Les chromophores classiques ne sont pas toujours efficaces en absorption à deux photons. Leur faible efficacité nécessite l'utilisation de fortes puissances laser et de grandes concentrations en colorants. Dans ce sens, la microscopie à deux photons in vivo requière le développement de nouvelles stratégies de marquage utilisant des chromophores spécialement dédiés à la microscopie à deux photons. Dans le cadre de collaborations avec des chimistes spécialisés dans la synthèse de molécules à forte section efficace d'absorption à deux photons, différents chromophores ont été synthétisés. Ces molécules organiques sont souvent hydrophobes et ne sont pas utilisables directement pour les applications en biologie. Le travail effectuer ici a consisté à encapsuler ces molécules dans des micelles de copolymères biocompatibles, les Pluronic. Les Pluronic sont des matériaux pouvant s'auto assembler en milieu aqueux sous forme de micelles et permettent de solubiliser des composés hydrophobes. Cette stratégie est déjà utilisé pour permettre de transporter différents composés hydrophobes dans les organismes vivants et a été utilisée ici pour transporter des chromophores ultrasensibles à deux photons dans le sang de manière à imager la vascularisation in vivo. / Classic fluorescent dyes are not necessary efficient in two-photon absorption. Their low two-photon absorption efficiency often requires high laser power and important dye concentrations. Therefore, new dyes and other administration strategies need to be developed specifically for intravital two-photon microscopy. In collaboration with chemists, specialized in the synthesis of molecules with a high two-photon absorption cross-section, different dyes have been synthesized. Most of these dyes are hydrophobic and not directly suitable for biological applications. The work presented in this thesis consisted of encapsulating hydrophobic molecules in biocompatible Pluronic block copolymers. In water, Pluronic unimers with hydrophobic and hydrophilic blocks self-assembled in hydrophilic micelles forming a hydrophobic core around the molecules. This strategy has been used already for the transport and delivery of different hydrophobic molecules in living organism. In the present study, this strategy has been transposed to transport ultra sensitive two-photon dyes in the blood plasma for deep vascular imaging in vivo.

A terapia fotodinâmica (TFD) atualmente é considerada uma modalidade terapêutica no fotoenvelhecimento, pois apresenta resposta satisfatória tanto no resultado estético como no tratamento e prevenção das lesões pré-malignas que geralmente acompanham a pele fotoenvelhecida. A destruição seletiva do tecido alvo ocorre através de uma reação química, onde se utiliza um agente fotossensibilizante que é exposto a uma determinada luz com comprimento de onda e na presença de oxigênio produz oxigênio \"singlet\" que é responsável pela citotoxicidade das células proliferativas e conseqüente morte celular. Foram estudadas 13 pacientes com fotoenvelhecimanto clínico que foram submetidas a três sessões de TFD-ALA tópico associado à luz vermelha (630nm) com intervalo quinzenal. Após 21 dias da ultima aplicação 12 pacientes apresentaram melhora do fotonvelhecimento; principalmente na coloração e textura da pele, clareamento das lesões de melanose solar, regressão da queratose actinica e melhora da flacidez. Houve também aumento do tecido colágeno e fibra elástica nas colorações de picrosirius e Weigert-oxona respectivamente. Em relação a modulação do sistema imune houve diminuição da população de linfócitos CD4 e CD8, aumento da interleucina-4 na epiderme e derme, diminuição da TNFα na epiderme e aumento na derme.A população de células de Langerhans e o INFγ não apresentaram alterações. A TFD-ALA associada à luz vermelha se mostrou eficaz no tratamento do fotoenvelhecimento clínico, no aumento do colágeno e fibras elásticas e na modulação do sistema imunológico. / PDT is nowadays considered one of the therapies for photo-aging, since it gives a satisfactory response for both, aesthetic result as well as treating and preventing pre-malignous lesions that are a frequent outcome of photo-aged skin. The selective destruction of target tissue occurs through a chemical reaction, by using a photosensitizing agent exposed to a certain light with a wave length which, in the presence of oxygen, produces singlet oxygen responsible for the citotoxidity of the proliferative cells and consequent cellular death. Thirteen pacients with clinical photo-aging have been studied, submitted to three sections of topic ALA-PDT associated with red light (630nm) at fortnight?s intervals.After 21 days of the last treatment, 12 patients showed improved photo-aging, specially regarding skin colour and texture, whitening of the solar melanosis lesions, regression of actinic cheratosis and improvement of flacidity. There also occured an increase of colagenous and elastic fibre tissue in the picrosirius and Weigert-oxona methods respectively.. Regarding the modulation of the immune system, there was a decrease of the lymphocytes CD4 and CD8 population, an increase of interleucin-4 in the epidermis and in the dermis, decrease of TNF-α in the epidermis and increase in the dermis. The Langerhans cells poulation and the INF-γ did not present alterations. The ALA-PDT associated to red light was effective for the treatment of clinical photo-aging, for colagenous and elastic fibres increase and in the imune system modulation.

O carcinoma epidermóide bucal (CEC) é uma neoplasia maligna com alta morbidade e mortalidade e de difícil tratamento. O tratamento convencional para o CEC inclui cirurgia e radioterapia, seguida ou não de quimioterapia. Apesar de serem amplamente difundidos, esses tratamentos podem ser ineficazes para alguns CECs resistentes. A terapia fotodinâmica (PDT) oncológica tem sido utilizada para o tratamento adjuvante do CEC bucal, principalmente nos casos menos invasivos e que necessitam de redução do tumor para a ressecção cirúrgica. Contudo, semelhantemente aos tratamentos convencionais, a PDT pode também induzir o aparecimento de populações celulares resistentes, fato já descrito para carcinoma cutâneo, adenocarcinoma de cólon e adenocarcinoma mamário. A hipótese de que células de CEC bucal possam desenvolver resistência à PDT ainda não foi testada. Portanto, o objetivo deste trabalho foi verificar se células de CEC bucal (SCC9) desenvolvem resistência a ciclos repetidos de PDT mediada pelo ácido 5- aminolevulínico (5-ALA-PDT) e avaliar se nesse processo ocorre modificação da expressão de marcadores relacionados a sobrevivência celular (NF?B, Bcl-2, iNOS, mTOR e Akt). Foi utilizada linhagem de células de CEC bucal (SCC9), submetida às seguintes condições: 1) Controle - células cultivadas sem nenhum tratamento; 2) ALA - células incubadas com 5-ALA (1mM durante 4 horas); 3) LED - tratadas com iluminação LED (630nm, 5,86J/cm2, 22,5J, 150mW, 150s); 4) PDT - tratadas com 5- ALA-PDT, com os protocolos do grupo ALA e LED combinados, gerando dose letal de 90%. Inicialmente foi realizado somente um ciclo de PDT, sendo avaliada a viabilidade celular em todos os grupos após 24, 48, 72 e 120h da irradiação. Também foi realizado ensaio de detecção da fragmentação de DNA (TUNEL) e análise por imunofluorescência da expressão das proteínas NF?B, Bcl-2, iNOS, pmTOR e pAkt nas células viáveis. Como resultado desse primeiro tratamento com 5-ALA-PDT, observou-se que as células sobreviventes ao tratamento apresentaram intensa marcação para pmTOR e exibiram potencial de crescimento durante o período analisado. Após esses ensaios, as células que sobreviveram a essa primeira sessão foram coletadas, replaqueadas e novamente cultivadas, sendo então submetidas a novo ciclo de 5-ALA-PDT. Esse processo foi realizado 5 vezes, variando-se a intensidade de irradiação à medida que se observava aumento na viabilidade celular. As populações celulares que exibiram viabilidade 1,5 vezes maior do que a detectada no primeiro ciclo PDT foram consideradas resistentes ao tratamento. Os mesmos marcadores analisados no primeiro ciclo de PDT foram novamente avaliados nas populações resistentes. Foram obtidas quatro populações celulares resistentes, com viabilidade de até 4,6 vezes maior do que a do primeiro ciclo de PDT e irradiação com LED que variou de 5,86 a 9,38J/cm2. A população mais resistente apresentou ainda menor intensidade de protoporfirina IX, maior capacidade de migração e modificação na morfologia nuclear. As populações resistentes testadas exibiram aumento na expressão de pNF?B, iNOS, pmTOR e pAkt, mas não da proteína anti-apoptótica Bcl- 2. Ensaio in vivo foi também conduzido em ratos, nos quais CEC bucal foi quimicamente induzido e tratado ou não com 5-ALA-PDT. Houve intensa expressão imuno-histoquímica das proteínas pNF?B, Bcl-2, iNOS, pmTOR e pAkt em relação ao controle não tratado, nas células adjacentes à área de necrose provocada pela PDT. Concluiu-se que as células de CEC bucal tratadas com 5-ALA-PDT a uma dose de 90% de letalidade desenvolveram viabilidade crescente após ciclos repetidos do tratamento, bem como exibiram superexpressão de proteínas relacionadas à sobrevivência celular, tanto in vitro quanto in vivo. Esses fatos, aliados à maior capacidade de migração, sugerem a aquisição de fenótipo de resistência à 5-ALAPDT. Esse aspecto deve ser cuidadosamente considerado no momento da instituição dessa terapia para os CECs bucais. / Oral squamous cell carcinoma (SCC) is a malignant tumor with high morbidity and mortality rates, and it is difficult to treat. Conventional treatment for oral SCCs includes surgery and radiotherapy that may be followed by chemotherapy. Although these treatments are widely used, they are ineffective against some resistant tumors. Oncologic photodynamic therapy (PDT) has been used as an adjuvant treatment for oral SCCs, especially in less invasive cases that require tumor reduction before surgical resection. However, like conventional treatments, PDT can induce the occurrence of resistant cell populations such as cutaneous carcinomas and colon and breast adenocarcinomas. The hypothesis that oral SCCs develop resistance to PDT has not yet been tested. Therefore, the aims of this study were to investigate whether oral SCCs (SCC9) develop resistance to several cycles of 5-aminolevulinic acidmediated PDT (5-ALA-PDT) and to determine whether the expression of markers associated with cell survival (NF?B, Bcl-2, iNOS, mTOR, and Akt) is altered during this process. An oral SCC (SCC9) cell line was used, which was subjected to the following conditions: 1) Control: cultured without any treatment; 2) ALA: incubated with 5-ALA (1 mM for 4 h); 3) LED: treated with LED light (630 nm, 5.86 J/cm2, 22.5 J, 150 mW, 150 s); and 4) PDT: treated with 5-ALA-PDT (with the protocols of the ALA and LED groups combined) generating a lethal dose of 90%. Initially, only one cycle of PDT was administered, and cell viability was determined in all groups 24, 48, 72, and 120 h after irradiation. Subsequently, the DNA fragmentation detection assay (TUNEL) and immunofluorescence analysis of the expression of proteins NF?B, Bcl-2, iNOS, pmTOR, and pAkt were performed on viable cells. The fraction of cells that survived the first treatment with 5-ALA-PDT exhibited intense staining for pmTOR and growth potential during the testing period. After these assays, the cells that survived the first cycle were collected, plated, and cultured and were subjected to another cycle of 5- ALA-PDT. This process was repeated five times at various irradiation intensities, and cell viability gradually increased. The cell populations that exhibited 1.5-times higher viability than that detected after the first PDT cycle were considered to be resistant to treatment. The markers analyzed after the first PDT cycle were again assessed in the resistant populations. Four resistant cell populations were obtained with a viability of up to 4.6-times higher than that of the first PDT cycle and LED light treatment, which varied between 5.86 and 9.38J/cm2. The most resistant population exhibited lower intensity of protoporphyrin IX, higher migration capacity, and changes in nuclear morphology. The resistant populations tested showed increased expression of pNF?B, iNOS, pmTOR, and pAkt, but not of the anti-apoptotic Bcl-2 protein. Moreover, an in vivo assay was conducted in rats; oral SCCs were chemically induced and treated with 5-ALA-PDT. The intensity of the immunohistochemical expression of proteins pNF?B, Bcl-2, iNOS, pmTOR, and pAkt in cells adjacent to the area with necrosis caused by PDT was higher than that observed in the untreated control. In conclusion, oral SCCs treated with 5-ALA-PDT at a lethal dose of 90% exhibited increasing viability after several treatment cycles and overexpression of proteins associated with cell survival both in vitro and in vivo. These results, together with the higher migration capacity, suggest the acquisition of the phenotype of resistance to 5-ALA-PDT. This aspect should be carefully considered when initiating this therapy for oral SCCs.

Ziel der vorliegenden Arbeit ist die photophysikalische Untersuchung Photosensibilisator-beladener Nanopartikel als Transportsysteme für aktives und passives Tumor-Targeting. Zu diesem Zweck wurden sowohl stationäre, als auch zeitaufgelöste spektroskopische Methoden angewandt. Der erste Teil beschäftigt sich mit der photophysikalischen Charakterisierung von Pheo-HSA-Nanopartikeln. Mittels stationärer und zeitaufgelöster Messungen konnte gezeigt werden, dass die Wechselwirkungen zwischen Phäophorbid a und den HSA-Nanopartikeln sehr stark ist. Diese Wechselwirkungen bewirken eine geringe Singulettsauerstoffquantenausbeute (0,07) in D2O verglichen mit dem von Phäophorbid a in Ethanol (0,52). Im Gegensatz dazu konnte nach der Inkubation in Jurkat- und HT-29-Zellen eine intrazelluläre Singulettsauerstoffgenerierung der Pheo-HSA-NPs nachgewiesen werden. Im zweiten Teil wurden mit den Photosensibilisatoren mTHPP and mTHPC beladene HSA- und PLGA-Nanopartikel untersucht. Es konnte gezeigt werden, dass die Photosensibilisator-Beladungsrate die photophysikalischen Eigenschaften der HSA- und PLGA-Nanopartikel stark beeinflusst. Für die HSA-Nanopartikel dominieren bei geringen Beladungsraten die Wechselwirkungen zwischen HSA und den Photosensibilisatormolekülen. Mit steigender Beladung spielen Wechselwirkungen zwischen den Photosensibilisatormolekülen eine zunehmende Rolle. Diese Wechselwirkungen verringern bei hoher Beladung der HSA-Nanopartikel die Generierung von Singulettsauerstoff. Auch für die PLGA-Nanopartikel konnte mit zunehmender Beladung ein verstärktes Singulettsauerstoffquenching nachgewiesen werden. Im dritten Teil dieser Arbeit wurden, für aktives Targeting von Tumorzellen, Oberflächenmodifizierte PLGA- und HSA-Nanopartikel untersucht. Die intrazellulären Singulettsauerstoffmessungen weisen auf eine erleichterte Aufnahme in Tumorzellen von Antikörper- und PEG-modifizierten HSA-Nanopartikeln in vitro hin. / The main goal of this PhD thesis is the photophysical investigation of biodegradable photosensitizer-nanoparticle carrier systems achieving passive and active tumour targeting strategies. For this purpose both steady state and time-resolved spectroscopic methods accompanied by data analysis were utilized. This work contains three main parts: First the photophysical properties of Pheo-HSA nanoparticles were compared to free pheophorbide a. Steady-state and time-resolved fluorescence experiments have already proved that the interaction between pheophorbide a and HSA nanoparticles is strong. This interaction leads to low singlet oxygen quantum yield (0.07) in D2O compared to free Pheo (0.52) in ethanol. But when incubated in Jurkat and HT-29 cell lines, Pheo-HSA nanoparticles have been proved to generate singlet oxygen inside cells. In the second part the well-known photosensitizers mTHPP and mTHPC were loaded to HSA- and PLGA- nanoparticles. It was found that the loading ratio determines the photophysical properties of both photosensitizer-loaded HSA and PLGA nanoparticles. For HSA nanoparticles, photosensitizer-nanoparticle interaction is the preferential mechanism in low loading ratio sample. But in high loading ratio sample, photosensitizer-photosensitizer interaction becomes the determining interaction. This interaction prevents singlet oxygen generation from high loading sample. For PLGA nanoparticles, high drug loading ratio also leads to a strong singlet oxygen quenching. At high drug loading ratio PLGA nanoparticles, some photosensitizer molecules may be localized deeply inside PLGA matrices and far away from surface. In the third part of this work, active tumour targeting behaviour achieved by surface modification of HSA and PLGA nanoparticles has been tested. Intracellular singlet oxygen measurement reveals that HSA nanoparticles, both with antibody and PEG surface modification have an enhanced targeting of tumour cells in vitro.

O carcinoma epidermóide oral (CEO) tem alta incidência e mortalidade no mundo, sendo frequentes casos de recidivas em função da resistência aos tratamentos convencionais. Essa resistência tem sido associada à presença de células tronco tumorais (CTT). A terapia fotodinâmica (PDT) tem sido utilizada nos casos de desordens orais potencialmente malignas (DOPM) e CEOs superficiais, com relativo índice de sucesso. Contudo, são relatados casos de recidivas, que sugerem certa resistência à PDT. O objetivo do presente trabalho foi avaliar se células de DOPM desenvolvem resistência à PDT mediada pelo ácido 5-aminolevulínico (5-ALA), tal qual ocorre com células de CEO expostas ao mesmo tratamento, bem como verificar se as células displásicas e neoplásicas orais exibem um fenótipo sugestivo de células tronco tumoral. Foram cultivadas células epiteliais displásicas orais (linhagem DOK) e células de CEO de língua (linhagem SCC9), divididas nos seguintes grupos experimentais: Grupo Controle - células sem tratamento; Grupo ALA - células tratadas com 5-ALA, com concentração e tempo de incubação igual ao do grupo PDT; Grupo LED - células tratadas com LED, com dosimetria igual ao do grupo PDT; Grupo PDT - células tratadas com PDT mediada pelo 5-ALA, subdivididas em duas gerações: geração 1 - células expostas a somente um ciclo de PDT; geração 4 - células expostas a quatro ciclos de PDT e que exibiram resistência (maior viabilidade) à dose inicial aplicada para a geração 1. Para esses grupos, foram realizados ensaios de formação de colônias e de invasão, bem como citometria de fluxo para verificar a expressão de CD44, ESA e p75NTR. Observou-se que todos os grupos expostos à PDT contendo células da linhagem DOK exibiram resistência à dose inicial de PDT, porém em intensidade menor àquela observada para a linhagem SCC9. As células resistentes à PDT de ambas as linhagens exibiram maior potencial de formação de colônias, mas somente a primeira geração de células exibiu maior potencial de invasão. Na análise de citometria de fluxo, observou-se que as células resistentes à PDT de ambas as linhagens exibiram superexpressão de CD44high/ESAhigh. A linhagem SCC9 exibiu ainda maior frequência de células CD44high/ESAlow e CD44high/p75NTR+, sugerindo a presença de uma subpopulação com alto potencial de invasão e resistente aos tratamentos convencionais. Não houve diferenças significativas na frequência de células ESAhigh/p75NTR+ em ambas as linhagens. Concluiu-se que as células displásicas orais desenvolvem resistência à PDT com intensidade menor se comparada às células SCC9. Em ambas as linhagens, as células resistentes exibiram um fenótipo sugestivo de células tronco tumorais. Esse fato deve ser considerado ao se instituir a PDT mediada pelo 5-ALA em lesões displásicas e neoplásicas orais que demandam múltiplos ciclos de tratamento. / Oral squamous cell carcinoma (SCC) has a high incidence and mortality in the world. There are frequent cases of relapses due to its resistance to conventional treatments. This resistance has been associated with the presence of cancer stem cells (CSC). Photodynamic therapy (PDT) has been used in cases of potentially malignant oral lesions (PMOL) and superficial SCC\'s, with a relative success rate. However, relapses are reported, which suggest some resistance to PDT. The aim of the present study was to evaluate whether PMOL cells develop resistance to PDT mediated by the 5- aminolevulinic acid (5-ALA), as occurs with SCC cells exposed to the same treatment, as well as to verify if dysplastic and oral neoplastic cells exhibit a suggestive phenotype of CSC. Oral dysplastic epithelial cells (DOK lineage) and CSC cells of the tongue (SCC9 lineage) were divided into the following experimental groups: Control Group - cells with no treatment; Group ALA - 5-ALA treated cells, with concentration and incubation time equal to that of the PDT group; LED group - LED light treated cells, with dosimetry equal to that of the PDT group; PDT group - cells treated with PDT mediated by the 5-ALA, subdivided into two generations: Generation 1 - cells exposed to only one PDT cycle; Generation 4 - cells exposed to four PDT cycles and exhibiting resistance (greater viability) to the initial dose applied for Generation 1. Colony forming and invasion assays, as well as flow cytometry to verify the expression of CD44, ESA and p75NTR were performed to the experimental groups. It was observed that all groups exposed to PDT containing DOK lineage cells exhibited resistance to the initial dose of PDT, but at a lower intensity than those observed for the SCC9 lineage. PDTresistant cells from both lineages exhibited increased potential for colony formation, but only the first generation of cells exhibited increased invasion potential. In flow cytometric analysis, PDT-resistant cells from both lineages exhibited overexpression of CD44high / ESAhigh. SCC9 lineage also exhibited higher frequency of CD44high / ESAlow and CD44high / p75NTR+ cells, suggesting the presence of a subpopulation with high invasion potential and resistant to conventional treatments. There were no significant differences in the frequency of ESAhigh / p75NTR+ cells in both lineages. It was concluded that the oral dysplastic cells develop resistance to PDT, with lower intensity when compared to SCC9 cells. In both lineages, the resistant cells exhibited a phenotype suggestive of CSC. This fact should be considered when establishing PDT mediated by the 5-ALA in oral dysplastic and neoplastic lesions that require multiple cycles of treatment.

La thérapie photodynamique (PDT) est une modalité de traitement des petites tumeurs accessibles à la lumière. Elle repose sur l'action combinée d'un photosensibilisateur qui, en présence d'oxygène et sous l'effet d'une irradiation lumineuse, induit la synthèse d'espèces réactives de l'oxygène cytotoxiques. L'effet tumoricide de la PDT se traduit par des dommages directs sur les cellules ainsi que des dommages indirects de la néovascularisation tumorale et une activation du système immunitaire. Dans cette étude, nous avons démontré dans une première partie l'intérêt de se baser sur la distribution intratumorale de la mTHPC et non pas sur les études de biodistribution pour l'optimisation des conditions de traitement par PDT et en particulier de l'intervalle drogue-lumière (IDL). Un co-ciblage des vaisseaux et du parenchyme tumoral via un fractionnement de l'administration de la mTHPC a permis d'obtenir un taux de guérisons de 100%. Cette efficacité a été corrélée à la potentialisation de la mort des cellules par apoptose et valorisée par son association à des dommages secondaires cutanés restreints. La stratégie de fractionnement de l'administration s'avère donc être très prometteuse dans un contexte clinique. Dans la seconde partie de cette étude, nous avons établi la redistribution de la mTHPC in vivo dans le modèle de la membrane chorioallantoïdienne de poulet (CAM) à partir de formulations liposomales (Foslip®, Fospeg®) et son impact sur les dommages vasculaires photoinduits par la PDT / Photodynamic therapy (PDT) is a therapeutic strategy for the treatment of small localized tumors accessible to the visible light irradiation. It is based on the combined action of photosensitizer (PS), light and molecular oxygen. Tumoricidal effect of PDT is triggered by direct damage of malignant cells and indirect vascular damage followed by an activation of the immune system. The present study investigates the relationship between photoinduced apoptosis in each compartment of interest (vascular versus neoplastic) and mTHPC-PDT treatment efficiency in function of the intratumoral distribution of mTHPC. The latter was defined by the drug-light intervals. In the first part, we demonstrated the importance of the intratumoral distribution of mTHPC to optimize photodynamic parameters. The fractionation of the PS administration permitted to obtain a tumor cure rate of 100% correlated to a massive apoptosis of pathological tissues. Moreover, this treatment strategy induced only limited skin damages and few inflammation which could be an advantage in clinical context. In the second part, we evidenced the mTHPC redistribution from liposomal formulations (Foslip®, Fospeg®) in vivo in the chick chorioallantoic membrane model (CAM) and its influence on photoinduced vascular damage

Der Gegenstand der vorliegenden Arbeit ist die photophysikalische Untersuchung von drei unterschiedlichen Photosensibilisator-Transportsystemen hinsichtlich ihrer Eignung für die photodynamische Therapie. In Fulleren-Pyropheophorbid-a-Konjugaten (P6, FHP6, FP6) finden nach Lichtabsorption effiziente Energietransferprozesse sowie exzitonische Wechselwirkungen unter den Pyropheophorbid a Chromophoren statt. Exzitonische Interaktionen limitieren das Potential der Komplexe zytotoxischen Singulett-Sauerstoff unter Lichtanregung zu generieren. Im Molekül FP6 wurde zusätzlich ein Elektrontransfer vom Pyropheophorbide a zum Fulleren beobachtet. FP6 ist daher für die photodynamische Therapie ungeeignet, kann aber aufgefaßt werden, als die Kombination eines Lichtsammelkomplex mit ladungsseparierendem Zentrum. Weiterhin wurde die Eignung von wasserlöslichen Zinkprotoporphyrin (ZnPP) Polymerkonjugaten als neuartige Photosensibilisatoren für die photodynamische Therapie von soliden Tumoren untersucht. Pegyliertes ZnPP (PEG-ZnPP) bildet mizellare Aggregate in Lösung, wobei die hydrophoben ZnPP Kopfgruppen starke exzitonische Wechselwirkungen unter Lichtanregung eingehen. Dies führt zu einer Unterdrückung der Generierung von Singulett-Sauerstoff in wässriger Lösung. Für ein Styren-Maleinsäure Copolymer, welches ZnPP nicht-kovalent inkorporiert (SMA-ZnPP), wurde ähnliche Resultate gefunden. In-vitro Experimente zeigten, dass die Mizelle nach Aufnahme in die Tumorzellen ihre Integrität verliert und ZnPP monomer freigesetzt wird. Unter Beleuchtung generiert SMA-ZnPP eine hohe Phototoxizität. Polymere Träger-Photosensibilisator Systeme erlauben die Entwicklung effizienter photodynamische Therapien unter der Voraussetzung, das die intramolekularen Transferprozesse unter den Chromophoren gezielt manipuliert werden durch das Trägersystem und somit vorteilhaft genutzt werden können. / The main objective of the thesis is the photophysical investigation of three different photosensitizer-carrier systems concerning the applicability of these macromolecules for photodynamic therapy. For hexapyropheophorbide a-fullerene [C60] molecular systems (P6, FHP6, FP6) it was found that after light absorption the pyropheophorbide a chromophores in all three compounds undergo very efficient energy transfer as well as partly excitonic interactions. The last process limits the potential of the compounds to generate cyctotoxic singlet oxygen under illumination. In the molecule FP6 a fast photoinduced electron transfer process from pyropheophorbide a to the fullerene moiety has been observed additionally. FP6 is inapplicable for PDT but this molecule can be considered as a combination of a light-harvesting system consisting of several separate pyropheophorbide a molecules and a charge-separating center. Furthermore the photosensitizing capabilities of water-soluble polymer conjugates of zinc protoporphyrin (ZnPP) as novel compounds for photodynamic therapy against solid tumors have been investigated. Pegylated ZnPP (PEG-ZnPP) forms micellar aggregates in solution whereas the hydrophobic ZnPP headgroups undergo strong excitonic interactions under illumination. This leads to suppression of singlet oxygen generation in aqueous solution. For a styrene-maleic acid copolymer incorporating ZnPP in a non-covalent fashion (SMA-ZnPP) similar results have been obtained. In-vitro experiments showed that the polymeric micelle is liberated after tumor cell uptake and ZnPP is released in monomeric form. Under illumination SMA-ZnPP generates strong phototoxicity. Polymer carrier-photosensitizer complexes will allow the development of more efficient PDT treatments under the precondition that the intramolecular transfer processes among photosensitizing molecules are selectively manipulated through the carrier system and therefore beneficially utilized.

A Terapia Fotodinâmica (TFD) é uma técnica para tratamento de câncer que usa um fotossensibilizador (FS) na presença de luz e oxigênio molecular gerando espécies altamente reativas de oxigênio que levam as células tumorais à morte. Porém a hidrofobicidade de alguns FSs podem induzir a agregação em sistemas biológicos, com redução da sua atividade fotodinâmica. A incorporação de FSs em sistemas nanocarreadores pode ser uma alternativa para superar este problema. O objetivo deste trabalho foi preparar e caracterizar dois FSs hidrofóbicos (Hipericina e Tetra-carboxiftalocianinade zinco) encapsulados em nanopartículas lipídicas sólidas (NLS) para um potencial uso em terapia fotodinâmica. Os FSs incorporados em nanopartículas lipídicas sólidas foram preparados utilizando a técnica de ultra-sonicação e a caracterização físico-química foi realizada. O tamanho médio das nanopartículas de hipericina e tetra-carboxiftalocianinade zinco foram de 153 e 245 nm respectivamente, índice de polidispersão de 0,28 para Hy-NLS e 0,29 para FtZnT-NLS. Uma das vantagens dos sistemas de encapsulação utilizando NLS é o alto valor de eficiência de encapsulação (EE%) e neste estudo foram obtidos valores de eficiência de encapsulação superior a 80% para a Hy-NLS e FtZnT-NLS. De modo a obter a eficiência fotodinâmica da Hy e FtZnT antes e depois do encapsulamento em NLS, as constantes de velocidade de foto-decomposição utilizando dois agentes captadores de 1O2 (1,3 Difenilisobenzofurano e ácido úrico) foram determinadas. As constantes de velocidade de foto-decomposição tiveram aumento significativo após o encapsulamento que ocorreu provavelmente devido a um aumento no tempo de vida do estado triplete causado pelo aumento da solubilidade. Hy-NLS e FtZnT-NLS apresentaram um aumento acima de 30% e 60% respectivamente na acumulação intracelular e uma melhoria na fototoxicidade correlacionado com o aumento da acumulação intracelular. Todas essas vantagens sugerem que hipericina e a tetra-carboxiftalocianinade zincoencapsuladas em nanopartículas lipídicas sólidas tem potencial para serem utilizadas em terapia fotodinâmica. / Photodynamic therapy (PDT) is a technique for treating cancer using a photosensitizer (PS) in the presence of light and molecular oxygen generating highly reactive oxygen species that lead to tumor cell death. The hydrophobicity of some photosensitizers can induce aggregation in biological systems, reducing its photodynamic activity. The incorporation of PSs in nanocarriers can be an alternative to overcome this problem. The aim of this work was to prepare and characterize two hydrophobic photosensitizers (Hypericin and Zinc tetra-carboxylicphthalocyanine) encapsulated in solid lipid nanoparticles (SLN) for potential use in photodynamic therapy. The PSs incorporated into solid lipid nanoparticles were prepared using the ultrasonication technique, and physico-chemical characterization was performed. The average size of the nanoparticles with hypericin and zinc tetra-carboxylicphthalocyanine was 153 and 245 nm respectively, the polydispersivity index of 0.28 to Hy-SLN and 0.29 to FtZnT-SLN. One of the advantages of encapsulation systems using SLN is the high value of encapsulation efficiency (EE %). In this study were obtained values of encapsulation efficiency greater than 80% for the Hy-SLN and FtZnT-SLN. In order to obtain the photodynamic efficiency of Hy and FtZnT before and after encapsulation in SLN, rate constants using photo-decomposition of two scavengers of 1O2 agents (1,3-Diphenylisobenzofuran and uric acid) were determined. The rate constants of photo-decomposition had significant increase after encapsulation which occurred probably due to an increase in the lifetime of the triplet state caused by the increased solubility. Hy-SLN and FtZnT-SLN showed an increase above 30% and 60% respectively in the intracellular accumulation and an improvement in phototoxicity correlated with increased intracellular accumulation. So, all these advantages suggest that hypericin and zinc tetra-carboxylicphthalocyanine encapsulated in solid lipid nanoparticles have potential to be used in photodynamic therapy.

As lesões potencialmente malignas orais (LPMO) constituem processos com chances de malignização e, portanto, o acompanhamento rigoroso e a retirada das lesões em situações de displasia são mandatórios. A terapia fotodinâmica (PDT) tem sido apontada como uma alternativa promissora e não invasiva para o tratamento dessas lesões. O princípio terapêutico da PDT envolve a geração de altos níveis de estresse oxidativo, pela associação de uma substância fotoativa com a energia eletromagnética e o oxigênio tecidual. É capaz de inviabilizar, através de cascatas de morte ainda pouco esclarecidas, células com alterações metabólicas significativas. A maioria dos trabalhos aponta a necessidade de várias sessões de PDT para erradicar as LPMO, porém o intervalo entre as sessões ainda é discutível. O objetivo deste trabalho foi estabelecer a relação anatomocronológica de marcadores de morte celular (caspase 3, beclin 1 e RIP 1 ) e de proteína de reparo do DNA durante o ciclo celular (PCNA) presentes após a PDT, com o intuito de verificar a cinética morte/proliferação celular e sugerir o intervalo de tempo entre as sessões de PDT mais adequado para a repetição da terapia. Para tanto, LPMOs foram induzidas por intermédio da aplicação tópica de 4-nitroquinolina-1-óxido (4-NQO) na mucosa lingual de ratos e posteriormente tratadas com PDT mediada pela administração tópica do ácido 5-aminolevulínico (5-ALA) e laser comercial (660nm, 90J. cm-2, 1000mW. cm-2). O efeito da PDT foi analisado nos tempos experimentais de 6h, 24h, 48h e 72h após a primeira sessão de PDT, e em 6h e 72h após uma segunda sessão. Nesses períodos, as línguas foram avaliadas clinica e histopatologicamente em relação ao percentual de redução das lesões induzidas e à morfologia do tecido, bem como por meio de análise imuno-histoquímica para PCNA; caspase 3 clivada (presente na apoptose), Beclin 1 (presente na autofagia) e RIP 1 (presente na necroptose). Foi determinada a porcentagem de células positivas para esses marcadores no epitélio da mucosa lingual. Não houve remissão completa das lesões nas duas sessões de PDT, mas a segunda sessão acarretou diminuição em torno de 50% no tamanho das lesões. O período de 6h após a PDT foi o que exibiu significativa atrofia epitelial, bem como a maior porcentagem de células positivas para todos os marcadores analisados, incluindo o PCNA, em ambas as sessões. Caspase 3, beclin 1 e RIP 1 exibiram significativa diminuição da expressão em 24h. O PCNA exibiu aumento significativo no período 72h, e nos dois períodos do segundo ciclo. Como não houve a presença de necrose, a expressão aumentada de RIP 1 foi associada ao processo de apoptose e autofagia. Concluiu-se que a PDT mediada pelo 5-ALA provocou aumento da expressão de caspase 3, beclin 1 e RIP 1 em LPMO nas primeiras 6h após a terapia. Nesse modelo de PDT, essas proteínas parecem interagir em mecanismos de morte por apoptose e por autofagia, mas não por necrose. Considera- se o intervalo de 24h como o mais adequado para novo ciclo com os presentes parâmetros, sem que se estenda além de 72h. / The potentially malignant oral lesions (PMOL) are processes with great chances for cancer transformation and therefore the close monitoring and removal of lesions in cases of dysplasia are mandatory. Photodynamic therapy (PDT) has been identified as a promising and noninvasive treatment for these injuries. The therapeutic principle of PDT involves the generation of high levels of oxidative stress, by association of a photoactive substance with electromagnetic energy and tissue oxygen. It can kill metabolically changed cells through cascades of death which are still unclear. Most studies indicate the need of several PDT sessions to eradicate PMOL, however the interval between sessions is not consensual. The aim of this study was to establish the anatomical and chronological relationship between cellular death biomarkers (caspase 3, beclin 1 and RIP 1 ) and a DNA repair protein during cell cycle (PCNA) present after PDT, aiming to check the kinetic death/cell proliferation and suggest the time interval between PDT sessions more suitable to repetition of the PDT. For this purpose, PMOLs were induced by 4-nitroquinoline-1-oxide (4-NQO) topical application on the lingual mucosa of rats and further treated with PDT mediated by topical administration of 5-aminolevulinic acid (5-ALA) and a commercial laser (Twin flex-MM Optics São Carlos-Brazil 660nm, 90J.cm-2,1000mW.cm-2).The effect of PDT was analyzed at 6h, 24h, 48h and 72h after the first session, and at 6h and 72h after a second session. In these periods, the tongues have been evaluated clinically and histopathologically regarding the percentage reduction of lesions induced and tissue morphology as well as by immunohistochemical analysis for PCNA, cleaved caspase 3 (present in apoptosis), Beclin 1 (present in autophagy) and RIP (present in necroptosis). The percentage of positive cells for these markers was determined in the epithelium of the tongue mucosa. There was no complete remission of lesions after two PDT sessions, but the second session resulted in a decrease of around 50% in lesion size. The period of 6 hours after PDT was the one in which significant epithelial atrophy was exhibited, as well as the highest percentage of positive cells for all tested markers, including PCNA in both sessions. Caspase 3, beclin 1 and RIP 1 exhibited a significant decrease of the expression at 24 hours. PCNA showed significant increase in the 72-hour period and after 6h and 72h from the second session. As there was no necrosis, the increased expression of RIP 1 has been linked to apoptosis and autophagy. It was concluded that PDT mediated by 5-ALA promoted increased expression of caspase 3, beclin 1 and RIP 1 at the PMOLs in the first 6 hours after therapy. For this PDT model, these proteins appear to interact with mechanisms of death by apoptosis and autophagy, but not necrosis. It is considered the range of 24h as the most suitable for another cycle with these parameters of PDT, which should not be extended beyond 72 hours.

Neste trabalho, descreve-se a síntese, caracterização e propriedades dos complexos derivados da tetra(3,4-piridil)porfirazina com os grupos [Ru(bipy)2Cl]1+. A conjugação eletrônica entre o resíduo piridínico e o anel da porfirazina promovem uma eficiente comunicação entre os grupos periféricos e o central, que é refletido no espectro de emissão e seu correspondente perfil de excitação. Esse tipo de comportamento revela um efeito antena no sistema H2TPyPzTRu, contrastante com as propriedades fotofisicas das porfirinas análogas TPyPRu, onde os grupos piridínicos exibem baixa interação eletrônica ao anel porfirínico. Apesar do forte acoplamento eletrônico entre os grupos perféricos e o central, as propriedades eletrônicas dos complexos de rutênio foram preservadas, exibindo potenciais redox muito próximos dos complexos livres e comportamento espectroeletroquímico típicos de complexos metálicos N-heterocíclicos. Esses aspectos levam a novas perspectivas relacionadas à estrutura dos compostos, pois são potencialmente interessantes para o estudo referente à formação de oxigênio singlete e para PDT. Outro direcionamento desta tese, foi o de explorar a geração de novas interfaces baseadas na formação de pares iônicos constituídos pelas espécies H2TPyPzTRu/CuTSPc em comparação com o filme da espécie catiônica H2TPyPzTRu. Através de medidas de espectroscopia de impedância eletroquímica, foram constatados mecanismos distintos de condução nos filmes formados, que pode ser ou um mecanismo misto envolvendo os complexos periféricos e o anel central da porfirazina, ou um mecanismo de condução eletrônica envolvendo somente o sistema de empilhamento π do anel central da porfirazina. / In this work, we describe the synthesis, characterization and properties of derived tetra(3,4-pyridil)porphyrazine complex containing four [Ru(bipy)2Cl]1+ groups. The electronic conjugation between the pyridinium moiety and the porphyrazine ring promote an efficient communication between the peripherical groups and central ring, which is reflected in the emission spectrum and related excitation profile. The observed behavior reveals an efficient antenna effect in the H2TPyPzTRu system. ln spite of the strong electronic coupling between the central and peripherical groups, the electronic properties of ruthenium complex were preserved, exhibiting redox potencials very close to those of free complexes. These aspects provided new perspectives of exploiting the compound strutures, particularly the oxygen singlet formation and PDT application. Another aspect focused in this investigation was the generation of new interfaces based on ion-pair formation of H2TPyPzTRu/CuTSPc, in comparison with its cationic species H2TPyPzTRu alone. By means of electrochemical impedance spectrocopy, it was shown that the conduction mecanisms in these films involve either the peripherical complex and the central porphyrazine ring.