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.

One of the greatest threats to human health is increasing antimicrobial resistance among pathogens, and finding alternatives for treatment of bacterial infections is of highest importance together with a more controlled use of antibiotics. Porphyrins and heme have both been shown to be a promising class of compounds for inactivation of bacteria; porphyrins by their excellent properties to act as a photosensitizer, and heme by its importance as an iron source during a bacterial infection in vertebrates. This thesis describes the development of analytical methods for the identification and determination of porphyrins and heme using liquid chromatography coupled to tandem mass spectrometry. Subsequently, these developed methods were applied to bacterial samples to investigate different culture conditions and additives effect to the intracellular porphyrin and heme composition. Singlet oxygen production of three naturally occurring porphyrins have been determined together with the photosensitivity for blue light and the porphyrin content in E. coli. Toothbrushes equipped with a LED, emitting light with a wavelength of 450 nm, were used in an eight week randomized clinical trial to investigate any positive periodontal effect of blue light. Porphyrin and heme content in Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were highly affected by the different cultivation conditions. The culture age of A. actinomycetemcomitans affected the porphyrin profile, while only small changes were observed for P. gingivalis during growth. A large change of the porphyrin profile could be observed when the bacteria were passaged onto a new growth medium. Additional porphyrins were detected and the total porphyrin content increased up to 28 times. These findings highlight the need for more standardized cultivation procedures when performing in vitro experiments. Heme content in Escherichia coli was affected when different additives related to biosynthesis of heme were added to the growth medium. The uptake of heme could be reduced with 52% when a compound that chemically looks similar to heme was added to the growth medium. Since heme acquisition is important for many pathogens, this could be a promising target for antimicrobial drugs. E. coli showed no sensitivity for 405 nm light using light doses up to 172.8 J/cm2 and only low concentrations of porphyrins could be quantified. By adding a porphyrin precursor to E. coli the intracellular concentration of porphyrins increased remarkably and a light dose of 57.6 J/cm2 reduced the bacterial number with &gt; 5 log10 steps. This shows that E. coli can be killed due to their endogenous porphyrins. In the clinical study we could see a weak trend that the 450 nm LED toothbrush possessed a phototherapeutic effect for three clinical indices. All indices were decreased in the intervention group, but there were no statistically significant difference compared to the control group. However, four inflammation markers were significantly decreased in the intervention group while only one decreased significantly in the control group. In conclusion, this thesis has shown that porphyrins and heme are produced endogenously in microorganisms and that the porphyrin profiles vary depending on culture conditions and different additives. Furthermore, porphyrins may be used as endogenous photosensitizers to inactivate bacteria, but more research is necessary to determine if there is a specific porphyrin that contributes more to the photosensitivity. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.</p>

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2018-06-19T15:58:48Z No. of bitstreams: 1 Nadua Apostolico.pdf: 2774487 bytes, checksum: c4de48bfb4f153970bee3d2f104e406e (MD5) / Made available in DSpace on 2018-06-19T15:58:48Z (GMT). No. of bitstreams: 1 Nadua Apostolico.pdf: 2774487 bytes, checksum: c4de48bfb4f153970bee3d2f104e406e (MD5) Previous issue date: 2015-12-16 / The sterilization step for acellular lungs prior to recellularization is important to ensure that they are free of any risk of transmitting infections from the donor to the recipient tissue/organ to be transplanted. However, there are no data on the mechanical effects induced in the lungs when subjected to a given dose of sterilization radiation through a 660-nm LED. The aim of this study was to evaluate the extracellular matrix (ECM) and lung mechanic parameters of decellularized lungs undergoing photodynamic therapy (PDT) with the purpose of sterilization, and also to perform microbiological analyses of lungs infected with C. albicans to determine the effectiveness of PDT. The lung mechanical evaluation parameters, Resistance (RL) and elastance (EL), exhibited no significant differences between groups. Also, there were no changes observed over time of irradiation, representing a maintenance of the viscoelastic behavior of the lung scaffold after 1h exposure to LED light. The ECM components remained virtually unchanged in the acellular lungs of both groups. We also showed that there was a reduction in fungi infection population after 45 minutes of PDT. However, more studies should be performed to establish and verify the effectiveness of PDT as a possible means for sterilizing lung scaffolds. / A esterilização de scaffolds pulmonares, antes de se iniciar a recelularização, é um fator importante visando assegurar que pulmões acelulares estejam livres de qualquer risco de transmissão de infecções a partir do doador para o receptor do tecido / órgão a ser transplantado. No entanto, não existem dados sobre os efeitos mecânicos induzidos nestes pulmões quando submetidos a terapia fotodinâmica. O objetivo deste estudo foi analisar a matriz extracelular (MEC) e avaliar parâmetros de mecânica pulmonar de pulmões descelularizados submetidos à terapia fotodinâmica (TFD) como possível esterilização, e também realizar análises microbiológicas de pulmões infectados por C. albicans para determinar a efetividade da TFD. Os valores obtidos para avaliação do comportamento da mecânica pulmonar, resistência (RL) e elastância (EL), não apresentaram diferenças significativas entre os grupos. Além disso, não foram observadas alterações ao longo do tempo de irradiação, o que representa uma manutenção do comportamento viscoelástico dos scaffolds de pulmão, após 1h de exposição a luz LED. Os componentes da matriz extracelular permaneceram praticamente inalterados nos pulmões acelulares de ambos os grupos. Também foi evidenciado que houve uma redução fungica após 45 minutos de PDT. No entanto, mais estudos devem ser realizados para estabelecer e verificar a eficácia do TFD como um possível meio para esterilizar scaffolds pulmonares.

Une des limitations de la thérapie photodynamique est la faible sélectivité des photosensibilisateurs (PS) pour les tissus tumoraux. La recherche de nouveaux PS plus sélectifs s’est orientée vers la synthèse de PS couplés à des motifs afins pour des récepteurs membranaires surexprimés dans certains cancers. Le récepteur à l’acide folique est surexprimé dans les carcinomes ovariens et des PS ont été conjugués à de l’acide folique. Des PS ont aussi été couplés à un peptide spécifique de neuropiline 1 surexprimé au niveau des cellules tumorales de médulloblastome. Une autre stratégie pour augmenter la sélectivité du traitement vise à produire les espèces réactives de l’oxygène spécifiquement au niveau du site tumoral. L’activité de clivage enzymatique de marqueurs biologiques surexprimés dans les zones tumorales est utilisée. Les gélatinases (MMP-2 et -9) et leur activateur MMP-14 sont connues pour jouer un rôle primordial dans l'angiogenèse tumorale et la croissance du glioblastome multiforme. Différents photodynamic molecular beacons (PMB), associant un PS et un quencher lié par un peptide substrat des gélatinases et MMP-14, ont été développés / One limitation of photodynamic therapy is the low selectivity of photosensitizers (PS) to tumour tissue. The search of new PS more selective began to focus on the synthesis of PS coupled with substrate specific of the membrane receptors overexpressed in certain cancers. The acid folic receptor is overexpressed in ovarian carcinomas and PS were conjugated with folic acid. PS were also coupled with a specific peptide of neuropilin 1 overexpressed in tumoral cells of medulloblastoma. Another strategy for increasing the selectivity of the treatment is to produce reactive oxygen species specifically at the tumor site. The activity of enzymatic cleavage of biomarkers overexpressed in tumour areas is used. The gelatinases (MMP-2 and MMP-9) and their activator MMP-14 are known to play a key role in tumour angiogenesis and the growth of glioblastoma multiform. Different photodynamic molecular beacons (PMB), composed of a photosensitizer and a quencher linked together by a peptide substrate of gelatinases or MMP-14, were designed.

Biofilme aeroterrestrischer Mikroorganismen stellen im nicht-medizinischen Bereich ein ständig größer werdendes Problem dar. Sie tragen ebenfalls wesentlich zur Verwitterung von Fassaden bei und verursachen durch die daraus resultierende Notwendigkeit der Entfernung des Biofilms und Instandsetzung der Fassaden hohe Kosten. Auch das von Biofilmen auf Innenwänden von Gebäuden ausgehende Gesundheitsrisiko für den Menschen ist schwerwiegend. Solarzellen sind aufgrund ihrer rauen Glasoberfläche ebenfalls ein bevorzugter Siedlungsort für Biofilme. Auf diesen können sie durch Verschattung die Effizienz der Solarzellen deutlich herabsetzen. Die Entfernung von Biofilmen erfordert vor allem den Einsatz von Bioziden, welche ihrerseits eine Belastung für die Umwelt darstellen und zudem oft nur begrenzte Wirksamkeit zeigen. Ziel dieser Arbeit ist daher, das Potenzial der Photodynamischen Inaktivierung (PDI) als alternative Methode zur Beseitigung oder zur Prävention der Bildung von Biofilmen zu bestimmen. Dazu wurden unterschiedliche Photosensibilisatoren (PS) bezüglich ihrer Phototoxizität auf im Rahmen dieser Arbeit etablierte phototrophe Modellkulturen in Suspension, subaquatischen und subaerialen Biofilmen untersucht. Neben der Entwicklung der Biomasse während der PDI wurde mittels spektroskopischer Methoden zudem die Aktivität der Kulturen sowie die EPS-Sekretion und die Biofilmbildung gemessen. Dabei konnte gezeigt werden, dass die kationischen PS PCor+ und TMPyP zur PDI phototropher Kulturen erfolgreich genutzt und die Biofilmbildung verhindert werden kann. Es wurde gezeigt, dass dieses Resultat ohne Aufnahme der PS in die Zellen erzielt wird. Dies ist bezüglich der Vermeidung einer möglichen Resistenzentwicklung der Mikroorganismen von entscheidender Bedeutung. Somit wurden mit dieser Arbeit die Voraussetzungen für die Entwicklung photodynamisch aktiver, antimikrobieller Oberflächenbeschichtungen zur Prävention des Wachstums aeroterrestrischer Biofilme geschaffen. / Biofilms of aeroterrestrial microorganisms are an ever-increasing problem in non-medical applications. They also contribute significantly to the weathering of facades and cause high costs due to the resulting necessity of removing the biofilm and repairing the facades. Furthermore, the health risk for humans caused by biofilms on interior walls of buildings is serious. In addition, solar cells are a preferred location for biofilms due to condensation on their rough glass surface. Growing there, they can significantly reduce the efficiency of the solar cells by shading, a problem which gains even more importance with increasing operating times of solar modules. In the removal of biofilms, biocides are prevalent. They are in turn a burden on the environment and often show only limited effectiveness. The aim of this work is therefore to determine the potential of Photodynamic Inactivation (PDI) as an alternative method to eliminate or prevent the formation of biofilms. Different Photosensitizers (PSs) were investigated with respect to their phototoxicity on phototrophic model cultures established in suspension, subaquatic and subaerial biofilms. In addition to biomass development during PDI, spectroscopic methods were used to measure culture activity, EPS secretion and biofilm formation. It was shown that the cationic PSs PCor+ and TMPyP can be successfully used for PDI of phototrophic cultures and that biofilm formation can be prevented. It has been shown that this result is achieved without uptake of PS into the cells. This is of decisive importance with regard to the avoidance of a possible resistance development in the microorganisms. Thus, this work created the prerequisites for the development of photodynamically active, antimicrobial surface coatings for the prevention of the growth of aeroterrestrial biofilms.

Den ökande koldioxidhalten i atmosfären har lett till ett ökat intresse för användning av solljus, ett exempel är inom kemi. Att använda ljus i kemi kan leda till nya och intressanta kemiska reaktioner och kan tillämpas i användningsområden som exempelvis fotokatalys och fotovoltaik. Genom att kombinera de kraftfulla egenskaperna av ljus i kemi med den höga selektivitet som karaktäriserar proteiner kan intressanta och kraftfulla maskinerier erhållas, så kallade fotoaktiva proteiner. Att studera de fotoinducerade beteendena i fotoaktiva proteiner är däremot icke-trivialt eftersom många utmaningar uppstår från ett experimentellt och teoretiskt perspektiv. Denna uppsats fokuserar på det teoretiska perspektivet. Simulationer av beteenden hos fotoaktiva proteiner leder till utmaningar relaterade till den stora variationen av tids- och längdskalor involverade, där allt ifrån ultrasnabba och lokaliserade excitationsprocesser till storskaliga strukturförändringar som utspelar sig på större tidsskalor. Detta, tillsammans med det faktum att det inte finns en "svart låda" som vi kan använda för nya teoretiska studier motiverar ett försiktigt tillvägagångssätt för teoretiska studier av fotoaktiva proteiner. I detta projekt görs en teoretisk undersökning av två nyligen framställda fotosensibiliserande proteiner (PSP2 och PSP3). De skapades för att härma huvudprocesserna av fotosyntesen i plantor. Fotosensibiliserande proteinerna skiljer sig med en enda aminosyra (position 203), som är en aspartat i PSP2 och en tyrosin i PSP3. Även fast proteinerna är strukturellt lika så uppvisar de olika fotoinducerade beteenden. Målet med denna studie är att klargöra för de mekanistiska detaljerna bakom dessa olikheter. Som ett första steg i att teoretiskt studera PSP2 och PSP3 upprättar vi ett protokoll för teoretisk förberedelse av proteinerna. Detta protokoll används sedan för att teoretiskt undersöka fotofysiken i dessa två proteiner med hjälp av kvantmekaniska/ molekylär mekaniska simuleringar. Våra resultat ger en första ordningens kartläggning av de exciterade tillstånden i de två proteinerna. Vi erhåller en intressant laddningsöverföring tillstånd i PSP3 som involverar tyrosinen och proteinets kromofor, med en energi lägre än det tillstånd ansvarig för absorption av ljus. Denna laddningsöverföring saknas i PSP2 och kan därför vara anledningen till de olika fotoinducerade beteendena i proteinerna. Detta kräver dock fortsatta studier av de två systemen. / The increase of carbon dioxide levels in the atmosphere have resulted in an increasing interest in using solar light for different purposes, one being chemistry. The introduction of light in chemistry can drive new and exciting chemical reactions, finding applications in many fields such as photocatalysis and photovoltaics. Combining the aspects of light in chemistry together with the high selectivity that characterizes proteins make for interesting and powerful machineries, so-called photoactive proteins. However, studying the behaviors of photoactive proteins is a non-trivial task and many challenges arise from both an experimental and a theoretical point of view. This thesis takes a theoretical perspective. The challenges associated with simulating photoactive protein behavior originate from the wide range of time and length scales involved, ranging from ultrafast and localized excitation processes to large-scale structural changes occurring on longer timescales. This, together with the fact that there is no black box that we can use for novel theoretical studies of photoactive proteins motivates a careful approach for theoretical studies of photoactive proteins. In this project, we conduct a theoretical investigation of two photosensitizer proteins (PSP2 and PSP3), recently engineered to capture the essence of plant photosynthesis. The photosensitizer proteins differ by only one residue (position 203), which is an aspartate in PSP2 while a tyrosine in PSP3. Although structurally similar, the proteins demonstrate different photoinduced behaviors. This study aims to shed light on the mechanistic details underlying these differences. As a first step to study PSP2 and PSP3 computationally, we develop a computational protocol for protein preparation. The protocol is then used for a theoretical investigation of the photophysics of the two proteins using quantum mechanics/molecular mechanics simulations. Our results provide a first mapping of the electronic-state manifold of the two proteins. We find an intriguing charge-transfer state in PSP3, involving the tyrosine and the protein chromophore, located below bright state responsible for light absorption. This state is absent in PSP2 and could therefore be the reason for the different photoinduced behavior of the two proteins. However, this requires further studies of the two systems.

Multimodal treatment adding immunotherapy and photodynamic treatment (PDT) to standard therapy might improve the devastating therapeutic outcome of glioblastoma multiforme patients. As a first step, we provide investigations to optimize dendritic cell (DC) vaccination by using PDT and ionizing radiation (IR) to achieve maximal synergistic effects. In vitro experiments were conducted on murine glioblastoma GL261 cells, primary DCs differentiated from bone marrow and T cells, isolated from the spleen. Induction of cell death, reactive oxygen species, and inhibition of proliferation by tetrahydroporphyrin-tetratosylat (THPTS)-PDT and IR were confirmed by WST-1, LDH, ROS, and BrdU assay. Tumor cargo (lysate or cells) for DC load was treated with different combinations of THPTS-PDT, freeze/thaw cycles, and IR and immunogenicity analyzed by induction of T-cell activation. Cellular markers (CD11c, 83, 86, 40, 44, 69, 3, 4, 8, PD-L1) were quantified by flow cytometry. Cytotoxic T-cell response was evaluated by calcein AM assay. Immunogenicity of THPTS-PDT-treated GL261 cells lysate was superior to IR-treated lysate, or treated whole cells proven by increased DC phagocytosis, T-cell adhesion, proliferation, cytolytic activity, and cytokine release. These data strongly support the application of PDT together with IR for optimal immunogenic cell death induction in tumor cell lysate used to pulse DC vaccines.

O presente trabalho tem como objetivo contribuir para o conhecimento, entendimento e desenvolvimento das células solares sensibilizadas por corantes. Assim foram abordadas rotas sintéticas com o fim de obter novas espécies supramoleculares constituídos por fragmentos que apresentam separadamente ótimas propriedades eletrônicas e eletroquímicas. A abordagem sintética feita é muito importante dado que dá as bases para sistemas supramoleculares como o apresentado nas perspectivas, nos quais estase visando diminuir os processos de recombinação e melhor aproveitamento do espectro solar através da estabilização do corante foto-oxidado e da transferência vetorial de elétron/energia. Na série de complexos apresentada e aplicada no presente trabalho realizou-se um estudo comparativo buscando determinar qual é o melhor sistema de injeção de elétrons na banda de condução do TiO2, obtendo uma diferença considerável entre os sistemas estudados, sendo assim o sistema com o ligante acido 2,2 -bipiridine-4,4 -dicarboxilico o complexo que melhor injeta elétrons na BC. / The present work aims to contribute to the knowledge, understanding and development of solar cells sensitized by dyes. Thus, synthetic routes were approached in order to obtain new supramolecular species constituted by fragments that separately present excellent electronic and electrochemical properties. The synthetic approach is very important since it provides the basis for supramolecular systems such as that presented in the perspectives, in which stasis is aimed at decreasing the processes of recombination and better utilization of the solar spectrum through the stabilization of the photo-oxidized dye and the vectorial transfer of electron /energy. In the series of complexes presented and applied in the present work, a comparative study was carried out to determine which is the best electron injection system in the conduction band of TiO2, obtaining a considerable difference between the studied systems, being thus the complex with the ligand 2,2 -Bipyridine-4,4 -dicarboxylic acid the best system that injects electrons into BC.

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.

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.