<p> Photodynamic therapy (PDn is an effective treatment option for various types of invasive tumors, the efficacy of which depends strongly on selective cell uptake and selective excitation of the tumor, which relies on proper dosage. The characterization of the fluorescence lifetimes of photosensitizers localized inside living cells may provide the basis for further investigation of in vitro PDT dosage measurements using time-domain spectroscopy and imaging. In this thesis, the fluorescence lifetimes of localized Photofrin ® and delta-aminolevulinic acid (ALA) induced protoporphyrin IX (PpiX) were investigated in living MAT-LyLu (MLL) rat prostate adenocarcinoma cells. Cells were incubated with the photosensitizers, and then treated with light under well-oxygenated conditions using a two-photon fluorescence lifetime imaging microscope (FLIM). Fluorescence lifetime images of these cells were recorded with average lifetimes of 5.5 ± 1.2 ns for Photofrin and 6.3 ± 1.2 ns for ALA-induced PpiX over 600 to 750 nm. Two channel FLIM revealed lifetimes of7.8 ± 0.5 ns for Photofrin® and 10.8 ± 1.7 ns for PpiX over 620 to 645 nm, while photoproducts observed on the second channel yielded lifetimes of 5.1 ± 0.4 ns over 650 to 670 nm for Photofrin® and 6.3 ± 1.0 ns over 670 to 690 nm for PpiX. Fluorescence lifetimes of both photosensitizers were found to be significantly shorter when localized in cells than when measured in solutions, suggesting that photosensitizers' lifetimes go through significant changes when bonded to intracellular components. These changes in lifetime also provide opportunities to quantitatively measure and monitor the binding states of the photosensitizers and their microenvironment, which may be used in real-time PDT dosimetry, as well as for diagnostic purposes. </p> / Thesis / Master of Applied Science (MASc)

Les dérivés de porphyrines sont impliqués dans de nombreux processus d'oxydoréduction. Ces composés conduisent à de nombreuses applications, dont la Thérapie Photodynamique (PDT). Il existe deux générations de dérivés de porphyrines qui sont actuellement remplacés par des composés de 3ème génération, plus actifs et entraînant moins d'effets secondaires. Ces composés sont capables de reconnaître spécifiquement et directement (par adressage) ou indirectement (par vectorisation) les cellules cancéreuses. Nous avons orienté notre travail vers la synthèse et l'évaluation biologique de composés tétrapyrroliques associés à des modules tels que des sucres ou des motifs peptidiques de type -RGD- qui permettent une reconnaissance spécifique des cellules cancéreuses, via les lectines ou les intégrines, respectivement. De plus, dans le cadre d'un programme européen Cost-Chemistry intitulé "New molecular systems with therapeutic applications in photodynamic therapy of cancer and microbial infections", nous avons étudié les propriétés photophysiques de nombreux photosensibilisants synthétisés par une équipe roumaine / Derived of porphyrins are tetrapyrrolic macrocycles involved in several redox processes. These compounds are used for different biological applications, like photodynamic therapy (PDT). Many teams in the world seek to synthesize compounds able to directly recognize specifically and (by targeting) or indirectly (by vectorization) cancer cells. These compounds are known as of 3rd generation. We are involved in the synthesis and the biological evaluation of tetrapyrrolic compounds associated to recognition and/or transport agents such as sugars or RGD-like peptide sequences. These moieties allow a specific recognition of the cancerous cells, via the lectins for the sugar moieties and the integrins for the RGD type moieties, respectively. Within the framework of an European Cost-Chemistry program entitled "New mo/ecu/ar systems with therapeutic applications in photodynamic therapy of cancer and microbia/ infections", we also studied the photophysical properties of photosensitizers synthesized by a Rumanian team

Posterior capsule opacification (PCO) is a major component of secondary cataract, a complication of current cataract surgery practice. This iatrogenic condition occurs in virtually all pediatric cases and to a lesser extent in adults. PCO correlates with the development in the latter half of the 20th Century of extracapsular cataract extraction (ECCE). In these surgeries, the lens capsule is left intact. During ECCE surgery a circular capsulotomy opening is created in the anterior lens capsule, and the cataractous, proteinaceous lens is removed, often via ultrasonic lens liquefaction i.e. phacoemulsification. The posterior, equatorial and remaining anterior portions of the sac-like capsule are left intact, permitting the insertion of an artificial lens into the emptied capsule. However, cells from the monolayer of epithelium on the inner surface of the capsule often begin to proliferate and migrate onto the normally cell-free inner surface of the posterior capsule, and may obscure the central axis of vision. Subsequently, a second surgery is necessary to create a small capsulotomy in the centre of the posterior capsule, usually employing an Nd:YAG laser. However, up to 5% of patients who have capsulotomies may then develop further serious, vision-threatening complications such as macular edema and retinal detachments. This thesis reports the photodynamic therapy (PDT) conditions required to prevent lens epithelial (LE) cell de novo proliferation and migration onto posterior lens capsules in a euthanized rabbit surgical model in order to predict parameters required to prevent PCO in humans. Experiments with primary in vitro cultures of human LE cells have shown rapid delivery of the photosensitizer benzoporphyrin derivative monoacid ring A (BPD-MA) and efficient killing with low light doses of 690 nm red light. Additional studies have shown the efficacy of various viscous agents in protecting the comeal endothelium. During model phacoemulsification ECCE surgeries, the use of hyaluronate viscoelastic carriers addressed the need for containment necessary for localized delivery of photosensitizer in the emptied capsule. Long-term monitoring of PDT-treated rabbit lens capsules in vitro has demonstrated a phototoxic effect including complete cell kill in this surgical model employing the prophylactic use of PDT.

The challenges in modern biological imaging applications are two-fold: (i) to develop better methods of imaging, and (ii) develop dyes that are suitable for these methods. This dissertation deals with the design and synthesis of dyes mainly by modification of known dyes to make them suitable for modern biological applications. Towards this aim, novel ways of derivatizing BODIPY dyes are explored. One method involves extending the conjugation via phenyl acetylene units, pushing fluorescence wavelengths near 600 nm. A different approach deals with C-H functionalization of BODIPY in which the fluors are functionalized with acrylate units, extending their fluorescence to the red. The BODIPY dyes developed are then incorporated in through-bond energy transfer cassettes. We examine the factors affecting energy transfer efficiencies by synthesizing analogs of the cassettes and also studying the electrochemical behavior of the donor and acceptor parts. The concept of through-bond energy transfer is incorporated into conjugated polymers by random incorporation of BODIPY dyes into polyfluorenes. The ideal ratio of fluorene to BODIPY parts was found to be 4:1. The BODIPY doping agents result in dispersed emissions when excited the polyfluorene polymers. Concurrently, the polyfluorene backbone acts as an energy harvester for the BODIPY dyes, in effect increasing their molar absorptivities. Finally the use of BODIPY dyes as photodynamic therapeutic agents was examined. We found that BODIPY dyes are efficient at producing reactive oxygen species when halogens are attached directly on the BODIPY core. Furthermore, the mechanism of cell death by using such agents was elucidated. Attachment of the most promising agent to polyglutamic acid is done to promote the EPR effect. Lastly we develop a potentially new type of PDT agent that absorbs strongly above 800 nm, permitting its use in deep tissue PDT.

Posterior capsule opacification (PCO) is a major component of secondary cataract, a complication of current cataract surgery practice. This iatrogenic condition occurs in virtually all pediatric cases and to a lesser extent in adults. PCO correlates with the development in the latter half of the 20th Century of extracapsular cataract extraction (ECCE). In these surgeries, the lens capsule is left intact. During ECCE surgery a circular capsulotomy opening is created in the anterior lens capsule, and the cataractous, proteinaceous lens is removed, often via ultrasonic lens liquefaction i.e. phacoemulsification. The posterior, equatorial and remaining anterior portions of the sac-like capsule are left intact, permitting the insertion of an artificial lens into the emptied capsule. However, cells from the monolayer of epithelium on the inner surface of the capsule often begin to proliferate and migrate onto the normally cell-free inner surface of the posterior capsule, and may obscure the central axis of vision. Subsequently, a second surgery is necessary to create a small capsulotomy in the centre of the posterior capsule, usually employing an Nd:YAG laser. However, up to 5% of patients who have capsulotomies may then develop further serious, vision-threatening complications such as macular edema and retinal detachments. This thesis reports the photodynamic therapy (PDT) conditions required to prevent lens epithelial (LE) cell de novo proliferation and migration onto posterior lens capsules in a euthanized rabbit surgical model in order to predict parameters required to prevent PCO in humans. Experiments with primary in vitro cultures of human LE cells have shown rapid delivery of the photosensitizer benzoporphyrin derivative monoacid ring A (BPD-MA) and efficient killing with low light doses of 690 nm red light. Additional studies have shown the efficacy of various viscous agents in protecting the comeal endothelium. During model phacoemulsification ECCE surgeries, the use of hyaluronate viscoelastic carriers addressed the need for containment necessary for localized delivery of photosensitizer in the emptied capsule. Long-term monitoring of PDT-treated rabbit lens capsules in vitro has demonstrated a phototoxic effect including complete cell kill in this surgical model employing the prophylactic use of PDT.

No description available.

Posterior capsule opacification (PCO) is a major component of secondary cataract, a complication of current cataract surgery practice. This iatrogenic condition occurs in virtually all pediatric cases and to a lesser extent in adults. PCO correlates with the development in the latter half of the 20th Century of extracapsular cataract extraction (ECCE). In these surgeries, the lens capsule is left intact. During ECCE surgery a circular capsulotomy opening is created in the anterior lens capsule, and the cataractous, proteinaceous lens is removed, often via ultrasonic lens liquefaction i.e. phacoemulsification. The posterior, equatorial and remaining anterior portions of the sac-like capsule are left intact, permitting the insertion of an artificial lens into the emptied capsule. However, cells from the monolayer of epithelium on the inner surface of the capsule often begin to proliferate and migrate onto the normally cell-free inner surface of the posterior capsule, and may obscure the central axis of vision. Subsequently, a second surgery is necessary to create a small capsulotomy in the centre of the posterior capsule, usually employing an Nd:YAG laser. However, up to 5% of patients who have capsulotomies may then develop further serious, vision-threatening complications such as macular edema and retinal detachments. This thesis reports the photodynamic therapy (PDT) conditions required to prevent lens epithelial (LE) cell de novo proliferation and migration onto posterior lens capsules in a euthanized rabbit surgical model in order to predict parameters required to prevent PCO in humans. Experiments with primary in vitro cultures of human LE cells have shown rapid delivery of the photosensitizer benzoporphyrin derivative monoacid ring A (BPD-MA) and efficient killing with low light doses of 690 nm red light. Additional studies have shown the efficacy of various viscous agents in protecting the comeal endothelium. During model phacoemulsification ECCE surgeries, the use of hyaluronate viscoelastic carriers addressed the need for containment necessary for localized delivery of photosensitizer in the emptied capsule. Long-term monitoring of PDT-treated rabbit lens capsules in vitro has demonstrated a phototoxic effect including complete cell kill in this surgical model employing the prophylactic use of PDT. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

No description available.

No description available.

Estudos com tratamento hipertérmico de tumores utilizando nanopartículas metálicas têm sido realizados durante as últimas décadas e mostram resultados bons quanto à remissão de tumores, por vezes chegando à cura completa. O mesmo acontece em relação aos tratamentos baseados em ação fotodinâmica de fotossensibilizadores. Tratamentos aliando a terapia hipertérmica com nanopartículas de ouro e a terapia fotodinâmica com diversos fotossensibilizadores tem efeito sinérgico e apresenta excelente potencial terapêutico, em que pese serem necessários mais estudos para que uma nova terapia conjunta possa ser implementada. A proposta deste trabalho foi investigar esse efeito sinérgico utilizando nanobastões de ouro complexados com fotossensibilizadores. Após a síntese dos nanobastões pelo método de seeding, a eficácia do tratamento fotodinâmico e da terapia hipertérmica, separadamente, foi investigada. A metodologia do recobrimento dos nanobastões por fotossensibilizador, em um primeiro momento, não logrou êxito com a porfirina, porém com a ftalocianina tetracarboxilada se mostrou mais eficaz. A taxa de fotodegradação da ftalocianina em solução foi investigada como parâmetro para a eficiência em geração de oxigênio singlete. Após centrifugação e lavagem das nanopartículas, no entanto, evidenciou-se por espectrofotometria que o fotossensibilizador não permaneceu aderido aos nanobastões. Em um segundo momento, optamos por recobrir os nanobastões por porfirinas tetrassulfonadas, com ou sem grupamentos metil-glucamina. Após o processo de recobrimento, essas ftalocianinas formaram complexos iônicos com o CTAB que recobre os nanobastões. Os complexos nanobastões-ftalocianinas foram analisados por microscopia eletrônica de transmissão e as taxas de geração de oxigênio singlete e de radical hidroxil foram investigadas. Além disso, foram utilizadas para testes in vivo e in vitro com células de melanoma melanótico (B16F10) ou amelanótico (B16G4F). As células tumorais em cultura ou os tumores em camundongos C57BL6 foram irradiados com luz em 635 nm e os tumores foram observados por 15 dias após o tratamento. Houve evidente aumento na geração de oxigênio singlete por ambos fotossensibilizadores, e maior geração de radicais livres por parte do fotossensibilizador metilglucaminado. O oposto ocorre com o fotossensibilizador sem metilglucamina. Houve, também, moderada citotoxicidade no escuro quando células foram incubadas com nanopartículas recobertas por ftalocianinas ou não. Quando ativados pela luz, os complexos ftalocianinas-nanobastões desencadearam um aumento de 5ºC no meio de cultura das células, e a morte celular observada foi extensa (91% para a linhagem B16G4F e 95% para a linhagem B16F10). Tanto os resultados in vitro quanto os in vivo indicam que as propriedades das ftalocianinas testadas são melhoradas significativamente quando elas estão complexadas aos nanobastões. Este é um estudo pioneiro por utilizar duas porfirinas tetrassulfonadas específicas e por utilizar o mesmo comprimento de onda para a ativação dos fotossensibilizadores e nanobastões. / Studies with hyperthermic tumor ablation using metallic nanoparticles have been performed on the last decades, and show promising results on tumor remission, sometimes achieving the complete cancer elimination. The same occurs regarding on treatments based on photodynamic activity of photosensitizer compounds. Studies indicate that those therapeutic interventions - hyperthermic therapy using gold nanorods and photodynamic activity with many photosensitizers - together can present a synergistic effect, and offer a great therapeutic potential, although more investigation needs to be performed before such approach could be implemented. We proposed to investigate the effect of the attachment of photosensitizers onto the surface of gold nanorods (well-characterized devices for hyperthermia generation). After nanorods synthesis through a seed-mediated method, the PDT and hyperthermia\'s efficacy was assessed separately. The method used for covering the gold nanorods with photosensitizers did not permit, in a first approach, the attachment of porphyrins onto the nanoparticles surface, but the attachment of tetrasulfonated phthalocyanines was more successful. The phthalocyanine\'s degradation rate was assessed as an indirect parameter of singlet oxygen generation. After centrifuging and washing the nanoparticles, we saw that the photosensitizers do not keep attached to the nanorods. On a second approach, we chose to recover the nanorods with two zinc phthalocyanines - with or without methyl-glucamine groups. After the recovering process, the phthalocyanines formed ionic complexes with the CTAB that is previously recovering the nanoparticles. The nanorod-phthalocyanines complexes were analyzed by TEM, and their singlet oxygen and hydroxyl radical generation yield were assessed. Furthermore, they were tested in vitro in melanoticB16F10 and amelanotic B16G4F melanoma cells, and in vivo. The tumor cells (in vitro) and the tumor tissue (in vivo) with nanoparticles were irradiated with laser (at 635 nm), and the tumor growth in mice was observed for 15 days after the laser irradiation. It is evident the increase in the singlet oxygen generation, and higher HPF activation for the glucaminated Pc, but the inverse for the other photosensitizer. It seems like there is a type I to type II switch on the action mechanism of the latter Pc. A mild cytoxocity was observed with the nanorods conjugated with photosensitizer in the dark, but when they are activated by light (and taken into account a 5ºC rise in the temperature because of the surface plasmon resonance from the gold nanorods), the cell killing is intense (91% for B16G4F cell line, and 95% for B16F10 cell line). Both in vitro and in vivo results indicate that the photodynamic properties of the phthalocyanines tested are enhanced when they are attached onto the nanorods surface. This is a novel study because we used two tetrasulfonated phthalocyanines and because we used the same wavelength to activate both the nanorods and the photosensitizers.