros kaki L.), apricot (Prunus armeniaca L.) and peache (Prunus persica L.), known for their global richness in these natural pigments. Three extraction processes were investigated: (i) the Soxhlet extraction based on the use of organic solvent at atmospheric pressure and used as reference, (ii) the accelerated solvent extraction (ASE) using organic solvent under high pressure, and (iii) the supercritical fluid extraction (SFE) using supercritical CO2 and ethanol as cosolvent. For these two last processes, a design of experiments (Surface Response Design) was used to identify the key factors and optimal extraction conditions of various carotenoids (pressure, temperature, flow, % cosolvent, time, number of cycles). Then, HPLC-PDA coupled with mass spectrometry (MS) enabled the identification and quantification of carotenoids from the extracts. Thus it was possible to compare the profiles in carotenoids content from each fruit as well as the performances of each extraction process. This study showed that the carotenoidic profile in the persimmon was the most interesting as compared to the profiles in the two other fruits. Extraction and purification of the carotenoids from persimmon by preparative high pressure liquid chromatography were then performed in order to have a sufficient amount of each carotenoid and sometimes of their conformational isomers. We finally performed a study of their photophysical properties (absorption, fluorescence emission, 1O2 inhibition) in order to evaluate their potential as 1O2 quencher in molecular construction such as a PMB

La nanomédecine activée à deux-photon est devenue l'un des principaux candidats à l'accomplissement de la sélectivité spatiotemporelle nécessaire pour la nanomédecine. En effet, la raison d'être de l'application médicale de nanotechnologie dans le domaine du traitement du cancer est de diminuer et supprimer les effets secondaires causés par les techniques actuelles telles que la chimiothérapie et la radiothérapie, à cause de leur manque de sélectivité. Parmi diverses nanoparticules (NPs), les nanoparticules de silice mésoporeuse (MSN) ont attiré une attention croissante dans la dernière décennie pour leur faible cytotoxicité, leur internalisation cellulaire et excrétion, et leur capacité de combiner de nombreuses fonctions à la fois pour le diagnostique et la thérapie de cancers via un seul nanovéhicule : l'ainsi appelée nanomédecine théranostique.Dans cette thèse, des MSN pour l'activation à un et deux-photon d'imagerie par fluorescence, de délivrance de principe actifs et d'acides nucléiques, et de photothérapie dynamique (PTD), seront présentées. Premièrement, le relargage contrôlé de molécules encapsulées dans des MSN fonctionnalisées avec des nanovalves est considéré par effet plasmonique. La photodégradation contrôlée de la silice soumise à l'effet photothermique de NPs d'or est ensuite étudiée. Deuxièmement, l'activation biphotonique est considérée pour la délivrance contrôlée de molécules anticancéreuses in-vitro par avec des nano-rotor et des nano-valves, ainsi que la fonctionnalisation de surface des NPs par des dérivés d'ammonium- azobenzene pour la délivrance d'acides nucléiques. Troisièmement, des MSN multifonctionnelles incorporant des photosensibilisateurs à deux-photon sont systématiquement étudiées en termes de leurs propriétés optiques et photophysiques; la sélection du meilleur matériau est suivie d'applications biomédicales in-vitro.De plus, deux types de nanomatériaux émergeant sont également élaborés pour la nanomédecine activée à deux-photon, des NPs de polysilsesquioxane pontés (BS) et d'organosilice mésoporeuse périodiques (PMO). Ces matériaux furent élaborés sans précurseur de silice (tétraéthoxysilane par exemple), et seulement à partir de bis- ou multi-organoalkoxysilane, afin d'obtenir le plus haut pourcentage de matière organique pour l'application ciblée. En conséquence, des NPs de BS et de PMO hybrides à base de disulfures se révélèrent être des outils biodégradables, et les NPs à base de photosensibilisateurs furent appliquées pour la PTD à deux-photon. Des NPs de BS et de cœur-coquille d'or-BS sont synthétisées pour d'efficaces imagerie et PTD à deux-photon, tandis que des NPs de PMO servirent de nano-plateformes théranostiques. En outre, diverse NPs de PMO multipodes à surface spécifique très élevées sont présentées pour la construction de structuration complexe à l'échelle nanométrique.Enfin, des nano-conteneurs d'MSN composées de cœur d'oxyde de fer (Fe3O4@MSN) sont décrites pour de multiples applications. D'une part, l'élaboration de NPs MSN (et PMO) magnétiques sensibles à deux-photon est étudiée en tant que perspective pour la délivrance de gène combinant l'imagerie par résonance magnétique. D'autre part, les conteneurs de Fe3O4@MSN sont misent en œuvre et appliqués pour la dépollution de métaux lourds via la fonctionnalisation d'un ligand de type acide diéthylène triamine penta acétique. L'augmentation de l'efficacité de la dépollution est étudiée par la fonctionnalisation de la surface extérieure et/ou des pores des Fe3O4@MSN
Two-photon actuated nanomedicine has become one of the main proponents for the achievement of the spatiotemporal selectivity needed for nanomedicine. Indeed, the raison d'être of the medical application of nanotechnology in the field of cancer treatment is to lower and suppress the side effects caused by current techniques such as chemotherapy and radiotherapy, due to their lack of selectivity. Among various nanoparticles (NPs), mesoporous silica nanoparticles (MSN) have attracted increasing attention over the past decade for their low cytotoxicity, cellular internalization and excretion, and the ability to carry multiple features for both the diagnosis and therapy of cancers in a single nanovehicle: the so-called theranostic nanomedicine.In this dissertation, I will describe MSN for one and/or two-photon-actuated fluorescence imaging, drug-delivery, gene delivery and photodynamic therapy (PDT). First, plasmonically-triggered cargo delivery via MSN nanovalves and designed mesoporous silica photodegradation is presented. Then, in-vitro two-photon-triggered drug delivery with azobenzene-functionalized MSN such as nanoimpellers and fluorescent nanovalves, along with preliminary studies of gene delivery via ammonium-functionalized nanoimpellers are discussed. Multifunctional MSN incorporating a two-photon photosensitizer are systematically studied in terms of the resulting optical and photophysical properties of the NPs, and then used for in-vitro biomedical applications.Furthermore, two kinds of emerging nanomaterials are also designed for two-photon actuated nanomedicine, bridged silsesquioxane (BS) and periodic mesoporous organosilica (PMO) NPs. These nanomaterials are elaborated without silica precursor (e.g. tetraethoxysilane) and solely with bis- or tetra-organoalkoxysilanes, thus providing materials with the highest organic content for the targeted applications. Consequently, disulfide-based hybrid BS and PMO NPs were elaborated as biodegradable nanomedical tools, and photosensitizer-based BS and PMO NPs were used for efficient in-vitro PDT. BS and gold-BS core-shells NPs are constructed for ultrabright two-photon imaging and efficient PDT, while two-photon functionalized PMO NPs serve as theranostic nanocarriers. Besides, versatile multipodal ethylene-benzene PMO NPs with very high surface areas are presented as a promising strategy for the design of structural complexities at the nanoscale.Finally, iron oxide core MSN shell (Fe3O4@MSN) nanocontainers are described for versatile applications. The design of two-photon-sensitive magnetic MSN and PMO core-shell nanovehicles is presented as a perspective for gene delivery and magnetic resonance imaging. Furthermore, Fe3O4@MSN containers are constructed for heavy metal removal of twelve of the most toxic metal ions through the diethylene triamine pentaacetic acid (DTPA) ligand. The enhancement of the pollutant removal efficiency is studied by selective surface and/or porous DTPA functionalizations

Ce projet de thèse avait pour but de synthétiser des molécules capables d’absorber la radiation Cherenkov (processus de CRET) et de les étudier en biologie pour développer les concepts d’imagerie par luminescence Cherenkov proche infrarouge (CLI-NIR) et de thérapie photodynamique Cherenkov (PDT-CR). L’utilisation de cette source d’énergie Cherenkov dans le domaine médical est relativement jeune (2009). En 2019, la synthèse de molécules spécialement dédiées pout une utilisation optimale du CR n’est pas encore répandue. Ainsi, une série de plateformes moléculaires ont été conçues, soit pour générer de la lumière dans une optique de diagnostic (fluorophores), soit pour générer des espèces réactives de l’oxygène dans une optique thérapeutique (photosensibilisateurs). Nous avons également proposé une autre utilisation biomédicale encore inédite de la radiation Cherenkov : l’activation de molécules pouvant libérer du monoxyde de carbone (photo-CORM).Il fut ainsi nécessaire de développer des fluorophores et des photosensibilisateurs associés à des antennes, pour leur permettre d’absorber au mieux la luminescence Cherenkov, dont le pic d’émission se situe dans la région de l’UV et du bleu du spectre électromagnétique. Les subphtalocyanines, les phtalocyanines et les cyanines 7 ont été choisies pour leurs propriétés de fluorescence et/ou photosensibilisatrices. Des antennes comme la coumarine et le pyrène ont été introduites sur ces plateformes.Les études photophysiques, en présence d’émetteurs Cherenkov, ont montré que certains « hybrides moléculaires » absorbent la luminescence Cherenkov et fluorescent dans le rouge/proche IR avec de bons rapports d’amplifications.L’étude de génération d’oxygène singulet et de phototoxicité sur cellules de mélanome murin en présence de radiation Cherenkov (à l’obscurité et en l’absence de tout autre rayonnement externe) a montré que d’autres « hybrides » sont de bons agents photosensibilisants.Ces travaux de thèse ont donc montré que le concept d’imagerie par luminescence Cherenkov proche infrarouge (CLI-NIR) permettrait d’obtenir un signal provenant d’une zone plus en profondeur par rapport à la CLI classique, et que la thérapie photodynamique activée par radiation Cherenkov (PDT-CR) permettrait de s’affranchir des contraintes de pénétrabilité tissulaire du faisceau d’irradiation en PDT classique
The aim of this thesis was to synthesize molecules capable of absorbing the Cherenkov radiation (the CRET process) and to study them to develop both concepts: near infrared Cherenkov luminescence imaging (NIR-CLI) and Cherenkov photodynamic therapy (CR-PDT). The use of such an energy source in the biomedical field is quite recent (2009). In 2019, the synthesis of molecules dedicated to an optimal use of the CR is not widespread. Hence, a series of molecules have been designed to achieve ether light emission for diagnostic purpose (fluorophores) or to achieve the generation of reactive oxygen species for a therapeutic purpose (photosensitizers). We have also suggested a new possible use of the Cherenkov radiation in the biomedical field: the activation of molecules able to release carbon monoxide (photo-CORM).The synthesis of fluorophores and photosensitizers bearing antenna have been carried out. Such antenna allows the resulting conjugates to absorb the Cherenkov luminescence, the emission of which is located in the UV/blue region of the electromagnetic spectrum. Subphthalocyanines, phthalocyanines and cyanines 7 have been chosen because of their fluorescence and/or photosensitizing properties. Antenna, such as coumarine or pyrene, have been introduced on these platforms.Photophysical studies, showed that our probes, in the presence of a Cherenkov emitter (such as [18F]-FDG, 18F-fluorodeoxyglucose), were able to absorb the Cherenkov light and subsequently emit photons in the red/NIR region leading to promising amplification of the radiance in this window.Also, in the presence of Cherenkov emitter (with no external light source), our probes appeared to be efficient photosensitizers because they generate singlet oxygen and lead to (Cherenkov-induced) photocytotoxicity on melanoma cells.These thesis studies showed that Near Infrared Cherenkov Luminescence Imaging (NIR-CLI) allowed to detect a signal coming from a deeper area compared to the classic CLI, and that Cherenkov induced Photodynamic Therapy (CR-PDT) allowed to overcome the penetrability issue coming from the irradiation beam in classic PDT

L'utilisation de nanoparticules comme vecteurs d'anticancéreux est de plus en plus étudiée afin de résoudre certains problèmes inhérents aux traitements conventionnels, tels que la faible spécificité des agents anticancéreux pour la tumeur, et le risque de provoquer des effets secondaires irréversibles. L'objectif de cette thèse a été d'étudier l'utilisation de nanoparticules de silicium poreux fonctionnalisées (pSiNP), biorésorbables et non toxiques in vivo, pour l'imagerie et la délivrance ciblée d'agents photosensibilisateurs pour la thérapie photodynamique (PDT) et pour la délivrance contrôlée d'acides nucléiques. Dans une première étude, différentes formulations à base de pSiNP, comportant une porphyrine et/ou un agent de ciblage des cellules cancéreuses, le mannose, ont été préparées. L'imagerie de fluorescence à 2-photon a montré que les nanoparticules étaient internalisées par les cellules plus efficacement lorsqu'elles étaient fonctionnalisées avec le mannose. L'efficacité photodynamique de ces systèmes a été démontrée in vitro sur plusieurs lignées cellulaires (cancer du sein (MCF-7) et cancer de la prostate (LNCaP)) sous irradiation biphotonique. Nous avons déterminé que l'effet PDT observé se produit par excitation des pSiNP, qui ensuite transfèrent leur énergie à la porphyrine greffée. En comparaison, une irradiation monophotonique entraîne une excitation directe de la porphyrine. Par ailleurs, des formulations encapsulant d'autres photosensibilisateurs tels que des complexes de ruthénium (II), ou des nanoparticules d'or ont été préparées et leur efficacité photodynamique a été également testée. Dans une deuxième étude, des pSiNP ont été préparées et fonctionnalisées avec des acides aminés (histidine, lysine et poly-L-Lysine) pour la complexation et la délivrance d'acides nucléiques (pDNA, siRNA). Les différentes formulations ont été testées en transfection cellulaire sur différentes lignées cellulaires (HEK, MCF-7). La délivrance photocontrôlée de siRNA sous irradiation biphotonique a été montrée pour des formulations fonctionnalisées via un ligand azobenzene photosensible
The use of nanoparticles as anticancer nanovectors is intensively studied in order to solve some inherent problems with conventional treatments, such as the low specificity of the anticancer agents for the tumor, and the risk of causing irreversible side effects. The goal of this thesis was to study the potential of functionalized bioresorbable and non-toxic porous silicon nanoparticles (pSiNP) for both, the imaging and the targeted release of photosensitizing agents for photodynamic therapy, as well as for the release of nucleic acids for gene therapy. In a first study, several pSiNP based formulations containing a porphyrin and/or a cancer cells targenting agent (mannose) were prepared. Two-photon fluorescence imaging showed that the nanoparticles were more efficiently internalized by the cells when they were functionalized with mannose. The photodynamic efficiency of these systems was demonstrated in vitro in several cell lines (breast cancer cell (MCF-7) and prostate cancer cell (LNCaP)) under 2-photons irradiation. We determined that the observed PDT effect occurs by exciting the pSiNP which then transfer their energy to the grafted porphyrin. In comparison, a 1-photon irradiation causes a direct excitation of the porphyrin. Furthermore, formulations encapsulating other photosensitizers such as ruthenium (II) complexes, or gold nanoparticles were prepared and their photodynamic efficiency was also tested. In a second study, pSiNP were prepared and functionalized with amino acids (histidine, lysine and poly-L-lysine) for the complexation and release of nucleic acid (pDNA, siRNA). The different formulations were tested in cellular transfection on different cell lines (HEK, MCF-7). The photo-controlled release of the siRNA under 2-photons irradiation was demonstrated for functionalized formulations via a photosensitive azobenzene ligand

Malgré des avancées considérables dans la détection et le traitement du cancer, cette maladie est à ce jour la deuxième cause de décès dans le monde. Ce projet de thèse a eu pour buts de concevoir et optimiser des agents d’imagerie pour le diagnostic et/ou la thérapie de divers cancers.Le premier axe de cette thèse a porté sur l’élaboration d’un agent d’imagerie bimodale TEP/fluorescence peptidique capable de cibler les récepteurs à la neurotensine, NTS1, surexprimés dans certains cancers. L'imagerie TEP permettrait une détection efficace des tumeurs et de leurs métastases, tandis que l'imagerie par fluorescence faciliterait l'évaluation des marges tumorales durant la chirurgie. Plusieurs composés ont été synthétisés, marqués au gallium-68 et étudiés en préclinique sur un modèle de cancer du pancréas. Des résultats très prometteurs ont été obtenus pour un des composés, faisant de lui un bon candidat pour le diagnostic et l’aide à la chirurgie du cancer du pancréas.Le second axe de recherche a été consacré à la conception d’un radiotraceur TEP ciblant NTS1. Une molécule nommée [177Lu]Lu-IPN01087 est actuellement évaluée dans des essais cliniques pour la radiothérapie ciblée et l'identification d'un compagnon diagnostic faciliterait la sélection des patients éligibles pour cette approche. Nous avons synthétisé et évalué in vivo sur un modèle de cancer colorectal différents radiotraceurs marqués au gallium-68, mettant en avant un potentiel candidat pour le diagnostic par imagerie TEP de cancers surexprimant NTS1.Enfin, la dernière partie de cette thèse est consacrée à l’élaboration d’un agent d’imagerie théranostique TEMP/PDT ciblant le récepteur EGFR. L’imagerie TEMP permettrait de diagnostiquer et stadifier les patients, tandis que la sonde PDT permettrait l’assistance à la chirurgie et la destruction des cellules tumorales. L’emploi d’une plateforme trivalente, la dichlorotétrazine, nous a permis d’introduire un agent chélatant l’indium-111 et un photosensibilisateur via une réaction de bioconjugaison site-spécifique sur un nanobody. Le conjugué obtenu a ensuite été évalué in vitro puis l’étude de la biodistribution et de l’efficacité de la thérapie photodynamique a été réalisée en préclinique
Despite significant advances in cancer detection and treatment, cancer is now the second leading cause of death worldwide. The aim of this thesis project was to design and optimize imaging agents for the diagnosis and/or therapy of various cancers.The first axis of this thesis focused on the development of a bimodal PET/fluorescence imaging agent capable of targeting neurotensin NTS1 receptors overexpressed in certain cancers. PET imaging would allow efficient detection of tumors and their metastases, while fluorescence imaging would facilitate the evaluation of tumor margins during surgery. Several compounds were synthesized, labelled with gallium-68 and studied in preclinical studies in a pancreatic cancer model. Very promising results were obtained for one compound, making it a good candidate for the diagnosis and fluorescence guided surgery of pancreatic cancer.The second line of research was devoted to the design of a PET radiotracer targeting NTS1. A molecule named [177Lu]Lu-IPN01087 is currently being evaluated in clinical trials for targeted radiotherapy. The identification of a diagnostic companion agent would facilitate the selection of patients eligible for this therapy. We synthesized and evaluated in vivo, in a colorectal cancer model, different gallium-68 labelled tracers, highlighting a potential candidate for the diagnosis by PET imaging of NTS1 overexpressing cancers.Finally, the last part of this thesis focused on the development of a theranostic SPECT/PDT imaging agent targeting the EGFR receptor. SPECT imaging would allow the diagnosis and staging of patients, while the PDT probe would facilitate the surgical resection of the tumor and eradication of cancer cells. We used a trivalent platform, dichlorotetrazine, that allowed us to introduce an indium-111 chelating agent and a photosensitizer via a site-specific bioconjugation reaction on a nanobody. The resulting conjugate was evaluated in vitro and the biodistribution and efficacy of the photodynamic therapy were investigated in preclinical studies

L'efficacité de la thérapie photodynamique (PDT) est confrontée à plusieurs verrous : les photosensibilisateurs (PSs) utilisés en clinique ne sont pas adaptés à la fenêtre thérapeutique, ils subissent un photoblanchiment lors du traitement, et leur nature organique pose des problèmes de solubilité en milieu biologique. Ils présentent également une faible sélectivité envers les tissus tumoraux à traiter. Dans le cadre de cette thèse, trois approches visant une amélioration de la PDT appliquée à la cancérologie ont été développées : la vectorisation, l'adressage, et l'optimisation de nouveaux PSs. La synthèse de structure PS/vecteur a permis d'élaborer des structures hydrophiles capables de vectoriser des PSs hydrophobes. Des porphyrines ont ainsi été greffées sur des dendrimères polyamidoamine (PAMAM) dissymétriques. La conservation des propriétés photophysiques des PSs après leur couplage au dendrimère a été mise en évidence. Des quantum dots (QDs), grâce à la modularité de leurs propriétés photophysiques et leur capacité à résister au photoblanchiment, ont été synthétisés et utilisés comme nouvelle classe d'agents photosensibilisants. Ces QDs ont été préparés afin d'être hydrophiles et utilisables dans la fenêtre thérapeutique de la PDT. Une étude in vitro des QDs couplés à l'acide folique a mis en évidence leur activité photodynamique. Des études réalisées par une approche de plans d'expérience a permis de hiérarchiser les facteurs expérimentaux en fonction de leurs impacts sur l'activité photodynamique. Nous avons en particulier montré une amélioration de la sélectivité des conjugués envers les cellules surexprimant le récepteur à l'acide folique.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária
A terapia fotodinâmica (PDT, do inglês, photodynamic therapy) é uma opção terapêutica recente e valiosa, dirigida para a destruição das células tumorais, com potencial para ser incluída no tratamento principal de combate ao cancro, bem como adjuvante de outras terapêuticas. Atualmente é considerada uma estratégia terapêutica de sucesso, clinicamente, aprovada para o tratamento de lesões potencialmente malignas (LPM) e malignas da cavidade oral. A PDT envolve a administração de um fotossensibilizador (PS, do inglês, photosensitizer) que, por si só, não possui efeito farmacológico. Contudo, este é, posteriormente ativado por irradiação, com luz visível de comprimento de onda (λ) adequado ao local a tratar. Com a foto-ativação do PS ocorre a produção de espécies reativas de oxigénio (ROS, do inglês, reactive oxygen species) que dão inicio à morte celular. O processo está associado com a indução de uma forte reação inflamatória local, potenciando a resposta imunitária e conduzindo à destruição efetiva das células tumorais. O efeito citotóxico ocorre, apenas, no local de ativação da luz e o PS acumula-se preferencialmente nas células malignas, pelo que esta terapêutica apresenta elevada seletividade e um baixo número de efeitos secundários. No contexto clínico, a PDT é utilizada em diversas áreas da medicina como a oftalmologia, a dermatologia e a oncologia, entre outras. No entanto, a sua utilização no tratamento do cancro ainda é limitada.
Photodynamic therapy (PDT) is a new and valuable therapeutic option, directed to the destruction of tumor cells, with the potential to be included in the primary treatment to combat cancer, as well as an adjunct to other therapies. A successful therapeutic strategy, clinically approved for the treatment of premalignant lesions (LPM) and malignant oral cavity is considered currently. PDT involves the administration of a photosensitizer (PS) which, by itself, does not possess pharmacological effect. However, this is subsequently activated by irradiation with visible light of wavelength (λ) appropriate to the site to be treated. With the photo-activation of the PS occurs the production of reactive oxygen species (ROS, English, reactive oxygen species) that give beginning to cell death. The process is associated with the induction of a strong local inflammatory response, enhancing the immune response and leading to the effective destruction of tumor cells. The cytotoxic effect occurs only in the light activation site and the PS accumulates preferentially in malignant cells, whereby this treatment has high selectivity and a low number of side effects. In the clinical context, PDT is used in many areas of medicine such as ophthalmology, dermatology and oncology, among others. However, their use in cancer treatment is still limited.

Porphyrins represent one of the oldest, most widely studied chemical structures, both in nature and in biomedical applications. Due to their tumor avidity and favorable photophysical properties, such as long wavelength absorption and emission, easy derivatization, high singlet oxygen quantum yield and low in vivo toxicity, porphyrins have found particular success for photodynamic therapy and fluorescence imaging of cancer. Additionally, they are excellent metal chelators, forming highly stable metallo-complexes, making porphyrins an efficient delivery vehicle for radioisotopes. Thus, there is great potential in the applications of these multi-modal porphyrin-based agents for cancer imaging and therapy. I have investigated the characteristics of various porphyrin-based probes and their potential application in different clinically relevant models. Here, I will discuss three types of porphyrin-based agents: 1) photodynamic molecular beacons (PPMMPB), 2) targeted peptide porphyrins (PPF) and 3) porphyrin-lipid nanovesicles, porphysomes. I will demonstrate that all of these porphyrin-based agents have potential clinical applications in various fields of cancer imaging and therapy. Although these three agents differ greatly, they all aim to increase the signal-to-background ratio of tumor to healthy tissue uptake of porphyrins, thereby increasing our ability to detect tumor tissue and better preserve healthy tissue during therapy.

This thesis work deals with different aspects of the chemistry of La(III) and Gd(III) complexes, their interaction with DNA and proteins, photo-induced cleavage of double-stranded DNA, photocytotoxic effect on cancer cells, cell death mechanism and cellular localization behaviour. Chapter I gives an introduction to the metal-based anticancer agents with special emphasis on clinically used drugs and the growing field of lanthanide therapeutics. An overview of the current strategies of cancer treatment, especially photodynamic therapy (PDT), is presented. Mode of small molecule-DNA interactions and the mechanistic aspects associated with DNA photodamage reactions and PDT effect are discussed with selected examples of compounds that are known to photocleave DNA on exposure to light of different wavelengths. A brief discussion on the various therapeutic applications of the lanthanide compounds is also made. Chapter II presents the synthesis, characterization, DNA binding, BSA binding, photo-induced DNA cleavage activity and photocytotoxicity of La(III) and Gd(III) complexes of phenanthroline bases to explore the UV-A light-induced DNA cleavage activity and photocytotoxicity of the complexes. Chapter III describes the synthesis, characterization, DNA binding, photo-induced DNA cleavage activity and photocytotoxicity of La(III) and Gd(III) complexes of phenanthroline bases with an aim to improve the design of the complexes to achieve better solution stability and DNA binding of the complexes. Chapter IV presents the synthesis, characterization, DNA binding, and UV-A light-induced DNA photocleavage activity and photocytotoxicity of La(III) and Gd(III) complexes of pyridyl phenanthroline bases with an objective to improve the photoactivity of the complexes by introducing an additional pyridyl group. Cell death mechanism and confocal microscopic studies are also carried out to gain more insight into the PDT effect caused by light in the presence of the complex. Chapter V describes the synthesis and characterization of La(III) and Gd(III) complexes of terpyridine bases and acetylacetonate to study the complexes as a new class of photosensitizers to explore their DNA photocleavage activity and photocytotoxicity in HeLa cells. Effect of attaching a glucose moiety to the acetyl acetone (Hacac) ligand has been studied. The cellular uptake behaviour of the La(III) pyrenyl-terpyridine complexes has also been investigated. Finally, Chapter VI presents the synthesis and characterization of curcumin and glycosylated curcumin La(III) and Gd(III) complexes having terpyridine base with an objective to study the photoactivated anticancer activity of the complexes in visible light. This chapter describes the visible light-induced DNA cleavage activity and photocytotoxicity of the complexes by exploiting curcumin and glycosylated curcumin as the photosensitizer ligands. Study on the cellular uptake behavior of curcumin La(III) complexes having pyrenyl terpyridine ligand is also presented. The references have been assembled at the end of each chapter and indicated as superscript numbers in the text. The complexes presented in this thesis are represented by bold-faced numbers. Crystallographic data of the complexes which are characterized structurally by single crystal X-ray crystallography are provided in CIF format in the enclosed CD (Appendix-I). Due acknowledgements have been made wherever the work described is based on the findings of other investigators. Any unintentional omission that might have happened due to oversight or mistake is sincerely regretted.

Photodynamic therapy (PDT) is a clinically approved and less invasive cancer treatment. It causes less damage to normal tissue compared to radiation or chemotherapy. The therapeutic procedure includes the application of a photosensitizing drug (PS) that is subsequently activated by monochromatic light of a specific wavelength. The photochemical reaction leads to the generation of highly toxic oxygen species (ROS) that induce oxidative stress in cells and ultimately promote cell death. The limitation of PDT is the development of resistance to PDT in some cancer cells. Several mechanisms are known to be involved in cellular defense against the cytotoxic effects of PDT, including the activation of antioxidant enzymes, drug efflux pumps, degradation of PS and overexpression of protein chaperons. However, the role of DNA repair in resistance of cancer cells to PDT has not been well studied. This project aimed to gain knowledge about the role of DNA repair and other potential novel mechanisms in acquiring resistance to photodynamic therapy, as well as to use this knowledge in the search for new resistance markers and new methods for sensitizing cancer cells to this type of treatment. For this purpose, PDT resistant cells were isolated from parental glioblastoma (GBM, U-87 MG) and cervical carcinoma (SKG-IIIa) cell lines following repetitive PDT cycles. Isolated resistant cells (U-87 MGR and SKG-IIIaR) were subjected to different approaches to characterize in terms of knowledge the degree of oxidative stress and DNA damage, cell cycle proliferation and course, protoporphyrin accumulation, epigenetic marks, expression and activity of DNA base excision repair (BER) enzymes and efficiency of DNA repair. Moreover, microarray and proteomics analysis was applied to explore novel mechanisms associated with the resistance to PDT in the glioblastoma cells. The results demonstrated that the resistance to PDT in U-87 MGR cell line may be a consequence of several mechanisms that can function independently or in collaboration. In U-87 MGR cells, a higher level of epigenetic changes, including DNA hypermethylation (5-mC and 5-hmC), upregulation of noncoding RNAs (SNORD family) and deregulation of some miRNAs expression, upregulation of histone H1F0 and overexpression of HDAC10 can cause striking effects in the regulation of gene expression engaged in the acquiring of resistance to PDT. Interestingly, some of these epigenetic marks are known to cause metabolic changes, cell cycle reprogramming, slower proliferation, and differentiation. Here, my data suggests that resistance to PDT in the glioblastoma cell line may be associated with reduced rate of some metabolic pathways, change in cell cycle regulation (the G1 phase elongation) and slower proliferation. U-87 MGR cells exhibited lower levels of porphyrins, both intracellular and in medium, including PpIX (PS for PDT), which, despite upregulation of drug efflux pumps, is most likely due to an impairment of the heme biosynthesis pathway. As expected, epigenetic changes also deregulate the expression of antioxidant enzymes, leading to lower oxidative DNA damage after PDT in the resistant cells than in the parental sensitive ones. Resistance to PDT in glioblastoma cells may also be associated with higher efficiency of the initial steps of the BER pathway and the repair of DNA breaks. Increased expression and higher activation of ATM and ATR kinases, main and primary signaling molecules of DNA damage response (DDR), were demonstrated in resistant U-87 MGR cells even before PDT treatment, suggesting that they are good targets for sensitization of resistant cells to PDT. As expected, resistant glioblastoma cells can be partially sensitized to PDT by inhibiting ATM kinase, which suggests that ATM has a crucial role in PDT resistance. This may be due to the role of ATM in extending the G1 phase (inducing the G1 checkpoint), and thus facilitating the rapid response to PDTmediated DNA damage in U-87 MG resistant cells. PDT resistant cervical carcinoma cell line, although shared some common features with glioblastoma resistant cells, differed in DNA repair. The cell cycle reprogramming, the G1 checkpoint, higher ROS scavenging activity and lower PpIX biosynthesis rate were common factors in both U-87 MGR and SKG-IIIaR cell lines. Compared to their parental line, PDT resistant SKG-IIIa cells overexpressed two important proteins involved in BER, APE1 and PARP1, and demonstrated increased activity of the major BER steps. This indicated that resistant cells could be sensitized to PDT by inhibiting the APE1 and PARP1 activity. Indeed both enzymes proved to be the target; however, the APE1inhibitor was more effective. These findings indicate a key role of the BER pathway in acquiring resistance to PDT in the cervical carcinoma cell line, which can be overcome by inhibiting two BER enzymes. In summary, data obtained during this project showed that, besides other mechanisms disclosed, DNA repair is the important mechanism of PDT resistance in both glioblastoma and cervical carcinoma cell lines. This study also suggests that the inhibitor of one of the major DDR kinase, ATM and inhibitors of the BER pathway (APE1 and PARP1) may become effective adjuvants that increase the effectiveness of PDT therapy in GBM and cervical cancer, respectively.
Terapia fotodynamiczna (ang. photodynamic therapy, PDT) jest klinicznie zatwierdzoną metodą walki z nowotworami charakteryzującą się niską inwazyjnością oraz brakiem poważnych efektów ubocznych. Opiera się na wykorzystaniu substancji fotouczulającej oraz światła widzialnego do niszczenia komórek nowotworowych. Mechanizm jej działania polega na wzbudzeniu fotouczulacza przez światło, a następnie jego interakcji z tlenem znajdującym się w komórce, co w konsekwencji prowadzi do powstania reaktywnych form tego pierwiastka (RFT) i stresu oksydacyjnego, a następnie śmierci komórek. Niestety skuteczność terapii fotodynamicznej jest niższa od innych metod leczenia nowotworów – chemioterapii i radioterapii. Wiązać się to może ze zjawiskiem nabywania przez komórki nowotworowe oporności na PDT. W obronę komórkową przed cytotoksycznymi skutkami PDT zaangażowanych jest kilka mechanizmów, w tym aktywacja enzymów antyoksydacyjnych, pompy usuwające leki, degradacja substancji fotouczulającej i nadekspresja białek opiekuńczych. Jednak rola naprawy DNA w oporności komórek rakowych na PDT nie została dobrze poznana. Niniejszy projekt miał na celu uzyskanie wiedzy na temat roli naprawy DNA i innych potencjalnych nowych mechanizmów w nabywaniu oporności na terapię fotodynamiczną, jak również wykorzystanie tej wiedzy w poszukiwaniu nowych markerów oporności i nowych metod uwrażliwiania komórek nowotworowych na ten rodzaj leczenia. W tym celu wyizolowano komórki oporne na PDT z linii komórek glejaka (GBM, U-87 MG) i raka szyjki macicy (SKG-IIIa) poprzez wielokrotne powtarzanie cykli PDT i dalszą hodowlę przeżywających komórek. Uzyskane nowotworowe linie oporne (U-87 MGR i SKG-IIIaR) scharakteryzowano pod kątem poznania stopnia stresu oksydacyjnego i uszkodzeń DNA, proliferacji i przebiegu cyklu komórkowego, akumulacji protoporfiryny, znaczników epigenetycznych, ekspresji i aktywności enzymów naprawy DNA przez wycinanie zasad (ang. base excision repair, BER) oraz wydajności naprawy DNA. Ponadto zastosowano analizę mikromacierzy i proteomikę w celu zbadania nowych mechanizmów związanych z opornością na PDT. Wyniki wykazały, że oporność na PDT w linii komórkowej U-87 MGR może być konsekwencją kilku mechanizmów, które mogą funkcjonować niezależnie lub współdziałać przy nabywaniu przez komórki oporności na PDT. W komórkach U87 MGR wyższy poziom zmian epigenetycznych, w tym hipermetylacja DNA (5-mC i 5-hmC), pozytywna regulacja niekodujących RNA (rodzina SNORD) i deregulacja ekspresji niektórych miRNA oraz nadekspresja histonu H1F0 i HDAC10 mogą mieć silny wpływ na regulację ekspresji genów zaangażowanych w nabywanie oporności na PDT. Co ciekawe, niektóre z tych znaczników epigenetycznych powodują zmiany metabolizmu, przeprogramowanie cyklu komórkowego, spowolnienie proliferacji i różnicowania. Moje dane sugerują, że oporność na PDT w linii komórkowej glejaka może być związana ze zmniejszonym tempem metabolizmu, zmianą regulacji cyklu komórkowego (wydłużenie fazy G1) i wolniejszą proliferacją. Komórki U-87 MGR wykazywały niższe poziomy porfiryn, zarówno wewnątrzkomórkowych, jak i w pożywce, w tym PpIX (lek fotouczulający stosowany w PDT), co, pomimo braku bardziej efektywnie działających pomp komórkowych usuwających lek, jest najprawdopodobniej spowodowane zaburzeniem szlaku biosyntezy hemu. Zgodnie z oczekiwaniami zmiany epigenetyczne również rozregulowują ekspresję enzymów antyoksydacyjnych, prowadząc do mniejszego uszkodzenia oksydacyjnego DNA po PDT w komórkach opornych w porównaniu do wrażliwych. Oporność na PDT w komórkach glejaka może być również związana z wyższą wydajnością początkowych etapów szlaku BER i naprawą pęknięć DNA. Zwiększoną ekspresję i wyższą aktywację kinaz ATM i ATR, nadrzędnych cząsteczek sygnałowych odpowiedzi na uszkodzenie DNA (ang. DNA damage response, DDR), wykazano w komórkach opornych nawet przed traktowaniem PDT, co sugeruje, że są one dobrymi celami uwrażliwiania komórek na PDT. Zgodnie z oczekiwaniami, oporne komórki glejaka można częściowo uwrażliwić poprzez hamowanie kinazy ATM, która odgrywa kluczową rolę w oporności na PDT. Może to wynikać z roli ATM w zatrzymywaniu cyklu komórkowego w fazie G1, a tym samym ułatwianiu szybkiej odpowiedzi na uszkodzenie DNA wywołane przez PDT w komórkach opornych. Mechanizm oporności na PDT w komórkach raka szyjki macicy, chociaż łączy pewne cechy z glejakiem, różni się w ścieżce naprawy DNA. Przeprogramowanie cyklu komórkowego, wydłużenie fazy G1, wyższa aktywność zmiatania RFT i słabsza biosynteza PpIX są wspólnymi czynnikami w obu badanych liniach komórkowych opornych na PDT. Komórki raka szyjki macicy oporne na PDT wykazały silniejszą ekspresję niektórych białek BER, takich jak APE1 i PARP1 oraz zwiększoną aktywność głównych etapów BER w porównaniu z komórkami wrażliwymi. Dane te sugerowały, że komórki oporne mogą być uwrażliwione na PDT poprzez zahamowanie aktywności APE1 i PARP1. Okazało się, że oba enzymy są dobrym celem, z tym, że inhibitor APE1 był bardziej skuteczny. Odkrycia te wskazują na na kluczową rolę szlaku BER w nabywaniu oporności na PDT przez linię komórkową raka szyjki macicy, którą można pokonać przez hamowanie głównych enzymów tego szlaku naprawy DNA. Podsumowując, wyniki uzyskane podczas realizacji tego projektu wykazały, że oprócz innych ujawnionych mechanizmów naprawa DNA jest ważnym mechanizmem oporności na PDT zarówno w liniach komórkowych glejaka, jak i raka szyjki macicy. Badanie to sugeruje również, że inhibitor jednej z głównych knaz DDR, ATM i inhibitory szlaku BER (APE1 i PARP1) mogą stać się skutecznymi adiuwantami, które zwiększą skuteczność terapii fotodynamicznej, odpowiednio w GBM i raku szyjki macicy.

Thesis (Ph.D.)--State University of New York at Buffalo, 2006.
Title from PDF title page (viewed on Mar. 21, 2007) Available through UMI ProQuest Digital Dissertations. Thesis adviser: Bellnier, David A. Includes bibliographical references.

According to the results, Pa-PDT showed inhibitory effect on MDA-MB-231 cells in vitro with an IC50 value of 0.5 muM at 24 h. Pa-PDT was demonstrated to activate intracellular mitogen activated protein kinases (MAPK) pathways via reactive oxygen species (ROS) production. Pa-PDT IS also believed to induce extracellular signal-regulated kinase (ERK)-mediated autophagy and endoplasmic reticulum stress. Pa-PDT in combination with Tamoxifen is demonstrated to exert a synergetic effect in inhibiting cancer growth. The combination treatment induces both intrinsic and extrinsic apoptosis. Regarding the direct cancer cell killing activity, two dimensional gel electrophoresis screening revealed that Pa-PDT regulates proteins which involve in human leukocyte antigen (HLA) class I-restricted antigen-processing machinery. This activation of antigen presentation was confirmed by Western blot analysis and immunostaining. Furthermore, a cross-presentation of antigen with HLA class I proteins and 70-kDa heat shock protein was found in Pa-PDT-treated cells, as shown by the fluorescent microscopic observation and immunoprecipitation assay. Moreover, the immunogenicity of breast cancer cells was increased by Pa-PDT treatment that triggered phagocytic activity by human macrophages. Our findings provide the first evidence that Pa-PDT can trigger both apoptosis and anti-tumour immunity.
Cancer is one of the most lethal diseases worldwide. Treatments of cancer comprise surgical intervention, radiotherapy or chemotherapy; however, their side effects are still need to be overcome. In order to search for anti-cancer treatments with milder side effects and higher efficiency, traditional Chinese medicine (TCM) has been investigated. Previous study in our laboratory reported that pheophorbide a (Pa), an active compound purified from Scutellaria barbata, combined with photodynamic therapy (PDT) approach produces anti-tumour effect in a wide range of human cancers. Because of the lack of protocols for curing late phase breast cancer, my project is to investigate the therapeutic potential of Pa-PDT and its action mechanism on human breast cancer. A human breast cancer cell line MDA-MB-231, which is estrogen receptor nude and resistant to a conventional breast cancer drug tamoxifen, was used as an in vitro tumour model in my study to mimic the late stage of breast cancer.
Pheophorbide a (Pa) has been proposed to be a potential photosensitizer for the photodynamic therapy of human cancer. However, the immunomodulatory effect of Pa, in the absence of irradiation, has not yet been investigated. The present study revealed that Pa possessed immunostimulating effect on a murine macrophages cell line RAW 264.7. Pa could stimulate the growth of RAW 264.7 cells with the maximal effect at 0.5 muM after 48 h of treatment, where MAPK family including c-Jun N-tenninal kinase (JNK), ERK and p38 MAPK were activated by Pa treatment in a dose-dependent manner. Moreover, the induction of interleukin-6 and tumour necrosis factor-a secretion, and the enhancement of phagocytic activity were observed in Pa-treated RAW 264.7 cells. The results were similar in Pa-treated human immune competent cells (e.g. CD4+ and CD14+ cells) at higher Pa concentrations (from 1 to 10 muM). The present work is the first report to demonstrate the potential immunomodulatory effects of Pa on immune competent cells, apart from its well-known anti-tumour activity.
Bui Xuan, Ngoc Ha.
"December 2010."
Advisers: Fung Kwok Pui; Wong Chun Kwok.
Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves 123-144).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.

This thesis encompasses the development and testing of an interstitial photodynamic therapy (iPDT) system for the treatment of prostate cancer. It begins with the optical characterization of a novel photosensitizer (SL-052) followed by a study of tissue optics as it applies to iPDT. The design and integration of a time-fractionated light delivery system with real-time spectral detection is then examined. An optical phantom test medium is formulated and in vitro system operation and testing is performed. Finally, in vivo experiments are performed on animal models with a focus on canine prostate iPDT. Unique optical results with dosimetric relevance are discovered and investigated. This includes metrics for optically measuring local in vivo SL-052 concentrations in real-time as well as novel oscillatory drug photobleaching and recovery behavior during time-fractionated light delivery.
Photonics and Plasmas

Dissertação de Mestrado em Química Medicinal apresentada à Faculdade de Ciências e Tecnologia
A terapia fotodinâmica (TFD) apresenta várias vantagens sobre as terapias convencionais contra o cancro. Combinando o uso de um fotossensibilizador (FS), irradiação de luz de um comprimento de onda específico para cada FS e oxigénio, esta alcança uma seletividade única pela produção localizada de oxigénio singleto e outras espécies reativas de oxigénio (ROS) dentro de células tumorais levando à sua destruição, traduzindo-se, consequentemente, em menos efeitos colaterais.Relativamente aos fotossensibilizadores, os macrociclos porfirínicos em geral, e as clorinas em particular, têm as características fotofísicas ideais para serem usados na TFD, pois possuem um rico padrão de bandas de absorção na região espectral do vermelho e infravermelho próximo (NIR), entre 650 e 850 nm. Esta faixa é conhecida como janela fototerapêutica, e equilibra a penetração profunda dos tecidos com o fornecimento de energia suficiente para excitar o oxigénio para o seu estado singleto (comprimentos de onda mais efetivos inferiores a 800 nm). Contudo, as clorinas que resultam da simples redução de uma das ligações duplas do anel porfirínico apresentam desvantagens quanto à sua estabilidade e síntese em várias etapas. Uma vez que foi demonstrado que as porfirinas podem atuar como dienófilos em reações de Diels-Alder e como dipolarófilos em ciclo-adições 1,3-dipolar, uma forma mais simples de produzir clorinas mais estáveis é via reações de cicloadição com porfirinas que possuam dois grupos atractores de eletrões nas posições vicinais beta. Além disso, é bem conhecido que a incorporação de átomos de halogénios (-F, -Cl) nas posições orto dos anéis fenilo na posição meso do macrociclo melhoram as propriedades fotofísicas e apresentam propriedades citotóxicas melhoradas devido a alterarem o balanço entre a fluorescência e o cruzamento intersistema. Esta dissertação foca-se, portanto, numa reação de cicloadição [8π + 2π] de 5,10,15,20-tetraquis(pentafluorofenil)porfirina (TPPP20) com o anião metil diazafulvénio, gerado por extrusão térmica de SO2 a partir de 2,2-dioxo-6,7-dimetil-1H,3H-pirazolo[1,5-c][1,3]tiazole-6,7-dicarboxilato. Esta reação produziu a 5,10,15,20-tetraquis(pentafluorofenil)clorina (TPPC20) de um modo seletivo, a qual revelou ser um molde surpreendentemente versátil para novos derivados de clorina, uma vez que o substituinte pentafluorofenil reage facilmente com nucleófilos através, por exemplo, de substituições nucleofílicas aromática (SNAr) nos átomos de fluor da posição para, de forma altamente seletiva e frequentemente ocorre com alto rendimento.Foi demonstrado, ainda, que a hidrofilicidade dessas clorinas é crucial para garantir alta citotoxicidade contra as células cancerígenas. Sabendo que uma estratégia aplicada no design de fármacos para obter compostos com as propriedades ideais para serem utilizados em meios biológicos e para melhorar a permeabilidade celular é a incorporação de porções de polietilenoglicol (PEG), decidiu-se preparar um novo tipo de pentafluorofenilclorinas de anel fundido PEGuiladas, explorando essas mesmas reações nucleofílicas de substituição aromática. Desta forma, estas estruturas não só possuem estabilidade química e estrutural, melhorada pela introdução de um anel fundido, mas também aumentam a sua hidrofilicidade. Estas características em associação com um padrão rico de bandas de absorção dentro da janela fototerapêutica, tornam estes compostos promissores agentes fotodinâmicos muito ativos.O objetivo desta tese foi, em última análise, esclarecer sobre os detalhes sintéticos, a caracterização estrutural e a avaliação da citotoxicidade destes agentes muito promissores para a TFD contra linhas celulares de cancro do esófago (OE19) e de melanoma (A375).
Photodynamic therapy (PDT) presents several advantages over conventional cancer therapies. Combining the use of a photosensitizer (PS), light irradiation of a specific wavelength for each PS, and oxygen, it achieves a unique selectivity by the localized generation of singlet oxygen and other reactive oxygen species (ROS) inside tumor cells leading to their destruction, which, consequently, translates in less side effects.When it comes to the PS, porphyrin type macrocycles in general, and chlorins, in particular, have the ideal photophysical characteristics to be used in PDT because they have a rich pattern of absorption bands in the red and near-infrared spectral region (NIR), between 650 and 850 nm. This is the range known as phototherapeutic window, which balances deeper penetration of tissues by providing enough energy to excite the oxygen to its singlet state (most effective wavelengths inferior to 800 nm). Their only drawback is the problems related to multi-step synthesis and stability. Since it has been demonstrated that porphyrins can act as dienophiles in Diels-Alder reactions and as dipolarophiles in 1,3-dipolar cycloadditions, a simple way to produce chlorins is via cycloaddition reactions over the porphyrins bearing two vicinal electron-withdrawing groups in beta positions, namely Diels-Alder 1,3-dipolar cycloadditions. In addition to that, it is well-known that the incorporation of a halogen atom (-F, -Cl) in the ortho positions of the phenyl rings in the meso position of the macrocycle improves the photophysical properties and changes the balance between fluorescence and intersystem crossing, enhancing its photo-induced cytotoxic properties. Therefore, this dissertation focused on a [8π + 2π] cycloaddition of 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (TPPP20) with diazafulvenium methide, generated by thermal extrusion of SO2 from 2,2-dioxo-1H,3H-pyrazol[1,5-c]thiazole. This reaction afforded 5,10,15,20-tetrakis(pentafluorophenyl)chlorin (TPPC20) in a selective fashion, which has revealed to be a surprisingly versatile template to new chlorin derivatives, since the pentafluorophenyl easily reacts with nucleophiles through, for example, nucleophilic aromatic substitution (SNAr) of the para-F atoms providing a simple and general access to functionalized meso-tetraarylchlorins containing electron-donating substituents in the p-position of their meso-aryl groups in a highly selective way and frequently with high yield. Furthermore, it was demonstrated that the hydrophilicity of these chlorins is crucial to ensure high cytotoxicity against cancer cells. A strategy applied in drug design to achieve compounds with the ideal properties to be used in biological media and to improve cell permeability is the incorporation of polyethylene glycol (PEGs) moieties.For that reason, it was decided to prepare a new type of PEGylated ring-fused (pentafluorophenyl)chlorins by exploring these nucleophilic aromatic substitution reactions. This way, the obtained structures do not only show enhanced chemical and structure stability by the introduction of a fused ring but also increased hydrophilicity. All these characteristics make these compounds promising very active photodynamic agents.The aim of this thesis was, ultimately, to offer synthetic details, structural characterization and cytotoxicity evaluation of these very promising PDT agents against oesophagus cancer cell line (OE19) and melanoma cells lines (A375).

Photodynamic therapy(PDT) has recently emerged as a promising new non-invasive treatment modality for a large number of neoplastic and non-neoplastic lesions. Photoexcitation of a photosensitizing drug in the tumor tissue causes generation of reactive oxygen species which results in cell death. The current porphyrinic photosensitizers suffer a wide range of drawbacks leading to the development of the chemistry of alternative photosensitizing agents in PDT. Among them, the 4d and 5d transition metal-based photosensitizers have been explored extensively with the exception of the 3d metal complexes. The objective of this thesis work is to design and synthesize photoactive iron(III) abd cobalt(III) complexes and evalutate their photonuclease and photocytotoxic potential. Bioessential 3d metal ions provide an excellent platform for metal-based PDT drug designing as because of its varied spectral, magnetic and redox properties, with its complexes possessing rich photochemical behavior in aqueous and non-aqueous media. We have synthesized binary iron(III) complexes as netropsin mimics using amino acid Schiff bases derived from salicylaldehyde/napthaldehyde and arginine/lysine. The complexes were found to be good AT selective DNA binders and exhibited significant DNA photocleavage activity. To enhance the photodynamic potential, we further synthesized iron(III) complexes of phenolate-based ligand and planar phenanthroline bases. The DNA photocleavage activity of these complexes and their photocytotoxic potential in cancer models were studied. ROS generated by these complexes were found to induce apoptotic cell death. Ternary cobalt(III) complexes were synthesized to study the effect of the central metal atom. The diamagnetic cobalt(III) complexes were structurally dissimilar to their iron(III) analogues. Although the Co(III)/Co(II) redox couple is chemically and photochemically accessible but the Co(III)-dppz complex, unlike its iron(III)-dppz analogue, exhibited selective damage to hTSHR expressing cells but not in HeLa cells. A structure-activity relationship study on iron(III) phenolates having modified dppz ligands was carried out and it was found that electron donating group on the phenazine unit and an increase of the aromatic surface area largely improved the PDT efficiency. Finally, SMVT targeted iron(III) complexes with biotin as targeting moiety were synthesized and the in vitro efficacy of the complexes was tested in HepG2 cells over-expressing SMVTs and compared to HeLa amd HEK293 cells. The complexes exhibited higher phytocytotoxicity in HepG2 than in HeLa and cells and HEK293 cells. An endocytotic mode of uptake took place in HepG2 cells whereas in HEK293 cells, uptake is purely by diffusion. This is expected to reduce the side-effects and have less effect on cells with relatively less SMVTs. In summary, the present research work opens up novel strategies for the design and development of primarily iron-based photosensitizers for their potential applications in PDT with various targeting moieties.

The present thesis deals with different aspects of the chemistry and photo-biology of various ferrocene-conjugates, their interaction with double helical DNA, DNA photocleavage and photo-enhanced cytotoxicity in visible light, localization and cellular uptake to study the mechanism of cell death. Phenyl analogues of the active complexes have been synthesized and used for comparison in biological assays. Chapter I presents an overview of cancer and its types, various treatments for cancer. A general overview on the Photodynamic Therapy, a new modality of light activated cancer treatment and its various possible mechanism of action, has been made. The promise of photoactivated chemotherapy is discussed with recently developed metal based antitumor agents. Biological applications of few ferrocene conjugates as anticancer and anti-malarial agents are discussed. The objective of the present investigation is also presented in this chapter. Chapter II presents the synthesis, characterization, structure, DNA binding, DNA photocleavage, photocytotoxicity and cellular localization of ferrocene-conjugated dipicolylamine oxovanadium(IV) complexes of curcumin. To explore the role of the ferrocenyl moiety the phenyl analogue of the ferrocenyl complexes is synthesized and used as a control for comparison purpose. Chapter III deals with the photo-induced DNA cleavage and photo-enhanced cytotoxicity of ferrocene-conjugated oxovanadium(IV) complexes of heterocyclic bases. The synthesis, characterization, structural comparisons, DNA binding, DNA photocleavage and photocytotoxic activity in visible light are discussed in detail. Chapter IV describes the synthesis, characterization and structure of ferrocene-conjugated oxovanadium(IV) complexes of acetylacetonate derivatives. The complexes are evaluated for DNA binding, DNA photocleavage and photocytotoxic activity in HeLa, MCF-7, 3T3 cells in visible light. The fluorescent nature of the complexes is used to study the cellular localization of the complexes and the mechanism of cell death induced by the complexes is also discussed. Chapter V presents the photocytotoxic effect of ferrocene-conjugated oxovanadium(IV) complexes of different curcuminoids in HeLa , HepG2 and 3T3 cells. Curcumin based fluorescence has been successfully used to study the cellular uptake and localization behavior of the complexes. The positive role of the ferrocenyl complex is evident from the ~4 fold increase in its photocytotoxicity compared to the phenyl analogue. The apoptotic mode of cell death is evident from nuclear co-staining using Hoechst dye. Chapter VI describes the synthesis, characterization and photochemotherapeutic efficacy of ferrocene conjugates of N-alkyl pyridinium salts. Mitochondria targeting property of ferrocene compound having n-butyltriphenylphosphonium group has been studied by JC-1 assay. FACS analysis showed significant sub G1/G0 phase cell-cycle arrest in cancer cells on visible light treatment. Finally, the summary of the dissertation and conclusions drawn from the present investigations are presented. The references in the text have been indicated as superscript numbers and compiled at the end of each chapter. The complexes presented in this thesis are represented by bold-faced numbers. Crystallographic data of the structurally characterized complexes are given in CIF format in the enclosed CD (Appendix-I). Due acknowledgements have been made wherever the work described is based on the findings of other investigators. Any unintentional omission that might have happened due to oversight or mistake is regretted. INDEX WORDS: Ferrocene conjugates Crystal structure DNA binding DNA photocleavage Photocytotoxicity Vanadium Cellular Imaging