In dieser Arbeit wurden spektroskopische und strukturelle Untersuchungen an chiralen und achiralen supramolekularen Nanoarchitekturen von J-Aggregaten achiraler Cyaninfarbstoffe durchgeführt. Als Modellsysteme wurden Tetrachlorobenzimidacarbocyanin- Farbstoffe mit unterschiedlichen 1,1´-Dialkyl- und 3,3´-Bis-acidoalkyl-Gruppen verwendet. Zur Charakterisierung der Nanostrukturen wurden statische spektroskopische Methoden - UV/Vis-Spektroskopie, Circulardichroismus (CD)- und Fluoreszenzspektroskopie -, Röntgenkristallstrukturanalyse,sowie kryogene Transmissionselektronenmikroskopie (Cryo-TEM) verwendet. Die delikate Balance der Wechselwirkungskräfte, wie z. B. hydrophobe Wechselwirkung, Dispersionswechselwirkung, sowie Wasserstoffbrücken, führt bei der J-Aggregation von strukturell ähnlichen achiralen Chromophoren zu "chiralen" Superhelices und achiralen nanoskopischen Bändern. Durch Kombination der hydrophoben und hydrophilen Eigenschaften von Tensiden mit den unikalen Eigenschaften von J-Aggregaten entstehen Nanoröhren und Vesikel. Diese Nanostrukturen sind daher vielversprechende Kandidaten für künstliche Lichtsammel- und Antennensysteme. / In this work spectroscopic and structural investigations were performed on chiral and achiral supramolecular nanoarchitectures of J-aggregates of achiral cyanine dyes. Tetrachlorobenzimidacarbocyanine dyes with different 1,1´-Dialkyl- and 3,3´-Bis-acidoalkyl- substituents were used as model systems. To characterize these nanoarchitectures static spectroscopic methods - UV/vis-spectroscopy, circular dichroism (CD)- and fluorescence-spectroscopy -, x-ray crystal structure analysis, as well as cryogenic transmission electron microscopy (cryo-TEM) were used. The delicate balance of intermolecular forces, like hydrophobic interaction, dispersion forces, as well as hydrogen bonds, leads by J-aggregation of structural similar achiral chromophores to "chiral" superhelices and achiral nanoscopic ribbons. By combination of the hydrophobic and hydrophilic properties of surfactants with the unique properties of J-aggregates nanotubules and vesicles are built. These nanostructures are hopeful candidates for artificial antenna and light harvesting systems.

Vasoaktives Intestinales Peptid (VIP) ist ein 28meres Neuropeptid, das eine Vielzahl biologischer Aktivitäten ausübt, welche durch die Bindung an die heptahelikalen Transmembranrezeptoren VPAC1 und VPAC2 vermittelt werden. VPAC1 ist auf der Oberfläche vieler Tumorzellen überexprimiert. Daher ist VIP gekoppelt mit Signalmolekülen wie z.B. Fluoreszenzfarbstoffen eine interessante Zielstruktur für die optische Detektion von Tumoren. Der Einsatz von VIP in der Tumordiagnostik ist jedoch aufgrund der schnellen proteolytischen Degradation stark limitiert. In der vorliegenden Arbeit wurden VIP-Analoga mit höherer in vitro und in vivo Stabilität identifiziert und synthetisiert. Hierfür wurde eine neue Synthesestrategie entwickelt, welche die hochparallele Herstellung von löslichen VIP-Farbstoff-Konjugaten auf Cellulosemembranen (Spotsynthese) erlaubt. Es wurden 533 N-terminal Carbocyanin-farbstoff-markierte VIP-Analoga synthetisiert, wobei jeder VIP-Rest durch alle übrigen 19 L-Aminosäuren ausgetauscht wurde (Substitutionsanalyse). Alle Analoga wurden mittels Durchflußzytometrie hinsichtlich ihrer Bindung/Internalisierung an VPAC1-überexprimieren-den Zellen getestet. Diese Ergebnisse führten zur Identifizierung von VIP Aminosäureresten, die für die Wechselwirkung mit VPAC1 essentiell sind und lieferten weiterhin Hinweise über eine vorwiegend helikale Struktur des Rezeptor-gebundenen VIPs. Durch Einbau des Farbstoffs an alle VIP-Reste wurden die für die Wechselwirkung mit VPAC1 günstigen Positionen ermittelt. In Kompetitionstudien und cAMP-Assays ausgewählter Farbstoff-markierter VIP-Konjugate wurde demonstriert, daß sowohl die Spezifität als auch die Produktion von cAMP mit Hilfe der Modifikation der Farbstoffposition gesteigert werden konnte. Für die in Rattenleber getestete metabolische Stabilität der VIP-Analoga zeigte die Farbstoffposition nur einen geringen Einfluß. Die metabolische Stabilität konnte jedoch durch eine einzige Modifikation an Position 8 (Asp8 ® Arg8) erhöht werden. Weiterhin wurde das [Arg8]-VIP-Analogon als Kontrastmittel in in vivo Imaging-Experimenten mit VPAC1-überexprimierenden Tumoren inokulierter Mäuse appliziert. Hierbei wurden die Ergebnisse der Stabilitätstests in Rattenleber bestätigt. Das N-terminal farbstoffmarkierte [Arg8]-VIP-Derivat zeigte gegenüber dem nativen N-terminal markierten VIP-Konjugat eine höhere Halbwertszeit in vivo. Darüber hinaus konnte mit [Arg8]-VIP ein höherer Fluoreszenzkontrast zwischen normalen und Tumorgewebe induziert werden. / Vasoactive Intestinal Peptide (VIP) is a 28meric neuropeptide with a broad range of biological activities which are mediated via binding to the heptahelical transmembrane receptors VPAC1 and VPAC2. Since VPAC1 is overexpressed on the surface of numerous tumour cells, VIP coupled with signal structures like fluorescence dyes is an interesting target for the tumour detection in the near-infrared range. The use of VIP in tumour diagnosis is limited due to the rapid proteolytic degradation and therefore suggests need for optimised VIP-analogues with enhanced stability. In the present work a complete substitutional analysis of VIP N-terminally labelled with carbocyanine dyes was performed. For that reason a new synthetic strategy has been developed, which allows the parallel production of soluble VIP-dye conjugates using the spot synthesis technique. The resulting 560 derivatives were tested for binding and internalisation using VPAC1-overexpressing cells by means of flow cytometry. Based on these results a VIP binding motif has been delineated, which facilitates the modification of VIP concerning stability enhancement and preservation of binding characteristics. Using a dye-walk the dye-coupling positions beneficial for cell binding were identified. Radioactive competitions studies and cAMP assays of selected dye-labeled VIP-conjugates demonstrated that specifity as well as production of cAMP or biological activity has been increased by alteration of the dye-position. For increasing metabolic stability of labelled VIP-analogues the dye-position showed little influence. But is has been shown that stability of VIP was increased by only one modification at position 8 (Asp8®Arg8). Furthermore [Arg8]-VIP-derivative has been used as a contrasting agent in in vivo-Imaging experiments with VPAC1-overexpressing tumours inoculated in mice. Here the results of stability test in rat liver extract were confirmed. N-terminally dye-labelled [Arg8]-VIP analogue revealed higher half-life-time in vivo towards N-terminally labelled native VIP-derivative. In addition the [Arg8]-VIP induced a higher tumour contrast between normal and tumour tissue.

Um Prozesse in biologischen Systemen auf molekularer Ebene zu untersuchen, haben sich vor allem fluoreszenzspektroskopische Methoden bewährt. Die Möglichkeit, einzelne Moleküle zu beobachten, hat zu einem deutlichen Fortschritt im Verständnis von elementaren biochemischen Prozessen geführt. Zu einer der bekanntesten Methoden der Einzelmolekülspektroskopie zählt die Fluoreszenz-Korrelations-Spektroskopie (FCS), mit deren Hilfe intramolekulare und diffusionsgesteuerte Prozesse in einem Zeitbereich von µs bis ms untersucht werden können. Durch die Verwendung von sog. Fluoreszenzsonden können Informationen über deren molekulare Mikroumgebung erhalten werden. Insbesondere für die konfokale Mikroskopie und die Einzelmolekülspektroskopie werden Fluoreszenzfarbstoffe mit einer hohen Photostabilität und hohen Fluoreszenzquantenausbeute benötigt. Aufgrund ihrer hohen Fluoreszenzquantenausbeute und der Möglichkeit, maßgeschneiderte“ Farbstoffe in einem breiten Spektralbereich für die Absorption und Fluoreszenz zu entwickeln, sind Cyaninfarbstoffe von besonderem Interesse für bioanalytische Anwendungen. Als Fluoreszenzmarker finden diese Farbstoffe insbesondere in der klinischen Diagnostik und den Lebenswissenschaften Verwendung. Die in dieser Arbeit verwendeten Farbstoffe DY-635 und DY-647 sind zwei typische Vertreter dieser Farbstoffklasse. Durch Modifizierung können die Farbstoffe kovalent an biologisch relevante Moleküle gebunden werden. Aufgrund ihres Absorptionsmaximums oberhalb von 630nm werden sie insbesondere in der Bioanalytik eingesetzt. In der vorliegenden Arbeit wurden die spektroskopischen Eigenschaften der Cyaninfarbstoffe DY-635 und DY-647 in biomimetischen und biologischen Modellsystemen untersucht. Zur Charakterisierung wurden dabei neben der Absorptionsspektroskopie insbesondere fluoreszenzspektroskopische Methoden verwendet. Dazu zählen die zeitkorrelierte Einzelphotonenzählung zur Ermittlung des Fluoreszenzabklingverhaltens, Fluoreszenz-Korrelations-Spektroskopie (FCS) zur Beobachtung von Diffusions- und photophysikalischen Desaktivierungsprozessen und die zeitaufgelöste Fluoreszenzanisotropie zur Untersuchung der Rotationsdynamik und Beweglichkeit der Farbstoffe im jeweiligen Modellsystem. Das Biotin-Streptavidin-System wurde als Modellsystem für die Untersuchung von Protein-Ligand-Wechselwirkungen verwendet, da der Bindungsmechanismus weitgehend aufgeklärt ist. Nach Bindung der Farbstoffe an Streptavidin wurde eine erhebliche Veränderung in den Absorptions- und Fluoreszenzeigenschaften beobachtet. Es wird angenommen, dass diese spektralen Veränderungen durch Wechselwirkung von benachbarten, an ein Streptavidintetramer gebundenen Farbstoffmolekülen und Bildung von H-Dimeren verursacht wird. Für das System Biotin-Streptavidin ist bekannt, dass während der Bindung des Liganden (Biotin) an das Protein eine Konformationsänderung auftritt. Anhand von zeitaufgelösten Fluoreszenzanisotropieuntersuchungen konnte in dieser Arbeit gezeigt werden, dass diese strukturellen Veränderungen zu einer starken Einschränkung der Beweglichkeit des Farbstoffes DY-635B führen. Liegt eine Mischung von ungebundenem und Streptavidin-gebundenem Farbstoff vor, können die Anisotropieabklingkurven nicht nach einem exponentiellen Verlauf angepasst werden. Es konnte im Rahmen dieser Arbeit gezeigt werden, dass in diesem Fall die Auswertung mit Hilfe des Assoziativen Anisotropiemodells möglich ist, welches eine Unterscheidung der Beiträge aus den zwei verschiedenen Mikroumgebungen ermöglicht. Als zweites Modellsystem dieser Arbeit wurden Mizellen des nichtionischen Tensids Tween-20 eingesetzt. Mizellen bilden eines der einfachsten Systeme, um die Mikroumgebung einer biologischen Membran nachzuahmen. Sind die Farbstoffe in den Mizellen eingelagert, so kommt es zu keiner Veränderung der Mizellgröße. Die ermittelten Werte des Diffusionskoeffizienten der mizellar eingelagerten Farbstoffe spiegeln demzufolge die Translationsbewegung der Tween-20-Mizellen wider. Die Beweglichkeit der Farbstoffe innerhalb der Tween-20-Mizellen wurde durch zeitaufgelöste Fluoreszenzanisotropiemessungen untersucht. Neben der „Wackelbewegung“, entsprechend dem wobble-in-a-cone-Modell, wird zusätzlich noch die laterale Diffusion der Farbstoffe entlang der Mizelloberfläche beschrieben. / To investigate processes in biological systems on a molecular level, particularly fluorescence spectroscopic methods have proven. The possibility to observe single molecules led to significant progress in the understanding of basic biochemical processes. Fluorescence correlation spectroscopy (FCS) is one of the most popular methods of single molecule spectroscopy and is a powerful technique for the investigation of intramolecular and diffusion-controlled processes on a µs to ms time scale. The photophysical characteristics of fluorescent probes are often strongly influenced by their microenvironment. For confocal microscopy and single molecule detection applications fluorescent dyes with properties, such as high photostability and high fluorescence efficiency are highly needed. Due to the high fluorescence efficiency and the high potential to design tailor-made fluorescence probes covering a wide spectral range in absorption and fluorescence, cyanine dyes are highly attractive as fluorescence probes for bioanalytical applications, such as clinical diagnostics and life sciences. The dyes DY-635 and DY-647 are two typical representatives of this class of dyes and can be covalently attached to biologically relevant molecules. Because of their excitation wavelength above 630nm these dyes are especially suited for bioanalytical applications. In this work the spectroscopic properties of DY-635 and DY-647 in biomimetic and biological model systems were studied by absorption and fluorescence spectroscopy techniques: time-correlated single photon counting to determine fluorescence decay behavior, fluorescence correlation spectroscopy (FCS) to observe diffusion and photophysical deactivation processes, and fluorescence anisotropy to study the mobility and rotational behavior of the dyes in the respective model system. The well characterized system biotin-streptavidin was used as a model system for protein-ligand interactions. Binding to streptavidin resulted in significant changes in the steady-state photophysical characteristics of DY-635B and DY-647. These spectral changes are attributed to dye-dye interactions and the formation of H-dimers. Previous studies have demonstrated, that binding of biotin alters the conformation of streptavidin. Based on the evaluation of time-resolved anisotropy data in this study it was shown that these structural changes result in strong hindrance of the rotational freedom of DY-635B. For mixtures of unbound and streptavidin-bound dyes the fluorescence anisotropy decay curves are found to be nonexponential. In this case the concept of an associated anisotropy were applied which allowed discrimination between contributions from different microenvironments. As a second model system, micelles of the nonionic surfactant Tween-20 were used. Micelles are one of the simplest systems to mimic the microenvironment of a biological membrane. Incorporation of the dyes had no effect on the micelle size. The diffusion coefficient of the dyes, obtained by fluorescence correlation spectroscopy (FCS), reflects the translational behavior of Tween-20 micelles. The mobility of the dyes in the Tween-20 micelles was studied by time-resolved fluorescence anisotropy. In addition to a „wobbling“ motion ccording to the wobble-in-a-cone model, a lateral diffusion of the dyes along the micelle surface is described.

Os corantes ciânicos com dois cromóforos possuem características espectrais e energéticas vantajosas para aplicação em Terapia Fotodinâmica (TFD) do câncer. Entretanto, sua fotoatividade contra neoplasias não foi ainda estudada nem in vivo nem in vitro. Nesta tese apresentamos os resultados dos estudos in vitro dos mecanismos da fotocitotoxicidade dos corantes ciânicos com dois cromóforos (BCD) com ângulos entre os cromóforos = 1800, 1500 e 900 contra células neoplásicas, com a finalidade de avaliar a potencialidade da aplicação dos BCD como fotossensibilizadores (FS) em TFD. Os estudos foram realizados em comparação com o fotossensibilizador Photogem®,que já está sendo aplicado em TFD. Foram estudados o efeito fototóxico, a distribuição intracelular do BCD e a contribuição de apoptose e necrose na morte celular induzida por ele. Além disso, foi realizada a busca da formulação farmacêutica adequada para aplicação tópica do BCD180. Nos estudos da fotocitotoxicidade foram utilizadas as células neoplásicas de melanoma murino B16F10, melanoma humano C8161, adenocarcinoma de colo retal humano HT29, leucemia T humano (Jurkat) e leucemia mielóde aguda humana Hl-60. A citotoxicidade foi estudada em função da dose da irradiação, da concentração do FS e do tempo de incubação das células com FS. Todos os compostos testados apresentaram baixa citotoxicidade no escuro, quando sob irradiação com luz visível (? > 600 nm) sua citotoxicidade aumentou consideravelmente. Observamos que para todos os tipos de células neoplásicas a fotocitotoxicidade dos BCD, depois de atingir seu máximo na variação do tempo de incubação, é igual ou ultrapassa a fotocitotoxicidade do Photogem® nas mesmas condições experimentais. O estudo comparativo do BCD180 e dos BCD150 e BCD90 mostrou que nas mesmas condições experimentais os dois últimos possuem fotocitotoxicidade maior do que o BCD180. O conjunto dos resultados obtidos mostra que os BCD? podem ser considerados promissores FS para TFD do câncer. Os estudos através de microscopia de fluorescência da distribuição intracelular do BCD180 e dos marcadores fluorescentes das mitocôndrias Mitotracker GreenTM e Rodamina 123 e do núcleo 4\',6-diamidino-2-phenylindole (DAPI) mostraram que o BCD180 se localiza preferencialmente na região das mitocôndrias. Os mecanismos da morte celular induzida pelo BCD180 foram analisados através do estudo da morfologia das células Jurkat, liberação da fosfatidilserina, liberação do citocromo c, ativação da caspase-3 e do efeito na citotoxicidade do BCD180 da proteína Bcl-2 (inibidor do citocromo c). A análise mostrou que a apoptose é a principal responsável pela morte celular induzida pelo BCD180 no escuro, enquanto que, sob irradiação luminosa, tanto a apoptose quando a necrose contribuem para a morte celular, e a contribuição da necrose aumenta com o aumento da concentração do BCD180 e do tempo de pós-irradiação. A apoptose ocorre, provavelmente, pela via intrínseca ou mitocondrial. Além disso, foram realizados os testes de permeação cutânea do BCD180 utilizando várias formulações farmacológicas e foi determinado que a mistura de 10% de monoleína em propilenoglicol possui melhores características entre todas as formulações testadas. / Cyanine dyes with two chromophores possess vantage spectral and energetic characteristics for application in Photodynamic Therapy (PDT) of cancer. At the same time, their photoactivity against neoplasias was not yet studied neither in vivo, nor in vitro. In this thesis, we present the results of in vitro studies of photocytotoxicity mechanisms of cyanine dyes with two chromophores (BCD) with angles = 180,150 and 90 between chromophores against neoplasic cells, with the objective to evaluate BCD potentiality to be applied as photosensitizers (PS) to Photodynamic Therapy (PDT). The studies were realized in comparison with photosensitizer Photogem®, which is already applied to PDT. The BCD? phototoxic effect, their intracellular distribution and contribution of the apoptosis and necrosis in the cell death induced by BCD were studied. Besides, the search of adequate pharmaceutical formulation for BCD180 topic application was realized. The neoplasic cell lines of melanoma B16F10 in mice, human melanoma C8161, human colon adenocarcinoma HT29, human T-cell leukemia (Jurkat) and human leukemia Hl-60, were used in the study of photocytotoxicity, which was studied as a function of irradiation dose, PS concentration and incubation time of cells with PS. All tested compounds demonstrated low cytotoxicity in the darkness, while under irradiation by visible light (? > 600 nm) their cytotoxicity considerably increased. It was observed that for all types of neoplasic cells BCD photocytotoxicity under the same experimental conditions is equal or exceeds that of Photogem® when reaches the maximum with the incubation time variation. The comparative study of BCD180 with BCD150 and BCD90 demonstrated that under the same experimental conditions two latter compounds possess photocytotoxicity exceeding that of BCD180. A set of the results obtained demonstrates that BCD can be considered as promising PS for PDT of cancer. The study of intracellular distribution of BCD180, and of mitochondria and nucleus fluorescence selective probes Mitotracker GreenTM, Rhodamine 123 and 4\',6-diamidino-2-phenylindole (DAPI) show that BCD180 is mostly localized in the region of mitochondria. The mechanisms of the cell death induced by BCD180 were analyzed in the study of Jurkat cells morphology, phosphatidyl serine and cytochrome c liberation, caspase-3 activation, and by protein Bcl-2 (cytochrome c inhibitor) effect on BCD180 cytotoxicity. The analysis demonstrated that apoptosis is the main responsible for the cell death induced by BCD180 in darkness, while under light irradiation both apoptosis and necrosis contribute to the cell death, and necrosis contribution increases with BCD180 concentration and post-irradiation time.The apoptosis is probably realized by an intrinsic or mitochondrial way. Besides, the tests of BCD180 cutaneum permeation were realized using various pharmacological formulations. It was determined that among all formulations tested, the mixture of 10% of monoleine in propylene glycol possesses the best characteristics.

No description available.

Os corantes ciânicos (CC) são compostos orgânicos que possuem uma estrutura facilmente variável, permitindo obter-se as características fotofísicas desejáveis. Devido a sua alta afinidade por estruturas biológicas, baixa citotoxicidade no escuro, alta solubilidade em meio aquoso e fotoatividade os CC são considerados compostos promissores para aplicações no tratamento do câncer por terapia fotodinâmica (TFD). CC possuem uma forte tendência de se agregar em meio aquoso, que modifica suas características fotofísicas, reduzindo os rendimentos quânticos de fluorescência e do estado tripleto, diminuindo assim sua eficiência em suas aplicações como sonda fluorescente e na TFD, todavia, a agregação aumenta a eficiência da conversão da sua energia de excitação em calor, que é importante para sua aplicação na terapia por hipertermia (HT). Sendo introduzido num organismo o CC se encontra no ambiente onde ele vai interagir com sais e estruturas nano-heterogêneas (membrana celular, ácidos nucléicos etc.), interações que podem influenciar na sua agregação. Nesse trabalho investigamos o fenômeno da agregação dos CC em suas interações com sistemas nano-heterogêneos naturais (DNA) e sintéticos (micelas) em função da sua própria estrutura, da estrutura destes sistemas e da composição da solução: as concentrações do corante e do sistema nano-heterogêneo e a força iônica. Entre os CC, escolhemos como modelos a Acridina Laranja (AL) e os corantes com dois cromóforos (BCD) que se diferem pelo ângulo formado entre seus cromóforos. Utilizamos técnicas espectroscópicas estacionárias e com resolução temporal de absorção óptica, fluorescência, espalhamento ressonante e dinâmico da luz e fotólise por pulso relâmpago. Descobrimos que em soluções aquosas homogêneas os sais induzem a agregação dos CC. No caso da AL, os sais suprimem sua fluorescência pelo aumento da agregação da AL e pela formação de um exciplexo entre a AL em seu estado excitado singleto e o ânion do sal. A interação dos CC com estruturas nano-organizadas é complexa. Observamos que na interação do CC com o DNA aparecem várias espécies em equilíbrio, tais como monômeros de CC livres e ligados ao DNA, agregados de CC ligados ao DNA e agregados de DNA ligados com os monômeros de CC. A ligação da AL ao DNA reduz a probabilidade do contato da AL com outras moléculas. Contudo, na presença do DNA os sais reduzem a agregação da AL devido à redução da constante de ligação da AL com o DNA. Na presença do dodecil sulfato de sódio (SDS), observamos que em baixas concentrações este estimula a agregação do CC. O aumento da concentração de SDS induz a desagregação do CC. Identificamos que os agregados dos CC com SDS apresentam uma dinâmica que pode perdurar por diversas horas. Durante esse período os agregados trocam suas formas H e J. Investigamos uma possível aplicação prática da agregação numa terapia de HT, identificando que a agregação protege o CC da fotodecomposição e aumenta a eficiência da geração de calor. Os resultados obtidos são importantes para avaliar o potencial de aplicação do CC como fotossensibilizadores em terapia fotodinâmica, fotohipertermia e sondas fluorescentes em diagnóstico por fluorescência. / Cyanine dyes (CD) are organic compounds that have an easily variable structure, thus allowing obtain desirable photophysical characteristics. Due to their high affinity to biological structures, low cytotoxicity in the dark, high solubility in aqueous medium and photoactivity the CD are promising materials for application as photosensitizers in cancer treatment by photodynamic therapy (PDT) and as fluorescence probes in fluorescence diagnostics (FD). CD have a strong tendency to aggregate in aqueous media, which modify their photophysical characteristics, reducing its fluorescence and triplet state quantum yields, thus decreasing their efficiency in applications in PDT and FD. At the same time, aggregation increases the probability of excitation energy conversion into heat, which is important for application in hyperthermia (HT) therapy. Being introduced into organism, CD will interact with salts and nano-heterogeneous structures (cell membrane, nucleic acids etc.). These interactions can affect its aggregation. In this work we have investigated the CD aggregation phenomenon at its interactions with natural (DNA) and synthetic (micelles) nano-heterogeneous systems in function of their own structure, structure of the nano-heterogeneous system and the solution characteristics like dye and nano-heterogeneous system concentrations and ionic strength. Among CD, we have chosen as models Acridine Orange (AO) and cyanine dyes with two chromophores (BCD) that differ by the angle between chromophores. Stationary and time-resolved optical absorption, fluorescence, resonant and dynamic light scattering spectroscopies and flash photolysis were used. We have found that in homogeneous aqueous solutions salts induce the CD aggregation. In the case of AO, the salts quench the AO fluorescence by increasing its aggregation and by forming an exciplex between the AO molecule in its singlet excited state and the salt anion. Interaction of CD with nano-organized systems is complex. We observed that at CD interaction with DNA there appear several species in equilibrium, such as CD monomers free and bound to DNA, CD aggregates bound to DNA and DNA aggregates bound to CD monomers. The aggregation of DNA molecules around AO monomers reduces the probability for AO contact with other molecules. In the presence of DNA salts reduce AO aggregation due to reduction of the AO binding constant to DNA. Sodium dodecyl sulfate (SDS) in low concentrations induces CD aggregation, while higher SDS concentrations stimulate CD disaggregation. The process of CD aggregation in the presence of SDS can continue for several hours. During this period, the form of aggregates may modify from H to J or from J to H depending on the dye structure. The irradiation of dye solutions with visible light increases the solution temperature. Aggregation protected CD from photodecomposition and increased heat generation. The results obtained may help in evaluation the potential of CD as photosensitizers in photodynamic therapy, photohyperthermia and fluorescent probes in fluorescence diagnostics.

This dissertation describes the advantages and feasibility of newly synthesized near-infrared (NIR) bis-heptamethine cyanine (BHmC) dyes for non-covalent labeling schemes. The NIR BHmCs were synthesized for biomolecule assay. The advantages of NIR BHmCs for biomolecule labeling and the instrumental advantages of the near-infrared region are also demonstrated. Chapter 1 introduces the theory and applications of dye chemistry. For bioanalysis, this chapter presents covalent and non-covalent labeling. The covalent labeling depends on the functionality of amino acids and the non-covalent labeling relies on the binding site of a protein. Due to the complicated binding process in non-covalent labeling, this chapter also discusses the binding equilibria in spectroscopic and chromatographic analyses. Chapter 2 and 3 evaluate the novel BHmCs for non-covalent labeling with human serum albumin (HSA) and report the influence of micro-environment on BHmCs. The interesting character of BHmCs in aqueous solutions is that the dyes exhibit non- or low-fluorescence compared to their monomer counterpart, RK780. It is due to their H-type closed clam-shell form in the solutions. The addition of HSA or organic solvents opens up the clam-shell form and enhances fluorescence. The binding equilibria are also examed. Chapter 4 provides a brief introduction that summaries the use of capillary electrophoresis (CE), and offers a detailed instrumentation that discusses the importance and advantage of a detector in NIR region for CE separation. Chapter 5 focuses on the use of NIR cyanine dyes with capillary electrcophoresis with near-infrared laser induce fluorescence (CE-NIR-LIF) detection. The NIR dyes with different functional groups show that RK780 is a suitable NIR dye for HSA labeling. The use of BHmCs with CE-NIR-LIF reduces signal noises that are commonly caused by the interaction between NIR cyanine dyes and negatively charged capillary wall. In addition, bovine carbonic anhydrase II (BCA II) is applied to study the influence of hydrophobicity on non-covalent labeling. Finally, chapter 6 presents the conformational dependency of BHmCs on the mobility in capillary and evaluates the further possibility of BHmCs for small molecule detection. Acridine orange (AO) is used as a sample and it breaks up the aggregate and enhances fluorescence. The inserted AO into BHmC changes the mobility in capillary, owing to the conformational changes by AO.

Os corantes ciânicos (CC) são compostos orgânicos que possuem uma estrutura facilmente variável, permitindo obter-se as características fotofísicas desejáveis. Devido a sua alta afinidade por estruturas biológicas, baixa citotoxicidade no escuro, alta solubilidade em meio aquoso e fotoatividade os CC são considerados compostos promissores para aplicações no tratamento do câncer por terapia fotodinâmica (TFD). CC possuem uma forte tendência de se agregar em meio aquoso, que modifica suas características fotofísicas, reduzindo os rendimentos quânticos de fluorescência e do estado tripleto, diminuindo assim sua eficiência em suas aplicações como sonda fluorescente e na TFD, todavia, a agregação aumenta a eficiência da conversão da sua energia de excitação em calor, que é importante para sua aplicação na terapia por hipertermia (HT). Sendo introduzido num organismo o CC se encontra no ambiente onde ele vai interagir com sais e estruturas nano-heterogêneas (membrana celular, ácidos nucléicos etc.), interações que podem influenciar na sua agregação. Nesse trabalho investigamos o fenômeno da agregação dos CC em suas interações com sistemas nano-heterogêneos naturais (DNA) e sintéticos (micelas) em função da sua própria estrutura, da estrutura destes sistemas e da composição da solução: as concentrações do corante e do sistema nano-heterogêneo e a força iônica. Entre os CC, escolhemos como modelos a Acridina Laranja (AL) e os corantes com dois cromóforos (BCD) que se diferem pelo ângulo formado entre seus cromóforos. Utilizamos técnicas espectroscópicas estacionárias e com resolução temporal de absorção óptica, fluorescência, espalhamento ressonante e dinâmico da luz e fotólise por pulso relâmpago. Descobrimos que em soluções aquosas homogêneas os sais induzem a agregação dos CC. No caso da AL, os sais suprimem sua fluorescência pelo aumento da agregação da AL e pela formação de um exciplexo entre a AL em seu estado excitado singleto e o ânion do sal. A interação dos CC com estruturas nano-organizadas é complexa. Observamos que na interação do CC com o DNA aparecem várias espécies em equilíbrio, tais como monômeros de CC livres e ligados ao DNA, agregados de CC ligados ao DNA e agregados de DNA ligados com os monômeros de CC. A ligação da AL ao DNA reduz a probabilidade do contato da AL com outras moléculas. Contudo, na presença do DNA os sais reduzem a agregação da AL devido à redução da constante de ligação da AL com o DNA. Na presença do dodecil sulfato de sódio (SDS), observamos que em baixas concentrações este estimula a agregação do CC. O aumento da concentração de SDS induz a desagregação do CC. Identificamos que os agregados dos CC com SDS apresentam uma dinâmica que pode perdurar por diversas horas. Durante esse período os agregados trocam suas formas H e J. Investigamos uma possível aplicação prática da agregação numa terapia de HT, identificando que a agregação protege o CC da fotodecomposição e aumenta a eficiência da geração de calor. Os resultados obtidos são importantes para avaliar o potencial de aplicação do CC como fotossensibilizadores em terapia fotodinâmica, fotohipertermia e sondas fluorescentes em diagnóstico por fluorescência. / Cyanine dyes (CD) are organic compounds that have an easily variable structure, thus allowing obtain desirable photophysical characteristics. Due to their high affinity to biological structures, low cytotoxicity in the dark, high solubility in aqueous medium and photoactivity the CD are promising materials for application as photosensitizers in cancer treatment by photodynamic therapy (PDT) and as fluorescence probes in fluorescence diagnostics (FD). CD have a strong tendency to aggregate in aqueous media, which modify their photophysical characteristics, reducing its fluorescence and triplet state quantum yields, thus decreasing their efficiency in applications in PDT and FD. At the same time, aggregation increases the probability of excitation energy conversion into heat, which is important for application in hyperthermia (HT) therapy. Being introduced into organism, CD will interact with salts and nano-heterogeneous structures (cell membrane, nucleic acids etc.). These interactions can affect its aggregation. In this work we have investigated the CD aggregation phenomenon at its interactions with natural (DNA) and synthetic (micelles) nano-heterogeneous systems in function of their own structure, structure of the nano-heterogeneous system and the solution characteristics like dye and nano-heterogeneous system concentrations and ionic strength. Among CD, we have chosen as models Acridine Orange (AO) and cyanine dyes with two chromophores (BCD) that differ by the angle between chromophores. Stationary and time-resolved optical absorption, fluorescence, resonant and dynamic light scattering spectroscopies and flash photolysis were used. We have found that in homogeneous aqueous solutions salts induce the CD aggregation. In the case of AO, the salts quench the AO fluorescence by increasing its aggregation and by forming an exciplex between the AO molecule in its singlet excited state and the salt anion. Interaction of CD with nano-organized systems is complex. We observed that at CD interaction with DNA there appear several species in equilibrium, such as CD monomers free and bound to DNA, CD aggregates bound to DNA and DNA aggregates bound to CD monomers. The aggregation of DNA molecules around AO monomers reduces the probability for AO contact with other molecules. In the presence of DNA salts reduce AO aggregation due to reduction of the AO binding constant to DNA. Sodium dodecyl sulfate (SDS) in low concentrations induces CD aggregation, while higher SDS concentrations stimulate CD disaggregation. The process of CD aggregation in the presence of SDS can continue for several hours. During this period, the form of aggregates may modify from H to J or from J to H depending on the dye structure. The irradiation of dye solutions with visible light increases the solution temperature. Aggregation protected CD from photodecomposition and increased heat generation. The results obtained may help in evaluation the potential of CD as photosensitizers in photodynamic therapy, photohyperthermia and fluorescent probes in fluorescence diagnostics.

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