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1

Ruiz, González Rubén. "New strategies in antimicrobial photodynamic therapy and singlet oxygen detection." Doctoral thesis, Universitat Ramon Llull, 2013. http://hdl.handle.net/10803/119556.

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Tres estratègies adreçades a aportar una major perspectiva en el camp de la teràpia fotodinámica antimicrobiana comprenen la primera part de la tesi. En una primera aproximació, sis fotosensibilitzadors catiònics, amb diferent càrrega elèctrica neta i estructura, han estat caracteritzats en dissolució i les seves propietats fotoinactivadores provades contra diferents tipus de microorganismes. En una segona aproximació, conjugats entre un fotosensibilitzador i el pèptid antimicrobià Apidaecina 1b han estat analitzats fotofísica i mecanísticament, i les seves propietats correlacionades amb la seva habilitat fotosensibilitzadora en bacteris. En l'última aproximació, dues proteïnes fluorescents –TagRFP i miniSOG- han estat expressades en E. coli i s'ha estudiat la seva fototoxicitat així com el seu mecanisme d'acció. A més, s'ha reevaluat i estudiat en detall la capacitat de miniSOG per fotosensibilitzar la formació d'oxigen singlet. En la segona part de la tesi, l'interès s'ha centrat en la detecció d'oxigen singlet. En la primera secció, s'ha avaluat la capacitat de detecció en dissolució d'una nova díada composta per un naftoxazol i una trampa química d'oxigen singlet. En l'última secció, es desenvolupa, prova i discuteix la viabilitat de nanopartícules de poliacrilamida com a plataforma potencial per a la detecció d'oxigen singlet en sistemes intracel•lulars.<br>Tres estrategias destinadas a aportar una mayor perspectiva en el campo de la terapia fotodinámica antimicrobiana componen la primera parte de la tesis. En una primera aproximación, seis fotosensibilizadores catiónicos, con distinta carga eléctrica neta y estructura, han sido caracterizados en disolución y sus propiedades fotoinactivadoras probadas contra diferentes tipos de microorganismos. En una segunda aproximación, conjugados entre un fotosensibilizador y el péptido antimicrobiano Apidaecina 1b han sido analizados fotofísica y mecanísticamente, y sus propiedades correlacionadas con su habilidad fotosensibilizadora en bacterias. En la última aproximación, dos proteínas fluorescentes –TagRFP y miniSOG- han sido expresadas en E. coli y se ha estudiado su fototoxicidad así como su mecanismo de acción. Además, se ha reevaluado y estudiado en detalle la capacidad de miniSOG para fotosensibilizar la formación de oxígeno singlete. En la segunda parte de la tesis, el interés se ha centrado en la detección de oxígeno singlete. En la primera sección, se ha evaluado la capacidad de detección en disolución de una nueva díada compuesta por un naftoxazol y una trampa química de oxígeno singlete. En la última sección, se desarrolla, prueba y discute la viabilidad de nanopartículas de poliacrilamida como plataforma potencial para detección de oxígeno singlete en sistemas intracelulares.<br>Three strategies addressed to give further scope on to the antimicrobial photodynamic therapy field comprise the first part of the thesis. In a first approach, six cationic photosensitisers varying in the net electrical charge and structure have been characterised in solution and their photoinactivation skills have been tested against different types of microorganisms. In a second approach, conjugates between a photosensitiser and the antimicrobial peptide Apidaecin 1b have been analysed in a photophysical and mechanistic way, and their properties have been correlated with their bacterial photoinactivation ability. In the last approach, two fluorescent proteins –namely TagRFP and miniSOG- have been expressed in E. coli and their phototoxicity has been studied and characterised mechanistically. Moreover, the capacity of miniSOG to photosensitise singlet oxygen formation has been revisited and studied in detail. In the second part of the thesis, focus has been shifted towards singlet oxygen detection. In the first section, the probing ability of a new dyad comprising a naphthoxazole moiety plus a singlet oxygen chemical trap has been evaluated in solution. In the last section, we develop, test, and discuss the feasibility of a polyacrylamide nanoparticle scaffold as a potential platform for singlet oxygen detection in intracellular systems.
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2

Ding, Rui. "Enhanced Singlet Oxygen Production from Metal Nanoparticle Based Hybrid Photosensitizers." University of Cincinnati / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1447690607.

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3

Ragàs, Amalrich Xavier. "Singlet Oxygen in Antimicrobial Photodynamic Therapy: Biological effects, Mechanistic Studies and Future Directions." Doctoral thesis, Universitat Ramon Llull, 2010. http://hdl.handle.net/10803/9302.

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En aquesta tèsi s'ha avaluat l'ús de la teràpia fotodinàmica antimicrobiana (APDT) com a alternativa als antibiòtics. Inicialment, s'ha demostrat in vitro i en un model de cremades en ratolins el potencial de les fenotiazines, i més concretament del nou blau de metilè, per inactivar un bacteri resistent a antibiòtics com Acinetobacter baumanii. A més, també s'ha investigat el potencial dels porficens aril catiònics com a agents fotosensibilitzadors per a APDT in vitro i in vivo, inactivant exitosament diferents tipus de bacteris gram positius i negatius, així com fongs. <br/><br/>Per altra banda, s'han estudiat les cinètiques de formació i decaïment d'oxigen singlet (1O2) en E. coli mitjançant un espectrofotòmetre ultrasensible a l'infraroig proper amb resolució temporal per sota del microsegon. Els resultats ens permeten profunditzar en el mecanisme de l'APDT, és a dir, en la localització del fotosensibilitzador (PS) en el bacteri i la reactivitat de l'1O2. Per un costat, l'entorn del PS canvia quan la distribució de les càrregues catiòniques varia. Per l'altre, en funció del lloc de formació, l'1O2 reacciona amb l'entorn proper o és capaç d'escapar fora de les cèl·lules. <br/><br/>En tercer lloc, també s'ha estudiat, mitjançant sondes fluorescents i tècniques amb resolució temporal, l'habilitat d'una proteïna fluorescent per produir espècies reactives d'oxigen, actuant com a agents fotosensibilitzadors genèticament codificats. Així, s'ha calculat per primera vegada el rendiment quàntic de formació d'1O2 obtenint un valor similar al del cromòfor sintètic de les GFPs. <br/>Finalment, s'ha investigat mitjançant l'espectrofotòmetre en l'infraroig proper l'efecte plasmó produït per films d'illes de plata sobre l'1O2, demostrant un increment màxim en la luminescència d'1O2 a 1270 nm al voltant de 30 vegades en alguns casos.<br>En esta tesis se ha evaluado el uso la terapia fotodinámica antimicrobiana (APDT) como alternativa a los antibióticos. Inicialmente, se ha demostrado, in vitro y en un modelo de quemaduras en ratón, el potencial de las fenotiazinas, y más concretamente del nuevo azul de metileno, para inactivar una bacteria resistente a antibióticos como Acinetobacter baumanii. Además, también se ha investigado el potencial de los porficenos aril catiónicos como agentes fotosensibilizadores para APDT in vitro e in vivo, inactivando exitosamente distintos tipos de bacterias gram positivas y negativas, así como hongos. <br/><br/>Por otro lado, se ha estudiado las cinéticas de formación y decaimiento de oxígeno singlete (1O2) en E. coli mediante un espectrofotómetro ultrasensible en el infrarrojo cercano con resolución temporal por debajo del microsegundo. Los resultados nos permiten profundizar en el mecanismo de la APDT, es decir, en la localización del fotosensibilizador (PS) en la bacteria y la reactividad del 1O2. Por un lado, el entorno del PS cambia cuando la distribución de las cargas catiónicas varía. Por otro lado, en función del lugar de formación, el 1O2 reacciona con el entorno cercano o es capaz de escapar fuera de las células. <br/><br/>En tercer lugar, también se ha estudiado, mediante sondas fluorescentes y técnicas con resolución temporal, la habilidad de una proteína fluorescente para producir especies reactivas de oxígeno, actuando pues como agentes fotosensibilizadores genéticamente codificados. Así, se ha calculado por primera vez el rendimiento cuántico de formación de 1O2 mostrando un valor similar al del cromóforo sintético de las GFP. <br/>Finalmente, se ha investigado mediante el espectrofotómetro en el infrarojo cercano el efecto plasmon producido por films de islas de plata sobre 1O2, observando un incremento máximo de la luminiscencia de 1O2 a 1270 nm alrededor de 30 veces en algunos casos.<br>In this thesis, the use of antimicrobial photodynamic therapy as alternative to antibiotics has been assessed. First, the potential of phenothiazinium dyes, and specifically new methylene blue, to inactivate multidrug-resistant strains has been demonstrated against Acinetobacter baumanii in vitro and in a mouse burn model. In this regard, it also has been investigated the potential of aryl cationic porphycenes as photosensitizing drugs in APDT in vitro and in vivo, successfully inactivating different gram-positive and negative bacteria, and fungal cells.<br/>Second, the kinetics of singlet oxygen (1O2) production and fate in E. coli have been investigated by means of an ultrasensitive near-infrared spectrometer with submicrosecond time resolution. The results allowed us to gain insight into the mechanism of APDT, i.e., the localization of the photosensitiser (PS) in the bacteria and the reactivity of 1O2. On one hand, the microenvironment of the PS changes when the distribution of the cationic charges changes. On the other hand, depending on the site of production, 1O2 reacts with the nearest microenvironment or is able to escape out of the cells. <br/>Third, the ability of fluorescent proteins (GFPs), as genetically-encoded photosensitisers, to produce reactive oxygen species has been studied by means of fluorescent probes and time-resolved techniques. Thus, for the first time, the quantum yield of 1O2 production of some of the studied proteins has been calculated showing a value similar to that of the synthetic GFP chromophore.<br/>Finally, the plasmonic effect of silver island films on 1O2 has been investigated using the near-infrared spectrometer, demonstrating a maximum enhancement of the 1O2 luminescence signal at 1270 nm ca. 30-fold in some cases.
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4

Ferreira, Joana Teles. "Ruthenium phythalocyanines as potential photosensitizers for singlet oxygen generation." Doctoral thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/22727.

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Doutoramento em Química<br>A Terapia Fotodinâmica combina luz, oxigénio molecular e um fotossensibilizador para a produção de espécies reativas de oxigénio, como 1O2 e radicais livres, os quais vão induzir stress oxidativo e, eventualmente, morte celular. Isto permite assim a destruição de células tumorais de uma forma não invasiva, seletiva e localizada, com efeitos secundários reduzidos. As ftalocianinas são compostos promissores para serem aplicados como fotossensibilizadores na produção de oxigénio singleto. No entanto, um inconveniente destes compostos é a sua reduzida solubilidade em meio aquoso. Isto pode ser ultrapassado através da sua funcionalização nas posições periféricas e/ou axiais com grupos hidrofílicos apropriados, como por exemplo hidratos de carbono e cadeias de poliéter. A introdução de ligandos axiais reduz a agregação das ftalocianinas em solução, melhorando a sua eficiência para produzir 1O2. Este trabalho descreve a síntese de Ftalocianinas de Ruténio (RuPcs) para serem utilizadas como fotossensibilizadores. Estas RuPcs estão funcionalizadas nas posições axiais com ligandos apropriados que proporcionam a solubilidade e/ou seletividade requeridas para a sua aplicação em Terapia Fotodinâmica. Neste sentido, foram sintetizados diferentes derivados de piridina e fosfina funcionalizados com grupos iónicos, cadeias de poliéter, hidratos de carbono ou com unidades de ácido fólico, as quais foram posteriormente coordenadas ao ião central de RuPcs. Adicionalmente, a solubilidade em água foi melhorada através da funcionalização da periferia das RuPcs com cadeias poliéter. Foram estudadas as propriedades físicas e fotofísicas, nomeadamente, a solubilidade em água e a capacidade de produzir oxigénio singleto, de todos os fotossensibilizadores preparados. Além disso, os compostos foram avaliados in vitro em células do cancro da bexiga com respeito à sua capacidade para serem internalizados por células cancerígenas a aos seus efeitos tóxicos, tanto no escuro como mediante ativação com luz.<br>The Photodynamic Therapy (PDT) combines light, molecular oxygen and a PS for the production of reactive oxygen species (ROS), such as 1O2 and free radicals, which will induce oxidative stress and, eventually, cell death. This allows for the non-invasive, selective and localized destruction of tumor cells with reduced side effects. Phthalocyanines (Pcs) are promising compounds to be applied as PSs for singlet oxygen generation. However, a major drawback of these compounds is their low solubility in physiological media. This can be overcome through functionalization of the macrocycle at the peripheral and/or axial positions with appropriate hydrophilic functions, such as carbohydrates and polyether chains. The introduction of axial ligands reduces their aggregation in solution, thus improving their 1O2 generation efficiency. This work describes the synthesis of Ruthenium Phthalocyanines (RuPcs) to be applied as photosensitizers (PSs) for singlet oxygen generation. These RuPcs are endowed with suitable axial ligands, providing the required solubility and/or selectivity. In this respect, several pyridine or phosphine-based structures bearing charged functions, polyether chains, carbohydrate units or folic acid units were synthesized and further coordinated to the central ion of RuPcs. In addition, solubility in water is enhanced through peripheral functionalization with polyether chains. The photophysical properties of the prepared PSs, namely their solubility in water and their ability to generate singlet oxygen were studied. Furthermore, the PSs were also evaluated in vitro in bladder cancer cells with respect to their capability to be internalized by cancer cells and their toxic effects, both in the dark and upon activation by light.<br>La Terapia Fotodinámica combina luz, oxígeno molecular y un fotosensibilizador para la producción de especies reactivas de oxígeno, como el 1O2 y radicales libres, los cuales inducen estrés oxidativo y, eventualmente, la muerte celular. Esto permite así la destrucción de células tumorales de una forma no invasiva, selectiva y localizada, con efectos secundarios reducidos. Las ftalocianinas muestran un gran potencial para su aplicación como fotosensibilizadores para la producción de oxígeno singlete. Sin embargo, un inconveniente de estos compuestos es su solubilidad reducida en medio acuoso. Esto puede superarse a través de su funcionalización en las posiciones periféricas y/o axiales con grupos hidrofílicos apropiados, como por ejemplo hidratos de carbono y cadenas poliéter. La introducción de ligandos axiales reduce la agregación de las ftalocianinas en disolución, mejorando su eficiencia para producir 1O2. Este trabajo describe la síntesis de Ftalocianinas de Rutenio (RuPcs) para su utilización como fotosensibilizadores en la producción de oxígeno singlete. Estas RuPcs están funcionalizadas en las posiciones axiales ligandos apropriados que proporcionan la solubilidad y/o selectividad requeridas para una aplicación en Terapia Fotodinámica. En este sentido, se sintetizaron diferentes derivados de piridina y fosfina funcionalizados con grupos cargados, cadenas poliéter, hidratos de carbono o con unidades de ácido fólico, las cuales fueron posteriormente coordinadas al ión central de RuPcs. Además, se mejoró la solubilidad de las RuPcs mediante la introducción de cadenas poliéter en las posiciones periféricas. Se estudiaron las propiedades físicas y fotofísicas, en particular, la solubilidad en agua y la capacidad para producir oxígeno singlete, de los fotosensibilizadores preparados. Además, todos los compuestos se evaluaron in vitro en células del cáncer de vejiga urinaria, con respecto a su capacidad para ser internalizados por las células cancerígenas y a sus efectos tóxicos, tanto en la oscuridad como mediante la activación con luz
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5

Torra, Nonell Joaquim. "Flavin-binding fluorescent proteins as genetically encoded singlet oxygen photosensitizers." Doctoral thesis, Universitat Ramon Llull, 2018. http://hdl.handle.net/10803/659081.

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Aquesta tesi profunditza en l'estudi de les proteïnes fluorescents que uneixen flavina com a fotosensibilitzadors d'oxigen singlet codificables genèticament per a la teràpia fotodinàmica. Els fotosensibilitzadors biològics són una alternativa poderosa als fàrmacs convencionals sensibles a la llum degut a la major especificitat front a cèl·lules sanes i a la capacitat d’acumular-se preferentment en orgànuls crítics, gràcies al control genètic de l'expressió cel·lular. Una nova família de proteïnes fluorescents que contenen mononucleòtid de flavina com a cromòfor ha despertat molt d’interès ja que produeixen majors quantitats d'oxigen singlet que la proteïna verda fluorescent i els seus derivats. En aquest treball s'han avaluat les propietats fotofísiques, fotosensibilitzants i antimicrobianes d'onze flavoproteïnes provinents de diferents organismes. Totes les proteïnes estudiades són capaces de produir oxigen singlet i la majoria són altament fototòxiques quan s'expressen en cèl·lules E. coli. Malgrat que comparteixen el mateix cromòfor, les propietats fotofísiques de les proteïnes difereixen notablement d'una a l'altra. Per exemple, algunes són fotosensibilitzadors eficients i són capaces de provocar la mort de cèl·lules bacterianes però fotoblanquegen ràpidament. D’altres produeixen petites quantitats d'oxigen singlet però exhibeixen una elevada fotostabilitat. Els resultats presentats en aquest treball amplien el ventall de flavoproteïnes fotoactives i proporcionen una guia útil per triar la millor opció per a cada aplicació. MiniSOG va ser la primera flavoproteïna desenvolupada racionalment amb l’objectiu de produir d'oxigen singlet. Des de llavors ha estat emprada en nombrosos estudis, però les seves propietats fotoquímiques són complexes i encara resten qüestions pendents de resoldre. Mitjançant estudis estructurals i espectroscòpics s’ha aconseguit racionalitzar la seva modesta producció d’oxigen singlet, dilucidar les transformacions que experimenta quan s’irradia amb llum i establir una base científica sòlida pel desenvolupament racional de nous fotosensibilitzadors d'oxigen singlet codificats genèticament. Així mateix, s'han caracteritzat nous mutants de miniSOG més eficients, així com heterodímers de flavoproteïnes que combinen propietats fotofísiques complementàries. També s’ha demostrat que miniSOG i certs mutants indueixen selectivament la mort de cèl·lules de mamífer quan s’il·luminen amb llum blava, i que és possible combinar proteïnes fotoactives amb reporters fluorescents de processos cel·lulars. L'aplicació de la teràpia fotodinàmica pel tractament del melanoma continua sent un dels principals reptes biomèdics. En aquest estudi, s’ha proposat utilitzar miniSOG per tractar cèl·lules de melanoma. Lamentablement, no ha estat possible comprovar l’expressió correcta de la proteïna fotoactiva, pel que no s’ha pogut avaluar la idoneïtat de la proposta ni extreure’n conclusions.<br>Esta tesis profundiza en el estudio de proteínas fluorescentes que unen flavina como compuestos fotosensibilizadores de oxígeno singlete codificables genéticamente para su uso en terapia fotodinámica. Los fotosensibilizadores biológicos son una poderosa alternativa a los fármacos convencionales sensibles a la luz debido a su mayor especificidad frente a células sanas y a su capacidad para acumularse en orgánulos críticos, gracias al control genético de la expresión celular. Una nueva familia de proteínas fluorescentes que encapsulan el mononucleótido de flavina ha despertado un gran interés por su mayor capacidad de generar oxígeno singlete en comparación con la proteína verde fluorescente y sus derivados. En este trabajo se han evaluado las propiedades fotofísicas, fotosensibilizantes y antimicrobianas de once flavoproteínas provenientes de diferentes organismos. Todas ellas producen oxígeno singlete y la mayoría son altamente fototóxicas una vez expresadas en células E. coli. Aunque comparten el mismo cromóforo, las propiedades fotofísicas difieren notablemente de una a otra proteína. Por ejemplo, algunas son fotosensibilizadores eficientes y destruyen las células bacterianas, pero fotoblanquean rápidamente. Otras producen pequeñas cantidades de oxígeno singlete, pero son más fotoestables. Los resultados obtenidos amplían el abanico de flavoproteínas fotoactivas disponibles y proporcionan una guía útil para elegir la mejor opción para cada aplicación. MiniSOG fue la primera flavoproteína racionalmente desarrollada para generar oxígeno singlete. A pesar de haber sido analizada en numerosos estudios, su fotoquímica es compleja y hay observaciones controvertidas e incógnitas que restan por resolver. Su caracterización estructural y espectroscópica ha permitido comprender los factores que limitan su modesta producción de oxígeno singlete, dilucidar las transformaciones que sufre bajo irradiación y establecer una base científica sólida para el desarrollo de nuevos fotosensibilizadores codificados genéticamente. Además, se han caracterizado nuevos mutantes de miniSOG más eficientes así como heterodímeros de flavoproteínas que combinan propiedades fotofísicas complementarias. También se ha demostrado que miniSOG y ciertos mutantes inducen selectivamente la muerte de células de mamífero cuando se iluminan con luz azul y que es posible combinar proteínas fotoactivas con reporteros fluorescentes de procesos celulares. La aplicación de la terapia fotodinámica para el tratamiento del melanoma sigue siendo uno de los principales desafíos en la biomedicina. En este estudio se ha propuesto la expresión de miniSOG para tratar celulas de melanoma. Sin embargo, no ha sido posible comprobar la expresión correcta de la proteína fotoactiva, por lo que no se ha podido evaluar la idoneidad del enfoque ni sacar conclusiones adicionales.<br>This thesis reports the study of flavin-binding fluorescent proteins as genetically encodable singlet oxygen photosensitizers in photodynamic therapy. Biological photosensitizers are a powerful alternative to conventional light-sensitive drugs owing to their superior targeting potential and localized accumulation in critical organelles, conferred by the genetic control of cell expression. A novel family of fluorescent proteins encasing flavin mononucleotide as the chromophore is gaining much attention since flavoproteins produce higher amounts of singlet oxygen than the proteins derived from the green fluorescent protein family. In this work, the photophysical, photosensitizing and antimicrobial properties of eleven flavoproteins derived from different organisms have been evaluated. All the proteins studied are capable of producing singlet oxygen and most of them are highly phototoxic when expressed in E. coli cells. Although they encase the same chromophore, their photophysical properties differ remarkably from one protein to another. For example, some of them are efficient photosensitizers and kill bacterial cells but show rapid photobleaching. Others produce little amounts of singlet oxygen but exhibit high photostability. The results presented herein expand the toolbox of photoactive flavoproteins and provide valuable guides for choosing the best candidate for a given application. MiniSOG was the first flavoprotein rationally developed for singlet oxygen applications. Since then, it has been a matter of intense research; however, its photochemistry is complex and a number of controversial observations and fundamental questions remain unexplained. The combination of structural and spectroscopic studies has allowed to rationalize its modest singlet oxygen production, elucidate the transformations that it undergoes upon photolysis and establish a sound scientific basis for the rational design and development of new genetically encoded singlet oxygen photosensitizers. Novel miniSOG mutants with improved photosensitizing properties have been characterized as well as flavoprotein heterodimers that combine complementary photophysical properties. It has also been demonstrated that miniSOG and selected mutants induce mammalian cell killing selectively upon light exposure and that it is possible to combine photoactive proteins with fluorescent cell reporters. The application of photodynamic therapy for the treatment of melanoma remains one of the main challenges in the biomedical sciences. In this study, miniSOG has been selected to test the suitability of the genetically encoded approach in melanoma cell lines. Unfortunately, it has not been possible to ascertain the correct expression of the photoactive protein, which has prevented further studies and the possibility of drawing conclusions.
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Andrasik, Stephen James. "Singlet Oxygen Generation Using New Fluorene-Based Photosensitizers Under One- and Two-Photon Excitation." Doctoral diss., University of Central Florida, 2007. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2210.

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Molecular oxygen in its lowest electronically excited state plays an important roll in the field of chemistry. This excited state is often referred to as singlet oxygen and can be generated in a photosensitized process under one- or two-photon excitation of a photosensitizer. It is particularly useful in the field of photodynamic cancer therapy (PDT) where singlet oxygen formation can be used to destroy cancerous tumors. The use of two-photon activated photosensitizers possesses great potential in the field of PDT since near-IR light is used to activate the sensitizer, resulting in deeper penetration of light into biological tissue, less photo-bleaching of the sensitizer, and greatly improved resolution of excitation. The synthesis and photophysical characterization of new fluorene-based photosensitizers for efficient singlet oxygen production were investigated. The spectral properties for singlet oxygen production were measured at room temperature and 77 K. Two-photon absorption (2PA) cross-sections of the fluorene derivatives were measured by the open aperture Z-scan method. The quantum yields of singlet oxygen generation under one- and two-photon excitation ([Phi][sub Delta] and 2PA[Phi][sub Delta], respectively) were determined by the direct measurement of singlet oxygen luminescence at &#8776; 1270 nm. The values of [Phi][sub Delta] were independent of excitation wavelength, ranging from 0.6 - 0.9. The singlet oxygen quantum yields under two-photon excitation were 2PA[Phi][sub Delta] &frac12[Phi][sub Delta, indicating that the two processes exhibited the same mechanism of singlet oxygen production, independent of the mechanism of photon absorption.<br>Ph.D.<br>Department of Chemistry<br>Sciences<br>Chemistry PhD
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7

Planas, Marquès Oriol. "Novel approaches to singlet oxygen photosensitization in the nano- and bio-era." Doctoral thesis, Universitat Ramon Llull, 2017. http://hdl.handle.net/10803/404841.

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En aquesta tesis es detallen noves aproximacions nano- i biomoleculars amb l’objectiu de millorar la fotosensibilització i detecció d’oxigen singlet en medi biològic. En primer lloc es presenta una nova reacció fluoro- i cromogènica pel marcatge de proteïnes i nanopartícules amb derivats de porficè. Concretament, la reacció de porficens isotiocianat amb amines primàries i secundaries generen 2-aminotiazolo[4,5-c]poricè derivats amb un desplaçament concomitant en els seus espectres d’absorció i emissió per més de 70 nm cap al vermell. Aquesta reacció inesperada ha estat racionalitzada en base a la ciclació espontània del derivat 9-tiourea porficè generat en la reacció per donar lloc a un tiazoloporficè fusionat. L’abast de tal reacció s’ha expandit a altres derivats de porficè com els 9-amidoporficens. Finalment, s’ha utilitzat porficens isotiocianat pel marcatge de biomolècules i nanopartícules, lliurant conjugats teranòstics al infraroig proper. En segon lloc s’ha estudiat l’efecte de la morfologia de les nanopartícules se sílice sobre la capacitat de producció i desactivació d’oxigen singlet per part de la protoporfirina IX. L’agregació del fotosensibilitzador en la superfície de la nanopartícula limita la producció d’oxigen singlet. Per altra banda, a mesura que augmenta la porositat del nanomaterial es possible detectar diferents poblacions d’oxigen singlet; una d’elles és capaç d’escapar de la superfície de les nanopartícules i de decaure en el dissolvent mentre que l’altra es manté atrapada dins dels porus de sílice. Els resultats revelen que controlar el grau d’agregació del fotosensibilitzador i la seva localització en la matriu de sílice són factors crítics a tenir en compte en la preparació de materials nanoporosos per a l’alliberació d’oxigen singlet. Finalment s’han dissenyat i estudiat fotosensibilizadors plasmònics capaços d’augmentar la producció i el decaïment radiant del oxigen singlet. Mitjançant tècniques de espectroscòpia estacionària i resoltes en el temps es demostra que el nucli de plata exerceix un efecte dual a través d’una absorció incrementada de llum i un augment en el decaïment radiant d’oxigen singlet, fet que es tradueix en un augment de la seva fosforescència. A més a més es demostra que l’augment en la producció i en el decaïment radiant d’oxigen singlet depèn enormement de la proximitat entre fotosensibilitzador i nanopartícula plasmònica. D’aquesta manera s’han identificat 3 règims diferents a mesura que el fotosensibilitzador s’allunya de la nanoestructura plasmónica, essent la zona de major augment aquella compresa en distancies entre 10 i 20 nm del nucli metàl·lic. Addicionalment s’han utilitzat les nanopartícules per el tractament fotodinàmic de bactèries Gram-positives i Gram-negatives, observant un augment tant de la seva activitat antimicrobiana com en el límit de detecció d’oxigen singlet.<br>En esta tesis se detallan nuevas aproximaciones nano- y biomoleculares para mejorar la fotosensibilización y detección del oxígeno singlete en medio biológico. En primer lugar se presenta una nueva reacción fluoro- y cromogénica para el etiquetado de proteínas y nanopartículas con derivados de porficeno. Concretamente, la reacción de porficenos isotiocianato con aminas primarias y secundarias genera 2-aminotiazolo[4,5-c]porficeno derivados con un desplazamiento concomitante en sus espectros de absorción y emisión por más de 70 nm. Esta reacción inesperada ha sido racionalizada en base a la ciclación espontánea del derivado 9-tiourea porficeno generado tras la reacción para dar lugar a un tiazoloporficeno fusionado. El alcance de dicha reacción ha sido expandido a otros derivados de porficeno como los 9-amidoporficenos. Finalmente, los porficenos isotiocianato han sido usados para el marcaje de biomoléculas y nanopartículas, dando nanoconjugados teranósticos en el infrarrojo cercano. En segundo lugar, se ha sido estudiado el efecto de la morfología de las nanopartículas de sílice sobre la capacidad de producción y desactivación de oxígeno singlete por protoporfirina IX. La agregación del fotosensibilizador en la superficie de la nanopartícula limita la producción de oxígeno singlete. Por otro lado, a medida que aumenta la porosidad del nanomaterial es posible detectar distintas poblaciones de oxígeno singlete; una de ellas capaz de escapar de la superficie de las nanopartículas y decaer en el disolvente mientras que la otra se mantiene atrapada dentro de los poros de sílice. Los resultados revelan que controlar el grado de agregación del fotosensibilizador y su localización en la red de sílice son factores críticos a tener en cuenta en la preparación de materiales nanoporosos para liberación de oxígeno singlete. Finalmente se han diseñado y estudiado fotosensibilizadores plasmónicos capaces de aumentar la producción y el decaimiento radiante del oxígeno singlete. Mediante técnicas de espectroscopia estacionaria y resuelta en el tiempo se demuestra que el núcleo de plata ejerce un efecto dual mediante la absorción incrementada de luz y el aumento del decaimiento radiante del oxígeno singlete, lo que se traduce en un mayor aumento de su fosforescencia. Además se demuestra que el aumento en la producción y en decaimiento radiante del oxígeno singlete depende enormemente de la proximidad del fotosensibilizador a la nanopartícula plasmónica. Así, se han identificado 3 regímenes distintos a medida que el fotosensibilizador se aleja de la nanoestructura plasmónica, situándose el máximo factor de aumento a distancias entre 10 y 20 nm del núcleo metálico. Adicionalmente, las nanopartículas plasmónicas se han usado en bacterias Gram-positivas y Gram-negativas, observando un aumento tanto de su actividad antimicrobiana como en el límite de detección de oxígeno singlete.<br>Different new molecular, bio- and nano-engineered approaches to enhance the photosensitization and detection of singlet oxygen in biological media are reported in this thesis. First, a novel fluoro-chromogenic click reaction for the labeling of proteins and nanoparticles with porphycene derivatives is presented. Reaction of porphycene isothiocyanate with primary and secondary amines yields 2-aminothiazolo[4,5-c]porphycene derivatives with a concomitant red-shift of their absorption and fluorescence emission spectra by more than 70 nm. This unexpected reaction has been explained on the grounds of a spontaneous cyclization of the initially-formed 9-thiourea porphycene derivative onto a fused thiazoloporphycene and its scope has been expanded to other porphycene derivatives such as 9-amidoporphycenes. Furthermore, porphycene isothiocyanates have been used to label biomolecules and nanoparticles yielding near-infrared theranostic nanoconjugates. Secondly, the role of the morphology of silica nanoparticles onto the production and deactivation of singlet oxygen by protoporphyrin IX has been investigated. Aggregation of the photosensitizer onto the surface of the nanoparticle limits the production of singlet oxygen. In addition, as the porosity of the nanomaterial increases, different populations of singlet oxygen can be detected; one population is able to escape from the nanoparticle surface and decay in the bulk solvent while the other is trapped inside the silica pores. Results reveal that controlling the aggregation state of the photosensitizer and its location is of crucial importance when preparing nanoporous materials for singlet oxygen delivery. Finally, plasmonic photosensitizers capable of boosting the production and radiative decay of single oxygen have been designed and studied. Via time-resolved and steady state spectroscopic techniques, we demonstrate the silver core exerts a dual role of enhancing both the production of singlet oxygen, through enhanced absorption of light, and its radiative decay, which in turn boosts singlet oxygen phosphorescence emission to a greater extent. Furthermore, we show both the enhancement of the production and the emission of singlet oxygen to be dependent on proximity to the plasmonic nanostructure. Three distinct regimes have been identified as the photosensitizer moves apart the plasmonic nanostructure, with greater enhancement at distances between 10 and 20 nm. Moreover, hybrid plasmonic nanoparticles can be delivered to both Gram-positive and Gram-negative bacteria boosting both photoantibacterial activity and detection limit of singlet oxygen in cells.
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8

Joyce, Lauren Elizabeth. "Ru(II), Os(II), and Rh2(II,II) Complexes as Potential Photodynamic Therapy Agents." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1291176129.

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9

Yukruk, Funda. "Water Soluble Green Perylenediimide (pdi) Dyes As Potential Sensitizers For Photodynamic Therapy." Phd thesis, METU, 2004. http://etd.lib.metu.edu.tr/upload/3/12605693/index.pdf.

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Photodynamic therapy has been established as one of the approaches for the treatment of various malignant tumors. While most of the reagents used for this purpose are porphyrin derivatives, there is a strong motivation for finding novel and better sensitizers. Perylenediimides are known for their photo- and chemical stability, but they do not have absorptions in the red end of the visible spectrum. However, recently reported green perylenediimides which have dialkylamino substituents on the perylene core, provide an alternative. To that end, we have designed and synthesized novel green perylenediimides with remarkable water solubility at neutral pH and absorption peaks beyond 650 nm. We demonstrated that on red-light excitation, singlet oxygen trap 1,3-diphenyl-iso-benzofuran is rapidly degraded. We also carried out cell culture experiments<br>an important parameter to be optimized for practical application as a novel photodynamic therapy agent was the excited dye toxicity to dark toxicity. Our results confirmed that these novel perylenediimides acted as sensitizers generating singlet oxygen and the initial in vitro biological experiments demonstrated their potential utility in photodynamic therapy.
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Atilgan, Serdar. "Water Soluble Distyryl-boradiazaindacenes As Efficient Photosensitizers For Photodynamic Therapy." Master's thesis, METU, 2006. http://etd.lib.metu.edu.tr/upload/12607601/index.pdf.

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Photodynamic therapy (PDT) is an emerging treatment modality for a range of disease classes, both cancerous and noncancerous. This has brought about an active pursiut of new PDT agents that can be optimized for the unique set of photophysical characteristics that are required for a succesful clinical agent. There are many reported or commercially available photosensitizers, but most have limitations, such as low photostability, or a limited usable range of solvent conditions. In this study, we introduced a novel class of extended conjugation water soluble boradiazaindacene dyes which are efficient singlet oxygen generators. These sensitizers have strong absorptions in the therapeutic window and have spectacular photoinduced cytotoxicity. In addition, they display no dark toxicity at the active concentrations. With these remarkable properties, they are likely to find applications as promising new reagents for photodynamic therapy.
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Dost, Zeynep. "Efficient Synthesis Of Novel Near Ir Emitting Distyrylboradiazaindacene Sensitizers For Photodynamic Therapy." Master's thesis, METU, 2006. http://etd.lib.metu.edu.tr/upload/2/12607377/index.pdf.

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Photodynamic therapy (PDT) is a noninvasive method of treating malignant tumors and age-related macular degeneration. Current practice of PDT is limited to a few functionalized porphyrins, however these compounds are not considered to be ideal drugs for use in PDT. Among the limitations, the most prominent is the low extinction coefficient of porphyrins in the body&amp<br>#8217<br>s therapeutic window. Therefore, there is a significant impetus to develop novel and better efficiency sensitizers for use in PDT. Boradiazaindacenes (BODIPY dyes or difluoroboradipyrrines) are well known fluorescent dyes. We discovered novel distyryl-derivatized boradiazaindacene dyes. These dyes have strong absorptions beyond 650nm. In order to transform these novel dyes into potential PDT reagents, bromine substituents were placed and then heavy atom effect was showed. We also demonstrated that on red-light excitation, singlet oxygen trap 1,3-diphenyl-iso-benzofuran is rapidly degraded.
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12

Jiménez, Banzo Ana María. "New insights in photodynamic theraphy: production, diffusion and reactivity of singlet oxygen in biological systems." Doctoral thesis, Universitat Ramon Llull, 2008. http://hdl.handle.net/10803/9310.

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S'ha estudiat la cinètica de fotosensibilització de l'oxigen singlet (1O2) en cèl·lules eucariotes en suspensió mitjançant un espectròmetre d'última generació amb resolució temporal per sota del microsegon. Els estudis revelen que la cinètica del 1O2 depèn del seu lloc de formació. Per una banda, la producció del 1O2 es més lenta en els lisosomes que en el nucli. Per altra banda, el 1O2 es capaç d'escapar de les cèl·lules quan es fotosensibilitza en el nucli, però es queda confinat al interior si es fotosensibilitza en els lisosomes. Malgrat que el temps de vida del 1O2 es troba en els microsegons, la desactivació principal ve donada per interaccions amb les biomolècules característiques de cadascú dels orgànuls. <br/><br/>La incertesa respecte a la producció de 1O2 en un orgànul determinat es pot eliminar mitjançant l'ús de fotosensibilitzadors modificats genèticament, ja que aquets poden ésser expressats selectivament. Amb aquesta finalitat, s'avaluen les propietats fotosensibilitzants de mutants de proteïna fluorescent verd (GFP). Algunes de les GFPs estudiades sensibilitzen la formació del 1O2 malgrat amb baixa eficiència. Els resultats obtinguts es comparen amb els del cromòfor de la GFP i mostren que l'estructura proteínica, a sobre de modular les propietats fotofísiques del cromòfor, també el protegeix de la desactivació col·lisional. <br/><br/>Finalment, s'estudien les propietats d'absorció bifotónica del 2,7,12,17-tetrafenilporficé i del seu complex de pal·ladi (II). L'eficiència de formació del 1O2 per part dels dos compostos, desprès de l'absorció simultània de dos fotons, es aproximadament 100 vegades superior a la dels seus anàlegs porfirínics, amb seccions d'absorció bifotòniques &#948; ~ 25 GM. Les excel·lents propietats d'aquestos compostos s'expliquen mitjançant arguments qualitatius i s'analitzen les seves perspectives de cara al seu ús en teràpia fotodinámica.<br>Se ha estudiado la cinética de fotosensibilización de 1O2 en células eucariotas en suspensión, usando un espectrómetro de última generación con resolución temporal por debajo del microsegundo. Los estudios revelan que la cinética del 1O2 depende de su lugar de formación. Por una parte, la producción de 1O2 es más lenta en los lisosomas que en el núcleo. Por otra parte, el 1O2 es capaz de escapar de las células cuando es fotosensibilizado en el núcleo, mientras que queda confinado en el interior si se fotosensibiliza en los lisosomas. A pesar de que el tiempo de vida del 1O2 se encuentra en los microsegundos, la desactivación principal viene dada por interacciones con las biomoléculas características de cada orgánulo. <br/><br/>La incertidumbre respecto a la producción de 1O2 en un orgánulo determinado puede ser eliminada mediante el uso de fotosensibilizadores modificados genéticamente ya que pueden ser expresados selectivamente. Con este fin, se evalúan las propiedades fotosensibilizantes de mutantes de proteína fluorescente verde (GFP). Algunas de las GFPs estudiadas sensibilizan la formación de 1O2 aunque con baja eficiencia. Los resultados obtenidos se comparan con los del cromóforo de la GFP y muestran que la estructura proteínica, además de modular las propiedades fotofísicas del cromofóro, también lo protege de la desactivación colisional. <br/><br/>Finalmente, se estudian las propiedades de absorción bifotónica del 2,7,12,17-tetrafenilporficeno y de su complejo de paladio (II). La eficacia de formación de 1O2 de ambos compuestos, tras la absorción simultánea de dos fotones, es aproximadamente 100 veces superior a la de sus análogos porfirínicos, con secciones de absorción bifotónica &#948; ~ 25 GM. Las excelentes propiedades de estos compuestos se explican mediante argumentos cualitativos y se analizan sus perspectivas de cara a su uso en terapia fotodinámica.<br>The kinetics of singlet oxygen (1O2) photosensitisation in human skin fibroblasts have been investigated by means of an ultrasensitive near-infrared spectrometer with submicrosecond time resolution. The results indicate that the 1O2 kinetics are site-dependent. On one hand, the production of 1O2 is slower in the lysosomes than in the nucleus. On the other hand, 1O2 is able to escape out of the cells when photosensitised in the nucleus, while 1O2 photosensitized in the lysosomes is confined. Despite showing a lifetime in the microsecond time domain, the decay of 1O2 is governed by interactions with the biomolecules within the organelle there it is produced. <br/><br/>The uncertainty as to the intracellular site of 1O2 production may be removed by the use of genetically-encoded photosensitisers, which can be expressed in any desired organelle. Towards this end, the ability of some fluorescent proteins (GFPs) to photosensitise 1O2 has been studied. Some of the studied proteins are able to produce 1O2 albeit with a very low quantum yield. The results obtained are compared to those of the synthetic GFP chromophore and indicates that the protein scaffold not only plays a role in modulating the photophysical properties of the chromophore but also has a protective function from collisional quenching. <br/><br/>Finally, the two-photon absorption properties of tetraphenylporphycene and its palladium (II) complex have been determined. These compounds are ca. 100-fold more efficient two-photon 1O2 photosensitisers than their isomeric porphyrin counterparts, with two-photon absorption cross sections &#948; ~ 25 GM. Qualitative symmetry-based arguments are provided to explain the excellent two-photon properties and the prospects for photodynamic therapy are discussed.
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Kandoth, Noufal. "Design, Synthesis and Characterization of Photoactivable Cyclodextrin-Based Nanoparticles for Multimodal Anticancer Therapy." Doctoral thesis, Università di Catania, 2013. http://hdl.handle.net/10761/1280.

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Abstract The general goal of this thesis is the development of new generation photoactivable smart nanomaterials that can be modulated into tremendous way to achieve multimodal anticancer and antimicrobial therapeutic actions. Here the cyclodextrins act as the building block for the development of the nanocarrier in order to convey the therapeutic payload to the desired site. With this goal in mind, we have developed several kinds of nanosystems and are discussed in each chapter. The entire dissertation is divided into four sections based on the types of materials and the sections are further divided into twelve chapters based on the functional behavior of each nanosystems.
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Ozkaya, Ahmadov Tevhide. "Development of Multifunctional Nanoparticles: From Synthesis to Theranostic Applications." University of Cincinnati / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1463130977.

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15

Kholikov, Khomidkhodzha. "Enhanced Singlet Oxygen Generation and Antimicrobial Activity of Methylene Blue Coupled with Graphene Quantum Dots as an Effective Photodynamic Therapy Agent." TopSCHOLAR®, 2018. https://digitalcommons.wku.edu/theses/3059.

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Growing resistance of bacteria towards antibiotics resulted in extensive research effort for development and application of new materials and techniques. Due to their unique properties, graphene quantum dots (GQDs) have attracted much attention and are a promising material with potential applications in many fields. One use of GQDs is as a photodynamic therapy agent that generates singlet oxygen. In this work, GQDs synthesized by focusing nanosecond laser pulses into a mixture of benzene and nickel(II) oxide were combined with methylene blue (MB) to eradicate Gram-negative Escherichia coli and Gram-positive Micrococcus luteus. Theoretical calculation of pressure evolution was calculated using the standard finite difference method. Detailed characterizations were performed with transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier-transform infrared (FTIR), UV-Visible (UV-Vis), and photoluminescence (PL) spectra. Furthermore, singlet oxygen generation from MB-GQD mixture was investigated by measuring the rate of 9,10-anthracenediyl-bis(methylene) dimalonic acid photobleaching at 400 nm. Combining MB with GQDs caused enhanced singlet oxygen generation, leading to improved bacterial deactivation rate. The (3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide) (MTT) assay was used to determine if GQDs in dark conditions caused human cellular side-effects and affected cancer and noncancer cellular viability. We found that even high concentrations of GQDs do not alter viability under dark conditions. These results suggest that the MB-GQD combination is a promising photodynamic therapy agent that may be useful when antibiotics resistance is present.
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Grabow, Wade William. "METHOD FOR DETERMINATION OF SINGLET OXYGEN QUANTUM YIELDS FOR NEW FLUORENE-BASED PHOTOSENSITIZERS IN AQUEOUS MEDIA FOR THE ADVANCEMENT OF PHOTODYNAMIC THERAPY." Master's thesis, University of Central Florida, 2004. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4445.

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Photodynamic therapy (PDT) has been investigated over the past three decades and is currently an approved therapeutic modality for skin cancer, the treatment of superficial bladder, early lung and advanced esophageal cancers, and age-related macular degeneration in a number of countries. In PDT, the absorption of light by a chromophore generates cytotoxic species such as reactive singlet oxygen, leading to irreversible destruction of the treated tissue. The measurement of the singlet oxygen quantum yield is an important determinant used to evaluate the efficiency of new photodynamic therapy agents developed in the laboratory, to screen potential photosensitizers in aqueous media.The singlet oxygen quantum yield is a quantitative measurement of the efficiency in which photosensitizers are able to use energy, in the form of light, to convert oxygen in the ground state to the reactive species singlet oxygen useful in photodynamic therapy. Singlet oxygen quantum yields of photosensitizers differ when measured in different solvents. The majority of the existing quantum yield values found in literature for various photosensitizers are documented with the sensitizers in organic solvents though values in aqueous media are more valuable for actual applications. Determination of accurate and precise quantum yield values in aqueous solution is a much more difficult problem than in organic media. Problems in aqueous solution arise primarily from the physicochemical properties of singlet oxygen in water. Singlet oxygen has a much shorter lifetime in water than it does in organic solvents, causing challenges with respect to quantitative detection of singlet oxygen.The ensuing pages are an attempt to explore the theory and document the procedures developed to provide the accurate measurement of singlet oxygen in aqueous media. Details of this experimental method and singlet oxygen quantum yield results of new compounds relative to established photosensitizers will be presented.<br>M.S.<br>Department of Chemistry<br>Arts and Sciences<br>Chemistry
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17

García, Díaz María. "Drug delivery in photodynamic therapy: From pharmaceutics to animal testing." Doctoral thesis, Universitat Ramon Llull, 2012. http://hdl.handle.net/10803/81987.

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S'ha estudiat el desenvolupament de fotosensibilitzadors i la seva formulació en teràpia fotodinàmica. S'han caracteritzat les propietats fotofísiques dels fotosensibilitzadors porficènics. S'han proposat diferents estratègies tals com la introducció de grups carboxilat en la perifèria o ions de metalls pesants en el nucli, per millorar el disseny de nous fotosensibilitzadors basats en el macrocicle porficènic. Entre ells, el temocè (m-THPPo), el porficè anàleg a la temoporfina, mostra excel•lents propietats fotofísiques, fotoestabilitat i alta eficàcia fotodinàmica. A causa de la seva alta hidrofobicitat, s'ha desenvolupat una formulació liposomal per a l'administració in vitro i in vivo del temocè. m-THPPo/DPPC/DMPG (1:67.5:7.5 relació molar) té alta eficiència d'encapsulació mantenint les seves propietats tant fotofísiques com a biològiques. El temocè liposomal va exhibir l'eficàcia fotodinàmica in vitro més alta per molècula internalitzada, sent un sistema d'administració de fàrmacs eficaç per a una estratègia in vivo dirigida a les cèl•lules tumorals. El temocè encapsulat en micel•les de Cremophor EL va mostrar una mínima internalització cel•lular. Consistentment, la formulació micel•lar va mostrar millor la resposta in vivo quan s'utilitza en un règim vascular. Amb la finalitat de minimitzar la internalització del fotosensibilitzador en les cèl•lules normals, es van proposar liposomes decorats amb lligands folat. Aquesta estratègia resulta en una internalització dues vegades major dels liposomes dirigits al receptor folat respecte a la corresponent formulació no específica. Finalment, han estat explorats nous models cel•lulars in vitro per a l'optimització dels processos amb oxigen singlet. Els cultius cel•lulars en 3D reprodueixen l'heterogeneïtat d'oxigen i fotosensibilitzador que està present en els teixits reals, proporcionant informació molt útil per interpretar i predir el resultat de la teràpia fotodinàmica. També s'ha demostrat la capacitat de desactivació de l'oxigen singlet d'antioxidants en un model ex vivo de pell porcina.<br>Se ha estudiado el desarrollo de fotosensibilizadores y su formulación en terapia fotodinámica. Se han caracterizado las propiedades fotofísicas de los fotosensibilizadores porficénicos. Se han propuesto diferentes estrategias tales como la introducción de grupos carboxilato en la periferia o iones de metales pesados en el núcleo, para mejorar el diseño de nuevos fotosensibilizadores basados en el macrociclo porficénico. Entre ellos, el temoceno (m-THPPo), el porficeno análogo a la temoporfina, muestra excelentes propiedades fotofísicas, fotoestabilidad y alta eficacia fotodinámica. Debido a su alta hidrofobicidad, se ha desarrollado una formulación liposomal para la administración in vitro e in vivo del temoceno. m-THPPo/DPPC/DMPG (1:67.5:7.5 relación molar) tiene alta eficiencia de encapsulación manteniendo sus propiedades tanto fotofísicas como biológicas. El temoceno liposomal exhibió la eficacia fotodinámica in vitro más alta por molécula internalizada, siendo un sistema de administración de fármacos eficaz para una estrategia in vivo dirigida a las células tumorales. El temoceno encapsulado en micelas de Cremophor EL mostró una mínima internalización celular. Consistentemente, la formulación micelar mostró mejor la respuesta in vivo cuando se utiliza en un régimen vascular. Con el fin de minimizar la internalización del fotosensibilizador en las células normales, se propusieron liposomas decorados con ligandos folato. Esta estrategia resulta en una internalización dos veces mayor de los liposomas dirigidos al receptor folato respecto a la correspondiente formulación no específica. Por último, han sido explorados nuevos modelos celulares in vitro para la optimización de los procesos con oxígeno singlete. Los cultivos celulares en 3D reproducen la heterogeneidad de oxígeno y fotosensibilizador que está presente en los tejidos reales, proporcionando información muy útil para interpretar y predecir el resultado de la terapia fotodinámica. También se ha demostrado la capacidad de desactivación del oxígeno singlete de antioxidantes en un modelo ex vivo de piel porcina.<br>The photosensitizer and formulation development in photodynamic therapy have been studied. They have been characterized the photophysical properties of new porphycene-based photosensitizers. Different strategies such as the introduction of carboxylate groups in the periphery or heavy metal ions in the core have been proposed for improving the design of novel photosensitizers based on the porphycene macrocycle. Among them, temocene (m-THPPo), the porphycene analogue to temoporfin, shows excellent photophysical properties, superior photostability and high photodynamic efficiency. Owing to its high hydrophobicity, a liposomal formulation has been developed for in vitro and in vivo administration of temocene. m-THPPo/DPPC/DMPG (1:67.5:7.5 molar ratio) yielded high encapsulation efficiency maintaining its photophysical and biological properties. Liposomal temocene exhibited the highest in vitro killing efficacy per uptaken molecule and they were an efficient drug delivery system for in vivo tumor cell targeting strategy. Temocene encapsulated in Cremophor EL micelles showed minimal cell internalization. Consistently, micellar formulation showed the best in vivo response when used in a vascular regime. In order to minimize the internalization of the photosensitizer in normal cells, liposomes decorated with folic acid ligands were proposed. This strategy leads to a 2-fold higher uptake of folate-targeted liposomes than the corresponding non-targeted formulation. Finally, new in vitro cellular models for a better optimization of singlet oxygen-involved processes were explored. 3D cellular cultures reproduced the oxygen and photosensitizer heterogeneity found in real tissues, providing useful information to interpret and predict the photodynamic therapy outcome. The singlet oxygen quenching ability of antioxidants in ex vivo porcine skin model has also been demonstrated.
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18

Rodríguez, Amigo Beatriz. "Light-sensitive nanocarriers for drug delivery in photodynamic therapy." Doctoral thesis, Universitat Ramon Llull, 2018. http://hdl.handle.net/10803/462210.

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Aquesta tesi aprofundeix en l’estudi de nanotransportadors com a sistemes de vehiculització i en alguns casos, alliberació de fotosensibilitzadors emprats en teràpia fotodinàmica. S’han fet servir dos nanotransportadors de naturalesa diferent: proteïnes i liposomes. En primer lloc s’ha investigat els complexos formats entre la hipericina i les proteïnes apomioglobina i β-lactoglobulina. S’han estudiat les característiques fisicoquímiques i fotofísiques, avaluant l’activitat antimicrobiana en front a bacteris gram-positius i gram-negatius. En ambdues matrius proteiques el fotosensibilitzador es troba majoritàriament en forma monomèrica, preservant les seves propietats fotofísiques i formant un complex estable. En el cas de la β-lactoglobulina s’estudia a més, la formació del complex amb l’adició d’un 20% de DMSO com a co-solvent, fet que millora les propietats fotofísiques en detriment de la capacitat antimicrobiana. Ambdós complexos proteics son efectius contra bacteris gram-positius però no contra gram-negatius. Per altra banda, es demostra que la hipericina incorporada a la cavitat de l’apomioglobina pot ser utilitzada en microscòpia de super-resolució STED. Amb aquesta tècnica es pot monitoritzar els llocs d’unió del fotosensibilitzador a la membrana dels bacteris. Així mateix, s’estudia l’ús de la β-lactoglobulina com a portador dual d’hipericina i àcid retinoic. En aquest últim sistema multi-component s’avaluen les propietats fotofísiques per a verificar la formació i estabilitat del complex. En segon lloc, es desenvolupa un nanovehicle per la seva aplicació en teràpia combinada en el qual s’incorporen fàrmacs quimioterapèutics convencionals amb agents fotosensibilitzants, per superar resistències i millorar l’eficàcia dels tractaments individuals. Amb aquest objectiu, s’han dissenyat i estudiat dues formulacions liposomals diferents, ambdues amb el mateix fotosensibilitzador però encapsulant diferents agents quimioterapèutics. Es preparen formulacions bimodals on s’incorporen els dos agents al mateix vehicle i els seus homòlegs unimodals, amb la incorporació única d’un dels dos agents. S’han avaluat les característiques fisicoquímiques, fotofísiques i fotobiològiques de les suspensions bimodals i unimodals. La lozalització subcel·lular demostra que cada principi actiu es localitza a orgànuls diferents desencadenant rutes de senyalització cel·lular diferents, eludint els possibles mecanismes de resistència. El tractament in vitro en cèl·lules cancerígenes amb aquests sistemes tenen un efecte prometedor, ja que com a mínim presenten un comportament additiu respecte els tractaments individuals. Finalment, s’ha avaluat el potencial de la vehiculització activa mitjançant la unió covalent d’un anticòs monoclonal a la superfície, el que millora lleugerament els resultats per una de les dues formulacions.<br>Esta tesis profundiza en el estudio de nanotransportadores como sistema de vehiculización y en algunos casos, liberación de fotosensibilizadores empleados en terapia fotodinámica. Se emplean dos nanotransportadores de naturaleza distinta: proteínas y liposomas. En primer lugar se han investigado los complejos formados entre hipericina y las proteínas apomioglobina y β-lactoglobulina. Se han estudiado las características fisicoquímicas y fotofísicas, evaluando la actividad antimicrobiana frente bacterias gram-positivas y gram-negativas. En ambas matrices proteicas el fotosensibilizador se encuentra mayoritariamente en forma monomérica, preservando sus propiedades fotofísicas y formando un complejo estable. En el caso de la β-lactoglobulina se estudia además, la formación del complejo con la adición del 20% de DMSO como co-solvente, lo que mejora las propiedades físicas pero sorprendentemente, empeora la capacidad antimicrobiana. Ambos complejos proteicos son efectivos contra bacterias gram-positivas, pero no contra gram-negativas. Además, se demuestra que la hipericina en la cavidad de la apomioglobina es capaz de realizar microscopía de super-resolución STED, mediante la cual se puede monitorizar los sitios de unión a las bacterias. Asimismo, se ha estudiado la β-lactoglobulina como portador dual de hipericina y ácido retinoico. En este último sistema multi-componente se evalúan las propiedades fotofísicas para verificar la formación y estabilidad del complejo. En segundo lugar, se desarrolla un nanovehículo para su uso en terapia combinada en el que se incorpora fármacos quimioterapéuticos convencionales con agentes fotosensibilizantes, para superar las resistencias y mejorar la eficacia de los tratamientos individuales. Con este objetivo, se han diseñado y estudiado dos formulaciones liposomales diferentes, ambas con el mismo fotosensibilizador, pero con diferentes agentes quimioterapéuticos. Se preparan las formulaciones bimodales con ambos agentes en el mismo vehículo además de sus homólogos unimodales, con la incorporación única de uno de los dos agentes. Se han evaluado las características fisicoquímicas, fotofísicas y fotobiológicas de las suspensiones bimodales y unimodales. La localización subcelular demuestra que cada principio activo se localiza en orgánulos diferentes desencadenando rutas de señalización celular diferentes, eludiendo los posibles mecanismos de resistencia. El tratamiento in vitro en células cancerígenas de estos sistemas tiene un efecto prometedor siendo al menos aditivo en comparación con los tratamientos individuales. Finalmente, se ha evaluado el potencial de la vehiculización activa mediante la unión covalente de un anticuerpo monoclonal en la superficie, lo que lleva a resultados ligeramente superiores para una de las dos formulaciones.<br>This thesis reports the study of nanocarriers as drug delivery systems for photosensitisers in photodynamic therapy. Proteins and liposomes are the two nanovehicles of different nature used for this purpose. Beginning with the proteins, the complexes formed between hypericin and the proteins apomyoglobin and β-lactoglobulin have been explored. The physicochemical and photophysical properties have been studied, as also assessing their photoantibacterial activity against Gram-positive and Gram-negative bacteria. In both protein scaffolds the photosensitiser is found mainly in monomeric form, preserving its fluorescence and singlet oxygen photosensitising properties and yielding a stable complex. In the case of β-lactoglobulin, the complex formation has also been tested with the addition of a 20% DMSO as a co-solvent, which improves the photophysical properties but surprisingly, worsens its antimicrobial activity. Both protein complexes are effective against Gram-positive but not against Gram-negative bacteria. Moreover, it has been proved that hypericin, inside the apomyoglobin cavity, can perform STED microscopy through which its localization in bacteria can be monitored. Additionally, the suitability of β-lactoglobulin as a dual carrier for hypericin and acid retinoic has also been exploited. In this last multi-component system, the photophysical properties have been evaluated to confirm the formation and complex stability. Secondly, a nanocarrier for its use in combined therapy has been developed, in which conventional chemotherapeutic drugs are combined with photosensitising agents to overcome resistance and improve the effectiveness of the individual treatments. For this purpose, two different liposome formulations have been designed and studied with a common photosensitiser but different anti tumour drugs. The bimodal formulations with both agents entrapped and their unimodal counterparts, having each drug loaded in separate liposomes, have been evaluated. The physicochemical, photophysical and photobiological properties of bimodal and unimodal suspensions have been studied. The subcellular localization shows different organelle accumulation by each agent, triggering different key signals transduction pathways, eluding the cellular resistance mechanisms. The treatment in vitro of these multi-component liposomes with cancer cells has a promising effect, since at least an additive outcome is observed when compared with the individual treatments. Finally, we have explored the potential of active targeting strategies by covalently linking a monoclonal antibody to the surface, leading to slightly greater outcomes for one of the liposomal formulations.
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19

Valentine, Ronan. "Biophysical aspects of photodynamic therapy." Thesis, University of St Andrews, 2011. http://hdl.handle.net/10023/2471.

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Photodynamic therapy (PDT) is a multimodality cancer treatment available for the palliation or eradication of systemic and cutaneous malignancies. In this thesis, the application of PDT is for the treatment of non-melanoma skin cancer (NMSC). While PDT has a well-documented track record, there are, at this time no significant indicators to suggest the superiority of one treatment regime over the next. The motivation for this work is to provide additional evidence pertaining to PDT treatment variables, and to assist in optimising PDT treatment regimes. One such variable is the treatment light dose. Determining the light dose more accurately would assist in optimising treatment schedules. Furthermore, choice of photosensitiser pro-drug type and application times still lack an evidence base. To address issues concerning treatment parameters, fluorescence spectroscopy – a valuable optical diagnostic technique – was used. Monitoring the in vivo PpIX fluorescence and photobleaching during PDT was employed to provide information pertaining to the progression of treatment. This was demonstrated by performing a clinical study at the Photobiology Unit, Ninewells Hospital and Medical School, Dundee. Two different photosensitiser pro-drugs – either 5-aminolaevulinic acid (ALA) or its methyl ester (MAL) – were investigated and based on the fluorescence and pain data recorded both may be equally suitable for topical PDT. During PDT, surface fluorescence is observed to diminish with time – due to photobleaching – although cancerous cells may continue to be destroyed deep down in the tissue. Therefore, it is difficult to ascertain what is happening at depth in the tumour. This raised the questions; How long after surface PpIX fluorescence has diminished is the PDT treatment still effective and to what depths below the surface is effective treatment provided? In order to address these important questions, a three-dimensional (3D) Monte Carlo radiation transfer (MCRT) model was used to compute the light dose and the ¹O₂ production within a tumour, and the PpIX fluorescence emission from the tumour. An implicit dosimetry approach based on a single parameter – fluorescence photobleaching – was used in order to determine the ¹O₂ generation, which is assumed to be related to tissue damage. Findings from our model recommended administering a larger treatment light dose, advocating an increase in the treatment time after surface PpIX fluorescence has diminished. This increase may ultimately assist in optimising PDT treatment regimes, particularly at depth within tumours.
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20

Galland, Margaux. "Conception de sondes théranostiques moléculaires impliquand la PDT à excitation biphotonique." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSEN020.

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La thérapie photodynamique (PDT) est une technique thérapeutique qui permet un traitement localisé par irradiation lumineuse d’un photosensibilisateur (PS) grâce à la génération d’une espèce cytotoxique, généralement de l’oxygène singulet. Cependant, de nombreux PS sont également luminescents et les deux processus sont compétitifs. L’emploi de métaux de transition est connu pour améliorer le processus de PDT mais l’impact des ions lanthanides(III) en PDT est encore peu connu. Par ailleurs, l’utilisation de l’absorption biphotonique a de nombreux avantages parmi lesquels la possibilité d’exciter le PS dans la fenêtre de transparence biologique pour des applications en milieux biologiques.Les travaux de cette thèse visent à étudier quel est l’influence de la complexation d’un atome de lanthanide(III) à un PS sur la photophysique de désexcitation de ce dernier. Les complexes synthétisés et ceux étudiés ont montré que l’effet dépend du lanthanide(III). Il est ainsi possible, avec un choix judicieux du métal, de favoriser une voie de désexcitation par rapport à une autre. En particulier, l’ion Gd(III) se révèle avoir un effet bénéfique important pour la génération d’oxygène singulet et cet effet s’ajoute à celui que des atomes lourds comme le brome peuvent avoir. L’ion Yb(III) en revanche, favorise de manière générale le transfert d’énergie par effet d’antenne et la luminescence du lanthanide est alors le processus majoritaire. Enfin, l’emploi de Gd(III) complexé à un PS excitable à deux photons ouvre la voie à des agents théranostiques moléculaires combinant l’IRM en tant que fonction d’imagerie et la PDT pour la thérapie<br>Photodynamic Therapy (PDT) is a therapeutic technique which consists in generating a highly reactive species, generally singlet oxygen, by shining light on a photosensitizer (PS). However, many PS are also luminescent and both processes are competitive. The use of transition metals is well known to enhance the PDT effect, but little is known about the effect of lanthanide(III) metals.On the other hand biphotonic absorption has numerous advantages, among them the possibility to excite the PS in the so-called biological transparency window for biological applications.The aim of this PhD is to get a better comprehension of the effect of complexation of a lanthanide(III) atom with a PS on the photophysics and deactivation pathways of the latter. The synthesis and conducted studies of lanthanide complexes showed that the effect is dependent on which lanthanide(III) metal is used. Thus by choosing carefully the lanthanide metal, one can favor one deactivation pathway over another. In particular, the Gd(III) ion turns out to be very efficient in promoting singlet oxygen generation and its effect is additive to the already known positive effect of heavy atoms such as bromine. On the opposite, the Yb(III) ion mainly favors the energy transfer through the antenna effect and the complex preferentially emits light.Finally, using Gd(III) linked to a two-photon excited PS opens the path to molecular theranostic probes combining MRI as a imagery technique and PDT as a therapeutic one
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21

Ozlem, Suriye. "Towards Practical Applications For Molecular Logic Gates:." Master's thesis, METU, 2008. http://etd.lib.metu.edu.tr/upload/12609566/index.pdf.

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ABSTRACT TOWARDS PRACTICAL APPLICATIONS FOR MOLECULAR LOGIC GATES: &ldquo<br>AND&rdquo<br>LOGIC AS AN ADDITIONAL LAYER OF SELECTIVITY IN SINGLET OXYGEN RELEASE FOR PHOTODYNAMIC THERAPY &Ouml<br>zlem, Suriye M.S., Department of Chemistry Supervisor: Prof. Dr. Engin Umut Akkaya June 2008, 54 pages There have been many examples of individual molecular logic gates and molecular equivalents of more complex digital designs in recent years such as half adder, half subtractor, multiplexer. Neverethless, the unresolved issues of addressability and lack of communication between logic gates remain to be the Achille&rsquo<br>s heel for molecular logic gates. A few years ago we have demonstrated that appropriately decorated bodipy dyes can be very efficient generators for singlet oxygen, thus act as a satisfactory photodynamic agents. As a bonus, these dyes absorb very strongly at 660 nm which is considered to be within the therapeutic window of mammalian tissue. So, combining our earlier experience in molecular logic gates and rational design of photodynamic agents, we proposed a photodynamic therapy agent that would release singlet oxygen at a much larger rate when the cancer related cellular parameters are above a threshold value at the same location. Following the survey of the relevant literature for cancer related parameters, we decided that sodium ion concentration and pH (H+ concentration) could be very promising targets. In the tumor regions the pH can drop below 6 and the Na+ concentration is also significantly higher then normal tissues. As a result, in the proposed logic system the chemical inputs could be Na+ and H+. The system in fact is an automaton which is to seek higher concentration of both hydrogen and sodium ions, and release the toxic agent (singlet oxygen) only when both concentrations are high. Thus, the proposed logic gate is an AND logic gate, the output of which is singlet oxygen. Keywords: Photodynamic therapy, singlet oxygen, molecular logic gates, AND logic operation
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22

Negri, Laísa Bonafim. "Complexos rutênio-ftalocianinas como fotossensibilizadores para terapia fotodinâmica. Aspectos fotoquímicos e fotobiológicos." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/60/60136/tde-17042015-094616/.

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Síntese, aspectos estruturais, propriedades fotoquímicas, fotofísicas e fotoindução para avaliação citotóxica in vitro de derivados de [Ru (Pc)] (PC = ftalocianina) são descritos neste trabalho. O desenvolvimento ftalocianinas utilizando anéis macrocíclicos, substituídos e não substituídos simetricamente, coordenados com o centro metálico de rutênio permitiu a obtenção de diferentes compostos. Coordenação de ligantes derivados de óxido nítrico (NO) na posição axial do rutênio (II) permitiu obter [Ru(NO)(Pc)(NO2)], um complexo liberador de NO quando submetido ao processo de redução. Os complexos foram caracterizados por FTIR, UV-Vis, 1H RMN e espectrometria de massas. As propriedades fotoquímicas de complexos como ftalocianinas de rutênio (II) têm sido investigadas por fotólise a laser em diferentes comprimentos de onda. Na irradiação de luz em 660 nm em solução aquosa aerada, observamos o fotoprocesso com a produção de oxigênio singleto. Pode-se observar a relação do rendimento quântico de oxigênio singleto e dos fotossensibilizadores utilizados para sua produção. O oxigênio singleto para [Ru (Pc-R)] segue a ordem [Ru(Pc)]> [Ru (Pc-DCBz)]> [Ru(Pc-DMX)] com DCBz = ácido 4-(3,4-dicianofenoxi) benzóico e DMX = 4,5-bis (2,5-dimetilfenoxi) ftalonitrilo. Estudos fotobiológicos são destacados com particular enfoque na irradiação de luz em 660 nm com interesse para aplicações de terapia fotodinâmica (TFD). Neste contexto, temos também a avaliação citotóxica em linhagens de células B16F10 do efeito sinérgico entre NO e oxigênio singleto quando comparado ao complexo [Ru(Pc)], um composto que é apenas produtor de oxigênio singleto. O NO parece potencializar a ação de oxigênio singleto uma vez que em ensaios de viabilidade celular, o complexo [Ru(NO)(Pc)(NO2)] é pelo menos 40% mais ativo que [Ru(Pc)], considerando 8,93 J / cm2 como uma potência. Considerando apenas a produção de oxigênio singleto encontramos a espécie [Ru(Pc-DCBz)] mais citotóxica nas linhagens B16F10 e MCF-7, sob irradiação em 660nm. Com base em todos os estudos, concluímos que citotoxicidade é dependente tanto da localização subcelular quanto do rendimento quântico de oxigênio singleto. A citotoxicidade pode ser reforçada pela produção de NO, seguido por irradiação de luz na janela terapêutica.<br>Synthesis, structural aspects, photochemistry, photophysical and in vitro photoinduced cytotoxic properties of [Ru(Pc)] (pc = phthalocyanine) derivatives are described in this work. Use of an unsubstituted or symmetrically substituted phthalocyanine ring coordinated to the central ruthenium allowed to obtain different compounds. Coordination of nitric oxide (NO) derivative ligands on axial position of ruthenium(II) ion permitted to obtain [Ru(NO)(Pc)(NO2)] a NO deliver agent under reduction process. The complexes were characterized by FTIR, UV-Vis, 1H NMR and mass spectrometry. The photochemical properties of (phthalocyanine)ruthenium(II) like complexes have been investigated by laser photolysis at different wavelengths. At 660 nm light irradiation in aerated aqueous solution we observed photoprocesses with singlet oxygen production. We have addressed the relationship of the singlet oxygen quantum yield and the photosensitizers used in its generation. The singlet oxygen for [Ru(Pc-R)] follows the order [Ru(Pc)] > [Ru(Pc-DCBz)] > [Ru(Pc-DMX)] with DCBz = 4-(3,4-dicianofenoxy)benzoic acid and DMX = 4,5-bis (2,5-dimethylfenoxy)ftalonitrile. Photobiological studies are highlighted with particular focus on light irradiation on 660 nm with interest for photodynamic therapy (PDT) applications. In this way we also have compared the synergism effect of NO and singlet oxygen production with [Ru(Pc)] as a compound producer of singlet oxygen only by means of cytotoxicity in B16F10 cell line. The NO seems to leverage the singlet oxygen action once [Ru(NO)(NO2)(Pc)] cell viability is at least 40 % more active than [Ru(Pc)] considering 8.93 J/cm2 as a potency. Considering only singlet oxygen production we found [Ru(Pc-DCBz)] more cytotoxic specie in B16F10 and MCF7 cell lines, under irradiation in 660 nm. Based on all studies, we have concluded that citotoxycity is dependent on subcellular localization as well singlet oxygen quantum yield. The cytotoxicity could be enhanced by NO production followed by light irradiation on the therapeutic window.
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23

Tada, Dayane Batista. "Desenvolvimento de nanopartículas fotossensibilizadoras." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-31012008-080004/.

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No presente trabalho são apresentadas a síntese e a caracterização estrutural, fotofísica, fotoquímica e fotobiológica de nanopartículas contendo os fotossensibilizadores (FS) Azul de Metileno (AM) e Tionina. AM e Tionina foram incorporados nas nanopartículas sil-AM e sil-Tio pelo processo sol-gel. Nas nanopartículas Cab-Tio, Tionina foi ligada à superfície de sílica CabOsil® através de ligação covalente com reagentes bifuncionais. Todas as nanopartículas mostraram-se esféricas e com de diâmetro médio na faixa de 30 a 60nm. A imobilização dos FS induziu a agregação destes em extensões diferentes para cada tipo de nanopartícula. Foi observado que a maior presença de dímeros de FS leva à menor eficiência de geração de 1O2. Constatou-se que as nanopartículas sofrem pouca influência do meio, uma vez que os FS a elas ligadas não sofreram redução química por NADPH, nem supressão do estado tripleto por íons ascorbato e a supressão de fluorescência por íon brometo foi diminuída. Foi testado também o efeito do recobrimento destas nanopartículas com lipídios dioleilfosfatidil colina (DOPC) e fosfatidilglicerol (PG) e com Polietileno glicol (PEG). A adsorção das nanopartículas sobre membranas miméticas foi reduzida após os recobrimentos, resultado que foi explicado pelas interações de carga superficial (potencial zeta) e pela força de hidratação. As nanopartículas sil-AM e Cab-Tio apresentaram fototoxicidades in vitro, 38% e 20% maiores que os respectivos FS livres. A modificação das nanopartículas de sil-AM com lipídios e com PEG diminuiu a fototoxicidade das mesmas e no caso do recobrimento com lipídios levou ao aumento da toxicidade no escuro. Imagens de microscopia confocal mostraram que as nanopartículas com e sem recobrimento de lipídios entram em células B16. No caso das nanopartículas recobertas, observou-se um perfil de distribuição difuso por todo o citoplasma e no caso de nanopartículas sem recobrimento, estas encontraram-se em poucas regiões vacuolares do citoplasma. O perfil de distribuição homogênea por todo o citoplasma no caso de nanopartículas recobertas com lipídios pode ser o responsável pelo aumento de toxicidade no escuro. Concluiu-se que a ligação dos FS em nanopartículas com diferentes graus de agregação pode ser uma estratégia para obtenção de sistemas com capacidade modulada de geração de 1O2 e com reduzida susceptibilidade às composições do meio. As atividades fototóxicas das nanopartículas contra células B16 mostraram que estas podem ser úteis em Terapia Fotodinâmica de Câncer<br>In this work we present the synthesis and the characterization (structural, photophysical, photochemical and photobiological) of nanoparticles with incorporated photosensitizers (PS) Methylene Blue (MB) and Thionin. MB and Thionin were incorporated in sil-MB and sil-Th nanoparticles through sol-gel process. In the case of Cab-Th nanoparticles Thionin was linked to the surface of CabOsil® nanoparticles through cross-linking reactions. All nanoparticles were spherical and presented average diameter in the range of 30 to 60nm. Different extension of PS aggregation was observed in each nanoparticle. It was characterized that the higher the proportion of dimers to monomers the smaller the efficiency of singlet oxygen (1O2) generation. It was shown that nanoparticles can protect PS from external interferences, since NADPH did not reduce them, neither were their triplet state quenched by ascorbate ions. Besides, fluorescence quenching by bromide ions was reduced compared to free PS. The effect of covering the nanoparticles with lipids, i.e., di-oleil phosphatidylcholine (DOPC) and phosphatidylglycerol (PG), and with Polyethylene glycol was also tested. The nanoparticle adsorption over membrane mimics was reduced, which was explained by the interaction among surface charges (zeta potential) and by hydration forces. Sil-MB and Cab-Th nanoparticles presented in vitro phototoxicity 38% and 20% higher than the respective free PS. It was observed that the nanoparticle coating with lipids and with PEG reduced their photoxicity. Nanoparticles coated with lipids showed higher toxicity in the dark. Confocal fluorescence images of B16 cells showed that nanoparticles with or without lipid coating enter the cells. In the case of lipid-coated nanoparticles a diffuse distribution profile was observed and in the case of nanoparticles without coating, they concentrated in specific vacuolar regions of the cytoplasm. The homogeneous cytoplasmic distribution profile of lipid-coated nanoparticles can explain the increased toxicity in the dark. It has been concluded that immobilization of PS with different aggregation degrees is a strategy to obtain systems in which the modulated efficiency of 1O2 generation is not affected by the external medium. Finally, based on the observed in vitro phototoxicity activity against B16 cells, these systems can be useful in Photodynamic Therapy of Cancer
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24

Silva, Érika Ribeiro e. "Estudo da cinética e dos mecanismos da fototransformação de corantes ciânicos com dois cromóforos em interação com sistemas biomiméticos sob a ação da luz visível." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/59/59135/tde-02102015-164515/.

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A Terapia Fotodinâmica (TFD) é um método de tratamento em que um composto fotossensibilizador (FS) é introduzido no organismo do paciente e, posteriormente, a região de tratamento é irradiada com luz. A combinação de oxigênio, luz e FS produz espécies que geram um efeito curativo. Os FS utilizados atualmente, embora eficazes, apresentam desvantagens. Neste trabalho, foram estudados os corantes ciânicos com dois cromóforos ou biscianinas (BCD) que apresentam características favoráveis na aplicação como FS na TFD. O objetivo deste trabalho foi analisar os efeitos da concentração, da composição do ambiente e da própria estrutura de três BCDs, com ângulos entre os seus cromóforos de 180°, 150° e 90°, nas características das suas fototransformações em tampão e na interação com micelas de SDS e DNA. A fototransformação pode causar a perda da fotoatividade do FS e gerar fotoprodutos (FPs) que tanto podem ser tóxicos como fotoativos. Os processos de tratamento na TFD e os de fototransformação são paralelos. Isso permite utilizar o monitoramento do processo de fototransformação para determinar o grau do processo de tratamento em tempo real. Por esse motivo, também foi estudada a fototransformação dos FPs formados durante a irradiação. A análise da fototransformação foi realizada utilizando técnicas espectroscópicas de absorção óptica e de fluorescência estática. Mudanças nos espectros de absorção óptica dos corantes indicaram a formação de agregados. A agregação diminui a velocidade de fototransformação devido ao aumento da probabilidade de dissipação da energia de excitação por processos não radiativos. Não houve formação de agregados na irradiação dos FPs, pois estes possuem menor tendência de agregação em comparação com o próprio corante. Semelhanças nos espectros dos FPs dos três corantes indicaram que eles possuem a mesma estrutura do cromóforo. Na ausência do oxigênio a velocidade de fototransformação diminui. Este fato juntamente com a relação entre os tempos da fotólise e os rendimentos quânticos do estado tripleto dos BCDs sugeriram a participação do oxigênio singleto na fototransformação. Na presença de sistemas biomiméticos em altas concentrações, os corantes estão em suas formas monoméricas. Nestas interações, os corantes também sofreram fototransformação, com velocidades diferentes daquelas em tampão, com formação de fotoprodutos. Na fototransformação dos corantes na presença de micelas de SDS, sugerimos que as moléculas dos corantes ficam mais protegidas do contato do oxigênio. Por outro lado, na interação com o DNA, as moléculas dos corantes ligadas na superfície do DNA se tornam mais suscetíveis ao ataque do oxigênio, fazendo com que a fototransformação seja mais rápida.<br>Photodynamic therapy (PDT) is a treatment that a photosensitizer compound (PS) is inserted into the patient\'s body and, subsequently, the treatment area is irradiated with light. The combination of oxygen, light and FS produce species that generate a curative effect. The FS currently used, although can be effective, have disadvantages. In this study, the cyanine dyes with two chromophores (BCD) or biscyanines that have favorable characteristics in the application as FS in PDT were studied. The aim of this study was to analyze the effects of concentration, environmental composition and the structure of three BCDs, with angles between chromophores 180, 150 and 90 degrees, in the characteristics of phototransformation of the dyes molecules in phosphate buffer and in the interaction with SDS micelles and DNA. The phototransformation can cause the loss of the FS photoactivity and generate photoproducts (PPs) that are both toxic as photoactive. The treatment processes in PDT and phototransformation are parallel. It allows the use of phototransformation monitoring process to determine the degree of realtime treatment process. For this reason, it was also studied the phototransformation of PPs formed during irradiation. The phototransformation analysis was performed using optical absorption and fluorescence spectroscopies. Changes in the optical absorption spectra of the dyes indicated the formation of aggregates. The aggregation reduces phototransformation rate due to the increased probability of dissipation of the excitation energy by non-radiative processes. There was no aggregate formation on irradiation of PPs because they have less tendency to aggregate compared with the dye. Similarities in the spectra of the three PPs indicated that they have the same structure of the chromophore. In the absence of oxygen phototransformation rate decreases. This fact together with the relationship between photolysis time and the quantum yields of the triplet state of the BCDs suggested the involvement of singlet oxygen in their phototransformation. The dyes were in their monomeric form at high SDS and DNA concentrations. In these interactions, the dyes also were phototransformed, with different rates compared with phosphate buffer, with formation of photoproducts. In the phototransformation of dyes in the presence of SDS micelles, we suggest that the dye molecules were more protected from oxygen contact. On the other hand, in the interaction of dyes and DNA molecules, the dye molecules bound on DNA surface become more available to oxygen attack, and the phototransformation becomes faster.
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25

Gamelas, Sara Raquel Duarte. "Porphyrin derivatives and mRNA alternative splicing induced by photodynamic therapy." Master's thesis, Universidade de Aveiro, 2016. http://hdl.handle.net/10773/18633.

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Mestrado em Química<br>Neste trabalho é descrita a síntese de porfirinas regulares e N-confusas com o objetivo da realização de estudos acerca da sua eficiência como fotossensibilizadores (PSs) em terapia fotodinâmica (PDT) e a influência desta no splicing alternativo do mRNA. Os derivados porfirinicos foram preparados tendo como estrutura base a 5,10,15,20-tetraquis(1-metilpiridinio-4-il)porfirina. Inicialmente irá ser realizada uma breve introdução sobre os macrociclos tetrapirrólicos, nomeadamente sobre porfirinas. Segue-se o capítulo 2 onde há a descrição sintética dos derivados porfirínicos usados neste trabalho. Desta forma, através das condições estabelecidas por Lindsey foram sintetizadas duas porfirinas: 5,10,15,20-tetraquis(4-bromometilfenil)-2-aza-21-carboporfirina e a 5,10,15,20-tetraquis(4-bromometilfenil)porfirina e procedendo-se à sua cationização com piridina que originou os derivados 5,10,15,20-tetraquis(4-(piridinio-1-il-metil)fenil)-2-aza-21-carboporfirina e 5,10,15,20-tetraquis(4-(piridinio-1-il-metil)fenil)porfirina. A formação das porfirinas neutras ocorreu com baixo rendimento, mas a sua cationização foi conseguida com rendimentos acima dos 80%. Neste mesmo capítulo ainda são apresentadas as caracterizações fotofísicas de todos os derivados porfirínicos, nomeadamente fluorescência e geração de oxigénio singleto. As porfirinas regulares foram aquelas que demonstraram bons rendimentos quânticos de fluorescência e capacidade para gerar oxigénio singleto em DMF. No capítulo 3 são descritos todos os estudos biológicos realizados neste trabalho. Inicialmente foram realizados estudos de viabilidade celular em células MCF-7 e HeLa usando o teste com WST-1 e ATPliteTM e como PS de referência a 5,10,15,20-tetraquis(1-metilpiridinio-4-il)porfirina. Foram realizados estudos de concentração (0.1 μM-5.0 μM) e tempo de irradiação (0, 5, 10 e 15 min). A 5,10,15,20-tetraquis(4-(piridinio-1-il-metil)fenil)porfirina revelou ser o melhor PS, tendo sido selecionado um tempo de irradiação de 15 min e uma concentração até 5 μM para o estudos subsequentes. Os estudos de localização subcelular e uptake revelaram que a 5,10,15,20-tetraquis(4-(piridinio-1-il-metil)fenil)porfirina entra nas células mas não se localiza no núcleo como acontece com o controlo positivo. São ainda descritos estudos acerca do tipo de morte celular que as células podem sofrer depois da PDT com estes PSs. Por fim os estudos da influência da terapia fotodinâmica no splicing alternativo mostrou que a 5,10,15,20-tetraquis(4-(piridinio-1-il-metil)fenil)porfirina e a 5,10,15,20-tetraquis(1-metilpiridinio-4-il)porfirina parecem induzir um ligeiro splicing alternativo do mRNA.<br>In this work the synthesis of regular and N- confused porphyrins with the objective of assessing its efficiency as photosensitizers (PSs) in photodynamic therapy (PDT) and its influence in alternative splicing of mRNA is described. The porphyrin derivatives were prepared having as a template the 5,10,15,20-tetrakis(1-methylpyridynium-4-yl)porphyrin. Initially, a brief introduction about tetrapyrrolic macrocycles, namely about porphyrins will be done. Then, chapter 2 will follow with the synthetic description of the porphyrin derivatives used in this work. Through Lindsey conditions, two porphyrins were synthesized: 5,10,15,20-tetrakis(4-bromomethylphenyl)-2-aza-21-carbaporphyrin; 5,10,15,20-tetrakis(4-bromomethylphenyl)porphyrin and then we proceeded to their cationization with pyridine to give: 5,10,15,20-tetrakis(4-(pyridynium-1-yl-methyl)phenyl)-2-aza-21-carbaporphyrin and 5,10,15,20-tetrakis(4-(pyridynium-1-yl-methyl)phenyl)porphyrin. The formation of the neutral porphyrins occurred with low yield but its cationization was achieved with yields higher than 80%. In the same chapter, the photophysical characterization of all porphyrins, namely singlet oxygen production and fluorescence quantum yield were described. The regular porphyrins demonstrated good fluorescence quantum yields and ability to generate singlet oxygen in DMF. In chapter 3, all the biological assays done in this work are described. Initially, cellular viability studies were done in MCF-7 and HeLa cells using the WST-1 reagent and ATPliteTM and as PSs reference the 5,10,15,20-tetrakis(1-methylpyridynium-4-yl)porphyrin. Studies of concentration (0.1 μM-5.0 μM) and irradiation time (0, 5, 10 and 15 min) were also done. The 5,10,15,20-tetrakis(4-(pyridynium-1-yl-methyl)phenyl)porphyrin showed to be the best PS and 15 min irradiation and a concentration up to 5 μM for the following studies were selected. The subcellular localization and uptake studies revealed that 5,10,15,20-tetrakis(4-(pyridynium-1-yl-methyl)phenyl)porphyrin can enter the cells but it is not localized in the nucleus, contrary to 5,10,15,20-tetrakis(1-methylpyridynium-4-yl)porphyrin. The studies regarding type of cell death that cells undergo after PDT treatment were also assessed. At last, the 5,10,15,20-tetrakis(4-(pyridynium-1-yl-methyl)phenyl)porphyrin and 5,10,15,20-tetrakis(1-methylpyridynium-4-yl)porphyrin seemed to induce a small alternative splicing of mRNA.
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26

López, Chicón Gracia Patrícia. "Fotoinactivación de especies fungicas patógenas mediante hipericina, TMPyP y NMB como agentes sensibilizantes fotodinámicos." Doctoral thesis, Universitat Ramon Llull, 2013. http://hdl.handle.net/10803/101405.

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S'ha avaluat mitjançant estudis in vitro la viabilitat de la teràpia fotodinàmica antifúngica per al tractament clínic d'infeccions causades per les espècies fúngiques patògenes Candida albicans, Candida parapsilosis, Candida krusei i Trichophyton mentagrophytes. L'objectiu és optimitzar aquesta tècnica per proporcionar una alternativa als fàrmacs antifúngics utilitzats en els tractaments clàssics actuals i eradicar així les micosis superficials. S'ha avaluat la capacitat citotòxica de tres fotosensibilitzadors amb propietats fotoquímiques diferents, la hipericina, la 5,10,15,20-tetraquis(N-metil-4-piridil)-21H,23H-porfina i el clorur de 3,7-Bis(etilamino)-2,8-dimetilfenotiacina-5-io, induint tots la inactivació cel.lular sobre les diferents espècies fúngiques. Per avaluar si la la tècnica produeix danys significatius sobre les cèl.lules epidèrmiques humanes s'han realitzat estudis sobre queratinòcits i fibroblasts humans. S'han avaluat els mecanismes d'acció dels tres fotosensibilitzadors mitjançant estudis espectroscòpics i cinètics de formació i desaparició dels seus estats excitats i oxigen singlet. En el cas de l'hipericina també s'han descrit els intermedis que es formen en irradiar, caracteritzant l'accessibilitat dels triplets a l'oxigen.<br>Se ha evaluado mediante estudios in vitro la viabilidad de la terapia fotodinámica antifúngica para el tratamiento clínico de infecciones causadas por las especies fúngicas patógenas Candida albicans, Candida parapsilosis, Candida krusei y Trichophyton mentagrophytes. El objetivo es optimizar esta técnica para proporcionar una alternativa a los fármacos antifúngicos usados en los tratamientos clásicos actuales y erradicar así las micosis superficiales. Se ha evaluado la capacidad citotóxica de tres fotosensibilizadores con propiedades fotoquímicas distintas, la hipericina, la 5,10,15,20-tetraquis(N-metil-4-piridil)-21H,23H-porfina y el cloruro de 3,7-Bis(etilamino)-2,8-dimetilfenotiacina-5-io, induciendo todos la inactivación celular sobre las distintas especies fúngicas. Para evaluar si la la técnica produce daños significativos sobre las células epidérmicas humanas se han realizado estudios sobre queratinocitos y fibroblastos humanos. Se han evaluado los mecanismos de acción de los tres fotosensibilizadores mediante estudios espectroscópicos y cinéticos de formación y desaparición de sus estados excitados y oxígeno singlete. En el caso de la hipericina también se han descrito los intermedios que se forman al irradiar, caracterizándose la accesibilidad de los tripletes al oxígeno.<br>Has been evaluated by in vitro viability studies of photodynamic therapy for clinical fungal infections caused by pathogenic fungal species Candida albicans, Candida parapsilosis, Candida krusei and Trichophyton mentagrophytes. The goal is to optimize this technique to provide an alternative to the antifungal drugs used in current classical treatments and thus eliminate superficial mycoses. We assessed the cytotoxic capacity of three photosensitizers with different photochemical properties, the hypericin, 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine and 3,7-Bis(ethylamino)-2,8-dimethylphenothiazin-5-ium chloride, inducing all cell inactivation on different fungal species. To assess if the technique produces significant damage to human epidermal cells, studies with human fibroblasts and keratinocytes were carried. We evaluated the mechanisms of action of the three photosensitisers by spectroscopic and kinetic studies of formation and disappearance of excited states and singlet oxygen. In the case of hypericin have also been described intermediates formed by irradiating, characterized accessibility of triplet oxygen.
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27

Bastien, Estelle. "Conception et optimisation de nanoparticules dendrimériques photoactivables dans le cadre d’un traitement photodynamique." Thesis, Université de Lorraine, 2015. http://www.theses.fr/2015LORR0322/document.

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La thérapie photodynamique (PDT) est une modalité de traitement des cancers prometteuse, mettant en jeu une action combinée de l’oxygène moléculaire, de la lumière et d’un photosensibilisateur (PS). Néanmoins, les PSs utilisés souffrent d’une faible solubilité dans les milieux aqueux ainsi que d’un tumorotropisme limité qui sont des barrières à la réussite du traitement. Ainsi, actuellement, une attention particulière est portée au développement de nanoparticules (NPs) capables de pallier les défauts des PSs. Notre travail a consisté à étudier des dendrimères poly(amidoamine) (PAMAM), macromolécules polymériques tridimensionnelles, conjugués via une liaison covalente au PS, la Chlorine e6 (Ce6). Cette construction nous a permis de vectoriser 32 molécules de Ce6 par dendrimère. La production d’oxygène singulet et l’émission de fluorescence ont été modérément affectées par le greffage covalent de la Ce6 aux NPs. In vitro, les dendrimères PAMAM ont permis d’accroitre l’efficacité PDT de la Ce6 en potentialisant son internalisation cellulaire via un mécanisme actif d’endocytose. Néanmoins, l’efficacité PDT des NPs est limitée par la concentration locale élevée en Ce6 en périphérie des dendrimères qui réduit son rendement quantique en oxygène singulet moléculaire, espèce cytotoxique. Une libération de la Ce6 permettrait ainsi de potentialiser l’efficacité PDT des NPs en restaurant notamment les propriétés photophysiques de la Ce6. La suite de ce travail a été de concevoir une NP capable de libérer la Ce6 sous l’action d’estérases retrouvées dans les cellules. Leur caractérisation a permis de démontrer en solution que les propriétés photophysiques de la Ce6 étaient rétablies à la suite de son relargage des NPs. Cette dernière construction clivable est prometteuse pour de futures applications en PDT<br>Photodynamic therapy (PDT) is a modality of cancer treatment, involving a combined action of molecular oxygen, light and photosensitizers (PS). However, the PSs suffer from a low solubility in aqueous media and limited tumor accumulation, diminishing the treatment success. Presently, particular attention is paied to the development of dendrimer-based nanoparticles (NP) that are able to overcome the shortcomings of the PSs. The present study investigates the poly(amidoamine) dendrimer (PAMAM), a tridimensional polymeric macromolecule, covalently functionalized with the PS Chlorin e6 (Ce6). The singlet oxygen generation efficiency and fluorescence emission were moderately affected by the covalent binding of the Ce6 to the dendrimer. This construction allows the vectorization of 32 Ce6 molecules per dendrimer. In vitro, PAMAM dendrimers improve the PDT efficiency of Ce6 by promoting their cellular internalization via an active endocytosis mechanism. However, the PDT efficiency of NPs is limited by the high local concentration of Ce6 at the periphery of dendrimers decreasing production of singlet oxygen. Ce6 release could restore Ce6 photophysical properties and as such improve the PDT efficiency of NP. Thus, the next step of this work was to design a cleavable NP able to release the Ce6 under esterase activity. In solution the NP characterization demonstrated that the photophysical properties of Ce6 were recovered after their release from the NP. This cleavable construction displays promising perspectives for future PDT applications
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28

Boix, Garriga Ester. "Biodegradable Poly(D,L-lactide) and Poly(D,L-lactide-co-glycolide) Nanoparticles for Photodynamic Therapy." Doctoral thesis, Universitat Ramon Llull, 2016. http://hdl.handle.net/10803/368179.

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Aquesta tesi aprofundeix en l'estudi de les nanopartícules de poli-(D, L-làctid-co-glicòlid) (PLGA) o poli-(D, L-làctid) (PLA) i les seves nanopartícules homòlogues recobertes amb polietilenglicol (PEG) com a sistemes de vehiculització per fotosensibilitzadors emprats en teràpia fotodinàmica. En primer lloc s'ha investigat la influència de la matriu polimèrica i del recobriment superficial amb PEG sobre les característiques fisicoquímiques, fotofísiques i fotobiològiques de suspensions amb un fotosensibilitzador atrapat físicament. El recobriment amb PEG confereix una major estabilitat a les nanopartícules en medi biològic. L'oxigen singlet generat en les nanopartícules de PLGA resta confinat en el seu interior, mentre que la presència de PEG facilita la difusió de l'oxigen singlet al medi extern, així com un alliberament més ràpid del fotosensibilitzador. En ambdós casos, el fotosensibilitzador es localitza als lisosomes a l'interior de les cèl·lules i indueix la mort cel·lular per apoptosi, la qual cosa indica que ambdós tipus de nanopartícules alliberen el fotosensibilitzador dins de la cèl·lula. L'efecte fototòxic és tanmateix major i més ràpid per a les nanopartícules PEGilades d'acord amb les observacions d'oxigen singlet. El paper de l'estructura química del fotosensibilitzador en les propietats fotofísiques i fotobiològiques de les nanopartícules també s'ha investigat. Els fotosensibilitzadors no metal·lats s'agreguen dins de les nanopartícules, mentre que els metal·lats romanen en forma monomèrica. Les seves propietats químiques també influeixen en la seva localització i, en conseqüència, en les propietats fotofísiques de les nanopartícules quan hi estan units covalentment, encara que hi siguin fotofísicament actius. Els fotosensibilitzadors encapsulats són capaços d'induir mortalitat cel·lular encara que estiguin agregats dins de les nanopartícules, mentre que els que estan units covalentment no ho són. Aquest fet permet concloure que l'alliberament del fotosensibilitzador dins de la cèl·lula és crucial per aconseguir una resposta fotodinàmica, i que les nanopartícules de PEG-PLGA no són internalitzades per les cèl·lules sinó que alliberen el fotosensibilitzador en la superfície cel·lular, cosa que no passa quan el fotosensibilitzador s'uneix covalentment a les nanopartícules. Finalment, s'ha examinat el potencial de la vehiculització activa mitjançant la conjugació del pèptid cRGDfK a la superfície de les nanopartícules. La presència de PEG és essencial per aconseguir un major augment en la concentració cel·lular de fotosensibilitzador. No obstant això, aquesta major concentració no produeix una major fototoxicitat en comparació amb les nanopartícules no marcades, el que suggereix que una major internalització no és l'únic factor important en el resultat final de la teràpia fotodinàmica.<br>Esta tesis profundiza en el estudio de nanopartículas de poli-(D,L-láctido-co-glicólido) (PLGA) o poli-(D,L-láctido) (PLA) y sus homólogas recubiertas con polietilenglicol (PEG) como sistemas de vehiculización para fotosensibilizadores empleados en terapia fotodinámica. En primer lugar se ha investigado la influencia de la matriz polimérica y del recubrimiento superficial con PEG sobre las características fisicoquímicas, fotofísicas y fotobiológicas de suspensiones con un fotosensibilizador atrapado físicamente. El recubrimiento con PEG confiere una mayor estabilidad a las nanopartículas en medio biológico. El oxígeno singlete generado en las nanopartículas de PLGA permanece confinado en su interior, mientras que la presencia de PEG facilita la difusión del oxígeno singlete al medio externo, así como una liberación más rápida del fotosensibilizador. En ambos casos, éste se localiza en los lisosomas en el interior de las células e induce muerte celular por apoptosis, lo que indica que ambos tipos de nanopartículas liberan el fotosensibilizador dentro de la célula. El efecto fototóxico es sin embargo superior y más rápido para las nanopartículas PEGiladas de acuerdo con las observaciones de oxígeno singlete. El papel de la estructura química del fotosensibilizador en las propiedades fotofísicas y fotobiológicas de las nanopartículas también se ha investigado. Los fotosensibilizadores no metalados se agregan dentro de las nanopartículas, mientras que los metalados restan en forma monomérica. Cuando éstos se unen covalentemente a las nanopartículas, sus propiedades químicas influyen en su localización en las mismas y, en consecuencia, en las propiedades fotofísicas de la suspensión, aunque preservan sus propiedades fotofísicas. Los fotosensibilizadores encapsulados son capaces de inducir mortalidad celular aunque estén agregados dentro de las nanopartículas, mientras que los que están unidos covalentemente no lo son. Este hecho permite concluir que la liberación del fotosensibilizador dentro de la célula es crucial para lograr una respuesta fotodinámica, y que las nanopartículas de PEG-PLGA no son internalizadas por las células, sino que liberan el fotosensibilizador en la superficie celular, lo que no ocurre cuando el fotosensibilizador se une covalentemente a las nanopartículas. Finalmente, se ha examinado el potencial de la vehiculización activa mediante la conjugación del péptido cRGDfK a la superficie de las nanopartículas. La presencia de PEG es esencial para lograr un mayor aumento en la concentración celular de fotosensibilizador. Sin embargo, dicha mayor concentración no produce una mayor fototoxicidad en comparación con las nanopartículas no marcadas, lo que sugiere que una mayor internalización no es el único factor importante en el resultado final de la terapia fotodinámica.<br>This thesis reports the study of poly-(D,L-lactide-co-glycolide) (PLGA) and poly-(D,L-lactide) (PLA) nanoparticles and their poly-(ethylene glycol) (PEG)-coated counterparts as delivery systems for photosensitizers in photodynamic therapy. First, the influence of the polymeric matrix and of the PEG surface coating on the physicochemical, photophysical and photobiological properties of nanoparticle suspensions containing a physically entrapped photosensitizer has been studied. PEG coating confers a higher stability to the nanoparticles in biological media containing serum proteins. Singlet oxygen produced in bare PLGA nanoparticles remains confined within them, while PEG surface coating facilitates singlet oxygen diffusion to the external medium as well as a faster drug release from the nanoparticles. In both cases, the photosensitizer localizes in lysosomes and induces cell death by apoptosis. The phototoxic effect is superior and faster for PEGylated NPs, in agreement with the singlet oxygen observations. The role of the chemical structure of the photosensitizer on the photophysical and photobiological properties of the colloidal suspensions has been subsequently investigated. Free base photosensitizers aggregate when entrapped in the nanoparticles, whereas metallated ones are incorporated in monomeric form. When the photosensitizers are covalently conjugated to the nanoparticles, their chemical properties influence their localization in the nanoparticles and consequently, the photophysical properties of the suspension, although they remain photophysically active. Physically entrapped photosensitizers are able to induce cell mortality in cells even if they are aggregated in the nanoparticles, while covalently conjugated photosensitizers are not. This drives us to the conclusions that the delivery of the photosensitizer into the cell is crucial to achieve a photodynamic response, and that PEG-PLGA nanoparticles are not internalized by cells but they rather deliver their cargo at the cell surface, which does not occur when the photosensitizer is covalently bound to the nanoparticles. Finally, we have explored the potential of the active targeting strategy by conjugating the cRGDfK peptide to the surface of the nanoparticles. The presence of a PEG side chain is essential to achieve an enhanced increase in the photosensitizer delivery into the cell, but unfortunately, it does not render a higher phototoxicity to the cells compared to the non-targeted nanoparticles, which suggests that a higher internalization is not the only important factor in the final outcome of photodynamic therapy.
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29

Chen, Kuan. "Photophysical characterization and optimization of novel polymer based photosensitizer carrier systems for PDT." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2010. http://dx.doi.org/10.18452/16159.

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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.<br>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.
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30

Junqueira, Helena Couto. "Síntese e estudo das propriedades fotoinduzidas de derivados fenotiazínicos em sistemas biomiméticos." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-18122008-105257/.

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Neste trabalho são apresentados estudos do efeito de interfaces nas propriedades fotofísicas e fotoquímicas do azul de metileno (AM) e de derivados fenotiazínicos com o intuito de avaliar o potencial destes compostos como fotossensibilizadores (FS) em terapia fotodinâmica. As propriedades físico-químicas do AM foram estudadas em soluções de SOS e observou-se que a presença do AM em solução altera o equilíbrio entre as micelas de SOS, diminuindo o valor da concentração micelar crítica de 7mmolL-1 para 70&#181;moIL-1. A presença das micelas em solução também interfere nas propriedades do AM. Em baixas concentrações de SOS há formação de dímeros de AM, constatados pelo aumento da absorbância em 580nm e diminuição da emissão de fluorescência. A caracterização das espécies transientes mostrou a existência de moléculas de azul de metileno no estado triplete (3AM) e de oxigênio singlete em soluções com altas concentrações de SOS e a formação de espécies radicalares do AM em baixas concentrações do tensoativo. Esta observação sugere que o mecanismo fotoquímico do AM é dependente da sua concentração local próxima de interfaces carregadas. As interações do AM e de alguns de seus derivados fenotiazínicos (tionina, azure A e azure B) com vesículas e com células HeLa foram estudadas e em ambos os casos observou-se que as moléculas com estruturas assimétricas são incorporadas em maior extensão. Em estudos de fototoxicidade, os compostos assimétricos apresentaram maior nível de morte celular do que o verificado para os compostos simétricos. Entretanto, ao se considerar a incorporação em células, os compostos simétricos se mostraram mais eficientes por molécula. Foi desenvolvido um método para determinação do 10gP dos fotossensibilizadores (FS) por voltametria com microeletrodos que se mostrou reprodutível. Novos FSs assimétricos derivados do Azure A com duas caudas hidrofóbicas de 4, 6 e 8 carbonos (AzC4, AzC6 e AzC8) foram sintetizados. Esses novos compostos possuem eficiências de fluorescência semelhantes ao AM. O composto AzC4 apresenta eficiência de geração de oxigênio singlete bem próxima à do AM (0,56), enquanto os outros dois compostos têm uma eficiência de geração de oxigênio singlete cerca de duas vezes menor. Este comportamento se deve ao fato dos FSs com cadeias carbônicas maiores interagirem mais fortemente entre si, apresentando maior grau de agregação. Os compostos sintetizados apresentam maior incorporação em vesículas que os compostos anteriormente estudados, indicando que a assimetria da molécula favorece a incorporação, assim como, o comprimento da cadeia hidrofóbica. Resultados de incorporação em células HeLa, mostraram que o composto AzC4 interage mais facilmente com a membrana celulare apresenta maior nível de morte celular devido à sua maior incorporação.<br>The effect of interfaces on photophysical and photochemical properties of methylene blue (MB) and its derivatives was studied in this work, aiming to emploit their potencial as photosensitizers (PS) in photodynamic therapy.The presence of MB in SDS solutions affect the micelle equilibrium decreasing the apparent critical micelle concentration of SDS from 7mmolL-1 to 70&#181;moIL-1. The properties of MB were also affected. At low SDS concentrations the formation of MB dimers was detected by the increase in the absorption in 580 nm and decrease of fluorescence emission. The characterization of transient species of MB showed the existence of MB molecules in the triplet state and emission of singlete oxygen at large SDS concentration and the formation of MB radicals in small surfactant concentration. These observations suggest that the photochemical mechanism of MB depends on its local concentration close to charged interfaces.The interactions of MB as well as other phenotiazine derivatives with synthetic vesicles and HeLa cells were studied. In both cases, the PS with asymmetrical structure presented higher degrees of incorporation. Studies of phototoxicity showed that the PS with asymmetrical structures present higher degree of cell death than the symmetrical compounds. However, if one consider the degree of incorporation the symmetrical compounds are more efficient per molecule. A method to determinate logP by voltammetry with microelectrodes was developed, showing good repeatability. New asymmetric photosensitizers derived from Azure A with two hydrophobic chains of 4, 6 or 8 carbons (AZC4, AZC6 e AzC8) were synthesized in order to study structure/activity relationship. These new compounds presented similar fluorescence efficiency to MB. The oxygen singlet generation efficiency of AZC4 is similar to MB, while the efficiency of the other PSs was twice smaller. This behavior was explained in terms PS aggregation, due to their longer hydrophobic chains. The synthesized PSs presented larger degree of incorporation than the commercial PSs, showing the role of asymmetry and hydrophobicity in incorporation yields. Incorporation in HeLa cells showed that AZC4 interacts more strongly with cells than the other synthesized PSs due to the rigidity of plasmatic membrane. Under the same illumination conditions, AzC4 was the PS that presented higher degree of cell death as a result of its larger incorporation in cells.
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31

Pollum, Marvin. "Applying Fundamental Photochemistry to Drive Drug Development: The Photo-Dynamics and Reactions of Sulfur-Substituted Nucleic Acids." Case Western Reserve University School of Graduate Studies / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1481287737895585.

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32

Oliveira, Carla Santos de. "Propriedades fotoquímicas dos fotossensibilizadores cristais violeta e azul de metileno em sistemas microheterogêneos e em células cancerosas em cultura." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-12062008-135805/.

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As propriedades fotofísicas e fotoquímicas de cristal violeta (CV) foram investigadas em soluções isotrópicas e verificou-se que solventes com constante dielétrica pequena favorecem a formação do par iônico, já o aumento na viscosidade do meio restringe a movimentação rotacional dos anéis aromáticos, resultando em um aumento no tempo de vida de fluorescência e, portanto no rendimento quântico de fluorescência (&#934;f) (Oliveira 2002). Os experimentos com CV foram conduzidos em micelas reversas do tensoativo aniônico bis-2-etilhexil sulfoccinato de sódio (AOT) em isooctano. A localização interfacial do CV nas micelas reversas de AOT em valores da razão molar entre água e surfactante (W0)pequenos e grandes foram encontrados através da técnica de Ressonância Magnética Nuclear (RMN) de próton e de carbono 13. Utilizando-se espectroscopia UV-Vis identificou-se que pares iônicos de contato estão presentes a valores pequenos de W0 e com o aumento do W0 pares iônicos separados por solventes são as espécies que predominam em solução. A comparação da eficiência de fotodegradação de CV em micelas reversas de AOT em função do W0 indicou que a fotoreatividade é maior em baixos valores de W0 . Este efeito deve estar relacionado à restrição da movimentação dos anéis aromáticos de CV devido ao ambiente restrito no qual este se localiza na micela reversa de OAT a W0 pequenos. A formação de intermediários reativos foi verificada através de Fotólise de Relâmpago a Laser e Emissão no infra-vermelho próximo, indicando a presença de espécies triplete, radical e oxigênio singlete com valor de rendimento quântico menor que 1%. Os produtos de fotólise foram identificados por técnicas cromatográficas e espectroscópicas. Na presença de oxigênio, houve maior formação de cetona de Michler. Com baixa concentração de oxigênio, o produto observável foi leuco-CV. Destes estudos propomos o mecanismo de CV neste meio. Após os estudos com micelas reversas, células cancerosas HeLa foram empregadas para comparar fotoatividade do CV com o azul de metileno (MB). As proporções de CV e MB dentro das células são altas, incorporando 70% e 80% da concentração da solução de incubação, respectivamente. Com o aumento da concentração de MB, um favorecimento da formação de dímero foi identificada. Já CV não sofre agregação nas condições estudadas. Nenhum dos fotossensibilizadores estudados tem um efeito danoso sobre as células HeLa em concentrações abaixo de 10&#181;M. Após irradiação, MB causou uma diminuição de cerca de duas vezes maior na taxa de sobrevivência celular comparado com CV. A formação de formação de oxigênio singlete após incorporação dos fotossensibilizadores foi investigada. Há formação de oxigênio singlete em células incubadas com MB, já com CV a geração de oxigênio singlete é pouco significativa sugerindo um mecanismo radicalar. O processo de morte celular foi estudado por citometria de fluxo e verificou-se que MB induz apoptose depois da irradiação em células HeLa. A absorção de luz por ambos fotossensibilizadores é similar, o que indica que a diminuição na sobrevivência não se deve à diferença de absorção luminosa. As diferenças de sobrevivência observadas com células incubadas com CV e MB e irradiadas foram relacionadas às diferenças das propriedades fotoquímicas destes fotossensibilizadores. A localização celular de CV e MB em células foram caracterizadas por microscopia de fluorescência. Verificou-se que ambos localizam-se em mitocôndrias. O aumento na concentração de CV não alterou o seu perfil de localização. Já para MB ao aumentar a concentração de MB, observa-se que o mesmo localiza-se além das mitocôndrias, em lisossomos. A comparação das propriedades fotoquímicas e de localização foram consideradas para explicar as diferenças de atividade fotodinâmica do CV e do MB em células HeLa<br>The photophysical and photochemical properties of crystal violet (CV) were investigated in isotropic solutions and it was found that solvents with small dielectric constants favor the formation of the ion pair and that the increase in viscosity of the medium restricts the rotational movement of the aromatic rings, resulting in an increase in fluorescent lifetime and therefore in the fluorescence quantum yield (&#934;f) (Oliveira 2002). CV experiments were conducted in reverse micelles of the anionic tensoactive sodium bis-2-ethylhexyl-sulfosuccinate (AOT) in isooctane. The interfacial localization of CV in the AOT reverse micelles at low and high values of molar ratio between water and surfactant (W0 was found through the proton and carbon 13 Nuclear Magnetic Resonance techniques (NMR). Using UV-Vis spectroscopy, it was identified that contact ion pairs are present in low W0 values and with the increase in the W0 solvent separated ion pairs are the species that predominate in solution. The comparison of the photobleaching efficiency of CV in AOT reverse micelles as a function of W0indicated that the photoreactivity is high with low W0 values. This effect must be related to the restrict environment in which CV is located. The reactive intermediate formation was found through the Laser Flash Photolysis and Near Infra-Red Emission, indicating the presence of triplet, radical and singlet oxygen species with a yield quantum of less than 1%. The photolysis products were identified through the chromatographic and spectroscopic techniques. In the oxygen presence, there was high Michler ketone formation. With the low oxygen concentration, the observable product was leuco-CV. With these studies we hypothesized a mechanism of CV in these proposed media. After the reverse micelles studies, the HeLa cancerous cells were used, in order to compare the CV and methylene blue (MB) photoactivity. The CV and MB proportion inside the cell was high, reaching 70% and 80% of concentration of the incubation solution, respectively. With the increase of MB concentration, a favoring of dimmer formation was identified. CV does not suffer aggregation in the studied conditions. None of the studied photosensitizers has a damaging effect upon the HeLa cells in concentrations below 10&#181;M. After irradiation, MB caused a decrease about twice higher in the cellular survival rate compared to CV. The singlet oxygen formation after the photosensitizer incorporation was investigated. There is a singlet oxygen formation in the cells incubated with MB, though with the CV the singlet oxygen generation is significantly low suggesting the radicalar mechanism. The cellular death process was studied by Fluorescence Activated Cell Sorting and MB-induced apoptosis was found after MB irradiation in HeLa cells. The light absorption by both photosensitizers is similar, which means that the survival decrease is not because of the light absorption difference. The survival differences observed with the cells incubated with CV and MB and irradiated were related to the differences in the photosensitizer photochemical properties. The cellular location of the CV and MB in cells were characterized by fluorescence microscopy. Both photosensitizers are located in mitochondrias. An increase in the CV concentration does not alter its local profile. However, with an increase in the MB concentration, MB was located not only in mitochondrias but also in lysosomes. The comparison of the photochemical and localization properties was considered in order to explain the differences in the photodynamic activity of CV and MB in HeLa cells
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33

Christine, Thifanie. "Synthèses et propriétés de nouvelles structures à base de BODIPY pour l’imagerie bimodale TEP/IO et la génération d’oxygène singulet." Thesis, Bordeaux, 2020. http://www.theses.fr/2020BORD0239.

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L’imagerie médicale est un outil très important pour le diagnostic de nombreuses maladies. La tomographie par émission de positons (TEP scan) est l’une des techniques les plus employées de par sa forte sensibilité, mais le très court temps de demi-vie du radionucléide utilisé implique une synthèse rapide du traceur radioactif. L’imagerie optique (IO) évite l’utilisation d’éléments radioactifs, mais sa sensibilité limitée dans les tissus profonds peut entraver son application in vivo. Afin d’obtenir des sondes plus modulables en fonction du besoin du biologiste (études in vitro ou in vivo), le développement de nouveaux traceurs bimodaux est devenu un axe de recherche important. Dans ce contexte, de nouvelles molécules bimodales à base de BODIPY (fluorescentes), pouvant être marquées au 11C, ont été synthétisées. Celles-ci ont été conjuguées à des dérivés d’oestradiol pour cibler les cellules tumorales du cancer du sein. Des études in vivo pour évaluer la biodistribution des molécules marquées au 13C sont en cours. En parallèle, leur transformation en photosensibilisateurs pour la production d’oxygène singulet a été également envisagée pour une potentielle application dans le domaine de la thérapie photodynamique. D’autre part, plusieurs photosensibilisateurs à base de BODIPY ont été synthétisés et évalués dans une réaction de photooxygénation, et leur application à d’autres types de réactions est actuellement en cours<br>Medical imaging is a very important tool for the diagnosis of numerous diseases. The positron emission tomography (PET scan) is one of the most employed technique for its high sensibility, but the very short lifetime of the radionuclide used implies a very fast synthesis of the radioactive tracer.1,2 Optical imaging (OI) avoids the use of radioactive elements, but its limited sensibility in deep tissues can hamper its in vivo application. In order to obtain more modular probes depending on the need of the biologist (in vitro or in vivo studies), the development of new bimodal tracers is becoming an important research axis. In this context, new bimodal BODIPY-based molecules (fluorescent) that could be labelled by 11C (PET) have been synthesized. The different structures were conjugated to oestradiol to target the tumour cells of breast cancer. In vivo studies are being carried out to evaluate the biodistribution of the [13C]-labelled molecules. In parallel, their modification in photosensitizers for the production of singlet oxygen has also been considered for potential applications in the field of photodynamic therapy. On the other hand, several BODIPY-based photosensitizers have been synthesized and evaluated in a photooxygenation reaction, and their application to other types of reaction are in progress
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34

Epelde, Elezcano Nerea. "Matériaux Hybrides nanostructures photoactifs pour des applications optiques et biomédicales." Thesis, Pau, 2016. http://www.theses.fr/2016PAUU3007/document.

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Dans ce manuscrit, la synthèse et la caractérisation complète de différents matériaux hybrides dédiés à des applications dans le domaine optique ou thérapeutique sont décrites. Dans un premier temps, des systèmes macroscopiquement ordonnés sont obtenus par intercalation de colorants tels que le Styryl 722 ou la pyronine-Y dans plusieurs films à base d’argile de type smectite. Les films d’argile sont élaborés par spin-coating et les colorants intercalés par immersion des films dans les solutions de ces colorants. Les effets de l’argile sur les propriétés des colorants sont analysés en détail et leur orientation préférentielle dans l’espace inter-couches est étudié grâce à la réponse anisotropique des films en lumière linéairement polarisée. Dans la deuxième partie, la synthèse par chimie sol-gel de monolithes de silice de grande dimension contenant des colorants laser présentant une forte absorption et une émission de fluorescence dans le visible est abordée. Des colorants laser à l’état solide (SSDL) avec de bonnes stabilités photochimique, thermique et chimique sont ainsi proposés. Dans le troisième chapitre, la synthèse par voie sol-gel de nanoparticules de silice (NP) d’environ 50 nm de diamètre fonctionnalisées sur leur surface externe est ensuite décrite. Grâce à l’encapsulation de molécules de colorants fluorescents dans leur cœur et le greffage de photosensibilisateurs sur leur écorce, des nanoparticules biocompatibles adaptées à la bio-imagerie et la thérapie photodynamique (PDT) ont été préparées. Pour optimiser leurs performances, les propriétés photophysiques et plus particulièrement la production d’oxygène singulet d’une nouvelle série de photosensibilisateurs basés sur les chromophores de type PODIPY ont d’abord été étudiées en détail. A partir de ces résultats, des BODIPY particulièrement efficaces ont été greffés sur les nanoparticules de silice afin de les utiliser pour la PDT. Les propriétés photophysiques de ces matériaux ont été analysées par spectroscopie d’absorption et de fluorescence (stationnaire ou résolue en temps) et les rendements quantiques de production d’oxygène singulet déterminés par des méthodes directe (émission de luminescence de l’oxygène singulet à 1270 nm) ou indirecte (utilisation de sondes chimiques spécifiques à l’oxygène singulet). Par ailleurs les matériaux hybrides ont été complètement caractérisés par plusieurs techniques (SEM, TEM, XRD, XPS, IR, DLS, BET)<br>Along this manuscript different hybrid materials are synthesized and extensively characterized for several uses: from optical to therapeutic applications. First, by the intercalation of different dyes, styryl 722 and pyronine-Y into several smectite clay films, macroscopically ordered system are obtained. Clay films are elaborated by spin-coating technique and the dyes are intercalated by the immersion of clay thin films into dye solutions. The effect of clay on the dye properties is deeply analyzed and its preferential orientation in the interlayer space of the clay is studied by the anisotropic response of the films to the linear polarized light. Second, large silica monoliths with embedded laser dyes with strong absorption and fluorescence bands in different region of the Visible spectrum are attained by sol-gel chemistry to obtain solid-state dye laser (SSDL) with good photo, thermal and chemical stabilities. Third, silica nanoparticles (NP) with suitable size (50 nm) and functionalized external surface are also synthesised by sol-gel chemistry. Through the encapsulation of fluorescent dye molecules in their core and by the grafting of photosensitizers on their shell, biocompatible nanoparticles for bio-imaging and Photodynamic Therapy (PDT) applications are prepared. In order to optimize their properties, a careful investigation of the photophysical properties and mainly the singlet oxygen generation of a large range of new photosensitizers based on chromophores known as BODIPYs, is previously carried out. Based on these results, some efficient BODIPYs are selected for grafting on silica nanoparticles in order to use them for PDT. The photophysical properties of all these hybrid materials are analyzed by absorption and fluorescence (steady-state and time correlated) spectroscopies, and the singlet oxygen measurements are monitored by direct method (recording the singlet oxygen luminescence at 1270 nm) and by indirect method (using selective chemical probe). Moreover, the hybrid materials are fully characterized by several techniques such as, SEM, TEM, XRD, XPS, IR, DLS, BET
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35

Zaghdoudi, Khalil. "Optimisation de l’extraction des caroténoïdes à partir du persimmon (Diospyros kaki L.), de l’abricot (Prunus armeniaca L.) et de la pêche (Prunus persica L.) : étude photophysique en vue d’une application en thérapie photodynamique (PDT)." Thesis, Université de Lorraine, 2015. http://www.theses.fr/2015LORR0297/document.

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La thérapie photodynamique (PDT) est une technique utilisée cliniquement pour traiter certaines maladies de la peau, la dégénérescence maculaire liée à l’âge et certains types de cancer. Elle fait intervenir trois composants : une molécule photosensible ou photosensibilisateur (PS), la lumière et l’oxygène. Après administration du PS, celui-ci va se localiser plus ou moins sélectivement dans les zones tumorales où il est alors activé par irradiation lumineuse à une longueur d’onde et une puissance données. Ceci engendre la formation d’espèces réactives de l’oxygène (ROS) très réactives, dont l'oxygène singulet1 O2, qui entraînent la destruction des tissus tumoraux par nécrose ou apoptose. Afin d’améliorer la sélectivité du traitement, différentes pistes sont actuellement exploitées dont l’élaboration de « photodynamic molecular beacons » (PMB). Dans un PMB, le photosensibilisateur (PS) est associé via un peptide à un inhibiteur 1O2, appelé quencher. Ce quencher inhibe la formation d’1O2 tant que le composé n’a pas atteint sa cible. Une fois la zone cancéreuse atteinte, des enzymes spécifiques clivent le peptide, libérant ainsi le PS qui retrouve alors sa capacité à former de l’1O2. Trouver un couple PS/quencher adéquat reste un challenge en PDT. Les propriétés photophysiques particulières des caroténoïdes et leur aptitude à inhiber la production d’1O2 font de ces derniers des quenchers potentiellement utilisables pour l’élaboration de PMBs. Chez les plantes, les caroténoïdes (carotènes et xanthophylles) sont des pigments associés à la photosynthèse, qui ont deux rôles principaux : un rôle de collecteur de lumière et un rôle photoprotecteur en protégeant le(s) système(s) photosynthétique(s) contre les dommages photooxydatifs liés à une exposition trop intense à la lumière. Ceci s’opère, entre autre, via le cycle des xanthophylles. Cette aptitude à capter de l’énergie présente un intérêt potentiel à ne pas négliger dans la perspective de la conception de PMB utilisables en thérapie photodynamique. Dans le cadre de cette thèse en co-tutelle avec la Faculté des Sciences de Bizerte nous avons ciblé les caroténoïdes présents dans trois fruits produits en Tunisie à savoir les kakis (Diospyros kaki L.), les abricots (Prunus armeniaca L.) et les pêches (Prunus persica L.) connus pour leur richesse globale en ces pigments. Divers procédés d’extractions ont été étudiés : (i) L’extraction de type Soxhlet par solvants organiques à pression atmosphérique, utilisée comme référence, (ii) l'extraction accélérée par solvant organique (ASE : Accelerated solvent Extraction) effectuée sous pression, enfin (iii) l'extraction par CO2 supercritique avec l’éthanol comme cosolvant. Pour ces deux derniers procédés, une approche par plan d’expériences (surfaces de réponses) a été utilisée pour identifier les facteurs clé et les conditions optimales d’extractions de divers caroténoïdes (pression, température, débit, % de cosolvant, temps, nombre de cycles). L'analyse par chromatographie liquide à haute performance couplée à la détection UV-Visible et à la spectrométrie de masse a ensuite permis l'identification et la quantification des caroténoïdes présents dans les extraits obtenus, permettant ainsi de comparer les profils caroténoïdiques propres à chaque fruit et les performances de chaque procédé d’extraction. Cette étude ayant révélé un profil caroténoïdique particulièrement intéressant chez le kaki par rapport aux autres fruits, une extraction et une purification des caroténoïdes de ce fruit par chromatographie liquide haute pression préparative a ensuite été effectuée afin de disposer d’une quantité suffisante de chaque caroténoïde, et parfois de leurs isomères conformationnels, en vue de l’étude de leurs propriétés photophysiques (absorption, émission de fluorescence, inhibition d’1O2) et de l’évaluation de leur intérêt potentiel en tant que quencher d’1O2 dans un édifice de type PMB<br>Photodynamic therapy (PDT) is a clinically used technique for treating skin diseases, age-relatedmacular degeneration but mainly some types of cancer. PDT involves three components: a photosensitive molecule named photosensitizer (PS), light and oxygen. After administration of the PS, this one will be located more or less selectively in tumoral regions where it is activated by light irradiation at appropriate wavelength and power. This leads to the formation of highly reactive and cytotoxic reactive oxygen species (ROS), especially singlet oxygen, resulting in the destruction of the tumor by necrosis or apoptosis. To improve the treatment selectivity, different strategies are being exploited, one of which is the development of "photodynamic molecular beacons" (PMB). In PMB the photosensitizer is linked via a peptide to an inhibitor of 1O2 (quencher). This quencher inhibits the formation of 1O2 as long as the compound has not reached its target, namely cancer cells. In order to inhibit the toxicity of the PS in non-target cells and restore toxicity only close to the biological target, it is necessary to find an adequate PS/quencher couple. This remains a challenge for PDT. Carotenoids are interesting candidates due to their specific photophysical properties and ability to inhibit 1O2, which makes them potential quenchers for building PMBs. In plants, carotenoids (carotenes and xanthophylls) are pigments involved in the photosynthesis, in which they play two main roles: a light collecting role and a protecting role by preserving the photosynthetic systems against photoxydative damages induced by a too intense light exposure. This protection can for instance occur via the well-known xanthophylls cycle. This capacity to catch energy presents a potential interest that should not be neglected in the framework of the design of PMBs usable in photodynamic therapy. Within the framework as part of this PhD thesis in Cotutelle with the Faculty of Sciences of Bizerte, we focused on carotenoids from three fruits produced in Tunisia: persimmon (Diospyros kaki L.), apricot (Prunus armeniaca L.) and peache (Prunus persica L.), known for their global richness in these natural pigments. Three extraction processes were investigated: (i) the Soxhlet extraction based on the use of organic solvent at atmospheric pressure and used as reference, (ii) the accelerated solvent extraction (ASE) using organic solvent under high pressure, and (iii) the supercritical fluid extraction (SFE) using supercritical CO2 and ethanol as cosolvent. For these two last processes, a design of experiments (Surface Response Design) was used to identify the key factors and optimal extraction conditions of various carotenoids (pressure, temperature, flow, % cosolvent, time, number of cycles). Then, HPLC-PDA coupled with mass spectrometry (MS) enabled the identification and quantification of carotenoids from the extracts. Thus it was possible to compare the profiles in carotenoids content from each fruit as well as the performances of each extraction process. This study showed that the carotenoidic profile in the persimmon was the most interesting as compared to the profiles in the two other fruits. Extraction and purification of the carotenoids from persimmon by preparative high pressure liquid chromatography were then performed in order to have a sufficient amount of each carotenoid and sometimes of their conformational isomers. We finally performed a study of their photophysical properties (absorption, fluorescence emission, 1O2 inhibition) in order to evaluate their potential as 1O2 quencher in molecular construction such as a PMB
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36

Niedre, Mark Jonathan. "Development and validation of singlet oxygen luminescence-based photodynamic therapy dosimetry." 2004. http://link.library.utoronto.ca/eir/EIRdetail.cfm?Resources__ID=94728&T=F.

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"Ru(ii) Diimine Complexes Chromophores For Applications In Photodynamic Therapy: Singlet Oxygen Sensitizers And Substitutionally Photolabile Complexes." 2015.

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38

Jarvi, Mark. "Evaluation of Photophysical Methods for Photodynamic Therapy Dosimetry." Thesis, 2012. http://hdl.handle.net/1807/32746.

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In photodynamic therapy (PDT), the combination of light, photosensitizer and molecular oxygen generates reactive oxygen species, including singlet oxygen (1O2), which is regarded as the primary cytotoxin and effector with most clinical photosensitizers. PDT has gained some acceptance for the treatment of cancer and other conditions. However, its clinical utility and effectiveness has been limited by variability in treatment response and failure to integrate adequate treatment planning and dosimetry. Direct PDT dosimetry through the detection of ultra-weak near-infrared 1O2 luminescence emission at 1270 nm (SOL) collapses the complexity of PDT into a single parameter, the 1O2 concentration. Prior to the present studies, it was shown that SOL was well correlated with PDT response in vitro and in vivo under controlled experimental conditions. However, SOL detection is technically challenging because of the very low radiative probability of 1O2 (~ 10-8 in biological environments), dynamic background signals and limited sensitivity of suitable photodetectors in this wavelength region. A technologically simpler and less costly PDT dosimetry approach is to use photosensitizer photobleaching to estimate the 1O2 dose. The first objective in this thesis was to characterize the dynamics of SOL measurements, in particular the influence of oxygen depletion, in order to improve the quantification of SOL and its use as an accurate PDT dose metric. Subsequently, direct comparison of SOL and photobleaching-based dosimetry during in vitro PDT treatment with meso-tetra(hydroxyphenyl)chlorin (mTHPC) showed that SOL dosimetry is robust but that photobleaching-based dosimetry can fail under hypoxic conditions. However, the latter can be salvaged through the identification of a previously unreported 613 nm emission from mTHPC that indicates hypoxia. These studies were carried forward into an in vivo dorsal skin-fold window chamber tumor model, which showed promising initial correlation between 1O2 dose and tumor response. This work also identified SOL detection limitations and opportunities for further development. Additionally, SOL measurements were used as a ‘gold standard’ to evaluate novel activatable PDT beacons and a novel “PDT biodosimeter” based on STAT3 cross-linking. Future work includes further tumor dose-response studies, characterization of novel photosensitizing agents, improvement on signal detection and processing, and studies in normal human skin.
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Čermák, Pavel. "Příprava a fotofyzikální hodnocení tetrapyridoporyrazinů vhodných pro fotodynamickou terapii." Master's thesis, 2016. http://www.nusl.cz/ntk/nusl-351477.

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Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department: Department of Biophysics and Physical Chemistry Candidate: Pavel Cermak Supervisor: Assoc. Prof. Veronika Novakova, PhD. Title of Thesis: Preparation and photophysical evaluation of tetra-3,4- pyridoporphyrazines suitable for the photodynamic therapy Tetra-3,4-pyridoporphyrazines (TPyPz) are aza-analogues of phthalocyanines. Their large system of conjugated bonds enables them to absorb light in the red part of the absorption spectrum. Due to their ability to produce singlet oxygen, they can be potentially used as photosensitizers in photodynamic therapy (PDT). Its mechanism is based on co-functioning of three elements - photosensitizer, light and oxygen. Photosensitizer excited by light absorption transfers its energy into tissue oxygen, thus, creating cytotoxic singlet oxygen. This method is beneficial for its high selectivity, low toxicity, minimal invasion and fast effect. The aim of this work was to synthetize and study water-soluble TPyPz suitable for PDT. Water solubility was achieved by quarternized amines, forming of salts or using suitable delivery systems (hydrophilic emulsion). Hydrophilicity was also increased by introduction of hydrophilic non-charged substituents (OH). At first, appropriate precursors for...
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Lin, Yu-shan, and 林玉珊. "Search for the appropriate photosensitizers for photodynamic therapy-Study of the excited singlet oxygen lifetime and the location of the photosensitizers in OC2." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/09946061341399641489.

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碩士<br>國立中正大學<br>物理所<br>96<br>Photodynamic therapy (PDT) is a treatment for malignant neoplasm. This method involves three key components: a photosensitizer、light and oxygen. A photosensitizer can be excited by light of proper wavelengths in the visible or near infrared regimes. From ground singlet state, the photosensitizers are first excited to an excited singlet state, from which they undergo a few relaxation process including the desired intersystem to an excited triplet state. When oxygen molecules exist around, the photosensitizer on a triplet state can relax to the ground singlet state by transferring the excess energy to the oxygen and promote them to the excited singlet state. Singlet oxygen is very aggressive and readily to react with the surrounding cancer cells. As a result, these destructive reactions kill the cells by appoptosis or necrosis. In this thesis, we measured the lifetime of singlet oxygen promoted to the first excited singlet state by the solutions of Hematoporphyrin dissoveld in acetone, 5,10,15,20 – Tetrakis (1-methyl-4-pyridinio) porphyrin tetra (p-toluenesulfonate) dissoveld in heavy water and Rose Bengal dissoveld in heavy water. According to our measurements, solutions of higher concentration show shorter lifetime. Besides, we also used the fluorescence microscopy to identify the locations of the photosensitizers in human oral cancer cells (OC2). This helps to tell if the excited singlet oxygen molecules kill the cancer cells nearby.
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Stíbal, Adam. "Fluorescenční detekce kožních nádorů." Master's thesis, 2013. http://www.nusl.cz/ntk/nusl-327826.

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This thesis discusses the photodynamic therapy (PDT) and it's application for dia- gnostics of tumours. The PDT is based on production of reactive oxygen species (ROS) from the molecules of photosensitizers (PS). PS are preferentially accumulated in disea- sed tissues, where ROS are produced and cause selective destruction of the target tissue, while surrounding healthy tissue remains intact. The photodynamic diagnostics uses the fluorescence of PS for digital and spectral imaging. This thesis is focused on detection of tumours from spectral characteristics of protoporphyrin IX fluorescence induced in tis- sue by methyl-aminolevulinate. The main part of the research was realised on rats' scars in vivo, the solutions of homogenized mouse fibroblasts were also studied. The way to distinguish between diseased and healthy tissue was found using measurements of fluo- rescence bleaching kinetics.
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Oleś, Tomasz. "Badanie fotosensybilizującej efektywności pochodnych bakteriochlorofilu a, oraz mechanizmów fotogenerowania przez te barwniki tlenu singletowego i wodnych rodników." Praca doktorska, 2015. https://ruj.uj.edu.pl/xmlui/handle/item/45319.

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