A terapia fotodinâmica (TFD) tem sido utilizada no tratamento de lesões neoplásicas e não-neoplásicas. Sua base é a combinação de três elementos-chave: fotossensibilizador, oxigênio molecular e luz em comprimento de onda que excite o fotossensibilizador, levando-o a gerar espécies reativas de oxigênio que causam danos à estrutura celular. Portanto, a iluminação é um dos fatores essenciais para a indução da resposta adequada. Nos casos de lesões superficiais, a iluminação é facilmente obtida através da irradiação da superfície da mesma. Contudo, no tratamento de tumores sólidos ou invasivos, é preciso recorrer à TFD intersticial, na qual fibras ópticas são inseridas no tumor. No entanto, as diferenças no perfil de emissão de luz a partir das fibras difusoras, dependem do modo de fabricação, tamanho e propriedades, o que dificulta o estabelecimento de uma dosimetria de luz apropriada. Com este estudo, buscou-se contribuir no entendimento de como a luz, emitida por uma fibra difusora, se comporta no meio túrbido, assim como no tecido biológico. Dessa forma, buscou-se prever como a luz se propaga no meio e, assim, poder estimar a dose adequada de luz que se deve entregar ao tecido para que toda uma região seja irradiada. Para isto, foi utilizada uma fibra óptica com um difusor cilíndrico de 20 mm de comprimento emissor, acoplada a um laser de diodo em 630 nm e uma fibra óptica isotrópica de coleta, para medir a intensidade de luz emitida pelo difusor em várias posições. As medidas permitiram obter uma caracterização do perfil de emissão da fibra, sendo o ar o único meio de propagação da luz nesse caso. Posteriormente à obtenção do perfil, uma solução lipídica foi utilizada como phantom de tecido biológico. As fibras foram encapadas de modo a expor somente uma seção de 1 mm de comprimento delas. Com as fibras submersas na solução do phantom, foram realizadas medidas do campo de luz gerado por este elemento de 1 mm de comprimento do difusor. A partir da caracterização da emissão do elemento, foi possível recuperar a distribuição de luz gerada por todo o difusor utilizando composições ponderadas feitas a partir deste elemento. Estas composições apresentaram melhores resultados quando foi considerada a uma prévia caracterização da fibra na ponderação da reconstrução. A TFD foi realizada em fígado de ratos sadios para a análise de uma resposta real e, com o auxílio de ferramentas computacionais, foi possível reconstruir a necrose constituída pela irradiação da fibra toda, a partir da necrose gerada por um elemento difusor de 2 mm da fibra (obtido da mesma forma que o elemento de 1 mm), com resultados também otimizados com o uso da caracterização da fibra como base para a soma ponderada. Os resultados demonstraram que, através da caracterização do perfil de iluminação da fibra difusora e da distribuição de luz em meio túrbido, foi possível definir teoricamente um padrão de necrose semelhante ao observado no modelo animal. Portanto, a reconstituição do perfil obtida possui potencial para permitir melhorias no entendimento e na dosimetria de aplicações intersticiais de luz para TFD. / Photodynamic therapy (PDT) has been used for treatment of several tumor types, and presents best results for surface lesion. Light penetration on biological tissue is one limiting factor in PDT, interfering with the treatment of invasive or solid tumors. In those cases, a possible solution is to use interstitial PDT, in which optical fibers are inserted into the tumor. Cylindrical diffusers have been used for the application of interstitial PDT. However, differences in the diffuser light emission depend on the manufacturing process, size and optical properties of the fiber, which make it difficult to establish light dosimeter. This study aims to determine the distribution of light generated by a cylindrical diffuser in a turbid medium. A solution of lipid emulsion was used as an optical phantom. An optical fiber with a cylindrical diffuser of 2 cm in length was connected to a diode laser 630 nm, and the spatial distribution of light generated by the diffuser was measured by scanning a collector optical fiber. From the measurement of the light field generated by an element (1 mm long) of a 20 mm-long cylindrical diffuser, recovery of the distribution of light generated by the entire diffuser is expected. The results obtained so far show that it is possible to reconstruct the light field of a 20 mm-long cylindrical element diffuser by measuring the light emitted simultaneously by 20 elements of 1 mm. Then, the PDT was done in rat liver to analyze a real response and, with help of computational tools, a necrosis generated by irradiation of all fiber was reconstructed, using a necrosis produced by an element 2 mm long (likewise the element 1mm long). The results showed that knowing the illumination profile of a cylindrical diffuser and the light distribution in turbid medium, it was possible to redefine a shape of necrosis from as animal model theoretically. Therefore, the reconstruction of the profile obtained has potential to improve understanding and the light dosimeter in interstitial PDT.

Silicon nanoparticles are attractive candidates for biological, photovoltaic and energy storage applications due to their size dependent optoelectronic properties. These include tunable light emission, high brightness, and stability against photo-bleaching relative to organic dyes (see Chapter 1). The preparation and characterization of silicon nanoparticle based hybrid nanomaterials and their relevance to photovoltaic and biological applications are described. The surface-passivated silicon nanoparticles were produced in one step from the reactive high-energy ball milling (RHEBM) of silicon wafers with various organic ligands. The surface structure and optical properties of the passivated silicon nanoparticles were systematically characterized. Fast approaches for purifying and at the same time size separating the silicon nanoparticles using a gravity GPC column were developed. The hydrodynamic diameter and size distribution of these size-separated silicon nanoparticles were determined using GPC and Diffusion Ordered NMR Spectroscopy (DOSY) as fast, reliable alternative approaches to TEM. Water soluble silicon nanoparticles were synthesized by grafting PEG polymers onto functionalized silicon nanoparticles with distal alkyne or azide moieties. The surface-functionalized silicon nanoparticles were produced from the reactive high-energy ball milling (RHEBM) of silicon wafers with a mixture of either 5-chloro-1-pentyne in 1-pentyne or 1,7 octadiyne in 1-hexyne to afford air and water stable chloroalkyl or alkynyl terminated nanoparticles, respectively. Nanoparticles with the ω-chloroalkyl substituents were easily converted to ω-azidoalkyl groups through the reaction of the silicon nanoparticles with sodium azide in DMF. The azido terminated nanoparticles were then grafted with monoalkynyl-PEG polymers using a copper catalyzed alkyne-azide cycloaddition (CuAAC) reaction to afford core-shell silicon nanoparticles with a covalently attached PEG shell. Covalently linked silicon nanoparticle clusters were synthesized via the CuAAC “click” reaction of functional silicon nanoparticles with α,ω-functional PEG polymers of various lengths. Dynamic light scattering studies show that the flexible globular nanoparticle arrays undergo a solvent dependent change in volume (ethanol> dichloromethane> toluene) similar in behavior to hydrogel nanocomposites. A novel light-harvesting complex and artificial photosynthetic material based on silicon nanoparticles was designed and synthesized. Silicon nanoparticles were used as nanoscaffolds for organizing the porphyrins to form light-harvesting complexes thereby enhancing the light absorption of the system. The energy transfer from silicon nanoparticles to porphyrin acceptors was investigated by both steady-state and time-resolved fluorescence spectroscopy. The energy transfer efficiency depended on the donor-acceptor ratio and the distance between the nanoparticle and the porphyrin ring. The addition of C60 resulted in the formation of silicon nanoparticle-porphyrin-fullerene nanoclusters which led to charge separation upon irradiation of the porphyrin ring. The electron-transfer process between the porphyrin and fullerene was investigated by femto-second transient absorption spectroscopy. Finally, the water soluble silicon nanoparticles were used as nanocarriers in photodynamic therapeutic application, in which can selectively deliver porphyrins into human embryonic kidney 293T (HEK293T) cells. In particular, the PEGylated alkynyl-porphyrins were conjugated onto the azido-terminated silicon nanoparticles via a CuAAC “click” reaction. The resultant PEGylated porphyrin grafted silicon nanoparticles have diameters around 13.5 ± 3.8 nm. The cryo-TEM and conventional TEM analysis proved that the PEGylated porphyrin grafted silicon nanoparticle could form the micelle-like structures at higher concentration in water via self-assembly. The UV-Vis absorption analysis demonstrated that the silicon nanoparticle could reduce the porphyrin aggregation in water which can reduce the photophysical activity of porphyrin. In addition, the nanoparticle complex was capable of producing singlet oxygen when the porphyrin units were excited by light. The cell studies demonstrated that the silicon nanoparticle could deliver the porphyrin drugs into HEK293T cells and accumulate in the mitochondria where the porphyrin could serve as an efficient photosensitizer to kill the cells via mitochondrial apoptotic pathway. / acase@tulane.edu

Current treatment for spinal metastasis involves a multimodal approach, including bisphosphonates and radiation therapy. Yet, tumour response varies considerably, thus novel treatments or combination therapies are needed to treat these metastases while preserving stability and integrity of the spinal column. Photodynamic therapy (PDT) has been shown to be successful in destroying vertebral osteolytic tumours and enhancing vertebral structure, particularly in combination with bisphosphonates. This thesis aims to evaluate the longer-term effects of PDT alone and in combination with bisphosphonate or radiation therapy on healthy vertebrae, and the short-term effects of PDT combined with radiation therapy on healthy and metastatically-involved vertebrae. The benefits of PDT on vertebral structure, both at short-term and longer-term time-points, were greatest in combination with previous bisphosphonate therapy. Similar effects, to a lesser magnitude, were seen with PDT in combination with radiation therapy. This work supports future translation of PDT for the treatment of spinal metastases.

Current treatment for spinal metastasis involves a multimodal approach, including bisphosphonates and radiation therapy. Yet, tumour response varies considerably, thus novel treatments or combination therapies are needed to treat these metastases while preserving stability and integrity of the spinal column. Photodynamic therapy (PDT) has been shown to be successful in destroying vertebral osteolytic tumours and enhancing vertebral structure, particularly in combination with bisphosphonates. This thesis aims to evaluate the longer-term effects of PDT alone and in combination with bisphosphonate or radiation therapy on healthy vertebrae, and the short-term effects of PDT combined with radiation therapy on healthy and metastatically-involved vertebrae. The benefits of PDT on vertebral structure, both at short-term and longer-term time-points, were greatest in combination with previous bisphosphonate therapy. Similar effects, to a lesser magnitude, were seen with PDT in combination with radiation therapy. This work supports future translation of PDT for the treatment of spinal metastases.

Photodynamic therapy (PDT) is a promising approach used in the treatment of cancer, age related macular degeneration, psoriasis, and other diseases. Our research is focused on the discovery of new photonucleases for use in PDT. This study evaluates the photo-induced DNA cleaving abilities of a series of acridine and phenazine-based chromophores. The extended, aromatic ring systems of these compounds are expected to intercalate between adjoining base pairs in the DNA double-helix. Once irradiated, strand breakage, or nicking of plasmid DNA is achieved at micromolar concentrations of compound (pH 7.0 and 22 °C). Our scavenger experiments show that this process occurs as a result of direct electron transfer to oxygen and/or by means of energy transfer which results in the production of singlet oxygen. Three of the photonucleases being examined were designed to chelate metal. These exhibited increased levels of DNA photocleavage in the presence of copper(II).

Photosensitizing molecules and/or metal complexes that interact with DNA via intercalation and groove binding have potential applications as molecular structural probes, as footprinting reagents and in photodynamic therapeutics. To this regard, small molecules that bind to DNA and the energetics involved in these interactions, acridine-based therapeutics, photosensitization, photodynamic therapy, phenothiazine-mediated photosensitization, DNA photocleavage reaction mechanisms and photosensitizing metal complexes are introduced in Chapter I. Next, in Chapter II, the synthesis of a photonuclease consisting of a 3,6-acridinediamine chromophore attached to four metal-coordinating imidazole rings is described. The DNA photocleavage yields, emission quantum yields, and thermal denaturation studies by this acridine-imadazole conjugate in the presence of 16 metal salts are also reported. In Chapter III is the synthesis of a bisacridine covalently tethered to a copper(II)-binding pyridine linker. Additionally, DNA photocleavage studies as well as DNA binding affinity and binding mode(s) of this bisacridine incorporating the copper(II)-binding pyridine linker are examined. The syntheses, characterization, DNA photocleavage studies, DNA thermal denaturation, and viscometric measurements of three new phenothiazinium photosensitizers are described in Chapters IV and V. Collectively, markedly enhanced DNA photocleavage yields are observed in the presence of metals (Chapters II-III) or in comparison to a parent molecule, Chapters II and IV. DNA melting isotherms show higher levels of duplex stabilization with the acridines, specifically in the presence of several metals (Chapter II-III) as well as with the phenothiazine-based ligands (Chapters IV-V). Moreover, different DNA binding modes were observed depending on metal complexation (Chapter III) and nucleic acid structure (Chapter IV). Finally, Chapter VI describes a small project implemented as a National Science Foundation pedagogical laboratory exercise in which a non-invasive procedure for DNA isolation from human cheek cells was utilized with the polymerase chain reaction to amplify alleles encoding a single nucleotide polymorphism involved in normal human color vision.

S’estudien dues estratègies sintètiques per obtenir porficensfuncionalitzats per a l'aplicació a la teràpia fotodinàmica. La introducció de substituents en el macrocicle de porficè ha de permetre modular les propietats físico-químiques i potencialment el seu efecte terapèutic en teràpia fotodinàmica. Així, es preparen dues famílies de compostos: porficens 2,7,12,17-tetrasubstituïts i porficens 9-substituïts. Així, es desenvolupa una nova ruta sintètica orientada cap a la introducció de la diversitat estructural per a obtenir porficens La primera estratègia està orientada cap a la introducció de diversitat estructural a les posicions 2, 7, 12 i 17 dels porficens. Aquesta estratègia es basa en la preparació d'un intermedi comú, el 2,7-dibromo-4H,5H-bis(trimetilsililetoximetil)tieno[3,2-b:4,5-b'] dipirrol-3,6-dicarboxilat de dietil, des del que es pot introduir una varietat de restes en el porficè utilitzant reaccions catalitzades per pal•ladi com la reacció de Suzuki. Aquesta metodologia fa innecessari el desenvolupament d'una síntesi de novo per a cada substituent. L'aplicació d'aquesta metodologia ha permès sintetitzar tres arilporficens nous: el tetra(p-metoxifenil)porficè, tetra(m-isopropoxifenil)porficè i el tetra(m-hidroxifenil)porficè. Respecte la segona família de compostos, s’estudia la N- i O-derivatització en posició 9 del 2,7,12,17-tetrafenilporficè (TPPo). Així, es sintetitzen el 9-nitro-2,7,12,17-tetrafenilporficè i el 9-amino-2,7,12,17-tetrafenilporficè que es prepara per reducció del primer. També s’obté el 9-acetoxi-2,7,12,17-tetrafenilporficè a partir del TPPo. Finalment, s’estudien les principals propietats fotofísiques dels derivats porficènics per tal de determinar la seva aplicabilitat en la teràpia fotodinàmica. Aquest treball ha donat lloc a diverses publicacions que s'inclouen com a annex. / Se estudian dos estrategias sintéticas para la obtención de porficenosfuncionalizados para su aplicación en terapia fotodinámica. La introducción de sustituyentes en el macrociclo del porficeno ha de contribuir a modular las propiedades fisicoquímicas y potencialmente su efecto terapéutico en terapia fotodinámica. Así, se preparan dos familias de compuestos: porficenos 2,7,12,17 tetrasustituidos y porficenos 9-sustituidos. En primer lugar se desarrolla una nueva ruta sintética orientada hacia la introducción de diversidad estructural para la obtención de porficenosfuncionalizados en las posiciones 2, 7, 12 y 17. Esta estrategia está basada en la preparación de un intermedio común, el 2,7-dibromo-4H,5H-bis(trimetilsililetoximetil)tieno[3,2-b:4,5-b’]dipirrol-3,6-dicarboxilato de dietilo, a partir del cual es posible introducir una gran variedad de restos funcionalizados en el porficeno utilizando reacciones catalizadas por paladio como la reacción de Suzuki. Esta metodología evita desarrollar una síntesis de novo para cada sustituyente. Aplicando esta metodología se han sintetizado tres nuevos arilporficenos: tetra-(p-metoxifenil)porficeno, tetra(m-isopropoxifenil)porficeno. y tetra(m-hidroxifenil)porficeno. Respecto la segunda familia de compuestos, se estudia la N- y la O- derivatización en la posición 9 del 2, 7 12, 17-tetrafenilporficeno (TPPo). Se sintetiza 9-nitro-2,7,12,17-tetrafenilporficeno y el 9-amino-2,7,12,17-tetrafenilporficeno que se prepara por reducción del primero. Asimismo se sintetiza el 9-acetoxi-2,7,12,17-tetrafeni-porficeno a partir de 2,7,12,17-tetrafenilporficeno (TPPo). Finalmente, se caracterizan las principales propiedades fotofísicas de dichos derivados porficénicos con el fin de determinar su aplicabilidad en la terapia fotodinámica. Este trabajo ha dado lugar a varias publicaciones que se incluyen como anexo. / Two synthetic strategies to prepare functionalized porphycenes for their application in photodynamic therapy has been envisioned. The introduction of substituents on the macrocycle of the porphycene should modulate the physicochemical properties and potentially its therapeutic effect in photodynamic therapy. According to these two strategies two families of compounds have been prepared: 2,7,12,17-tetrasubstituted porphycenes and 9-substituted porphycenes. The first strategy is based on the preparation of a common intermediate, the diethyl 2,7-dibromo-4H,5H-bis(trimethylsilyletoximethyl)thieno[3,2-b:4,5-b']dipyrrole-3,6-dicarboxylate. A variety of substituents were introduced in the porphycene using palladium catalyzed reactions such as the Suzuki reaction. This methodology avoids developing a de novo synthesis for each substituent. This new synthetic route gives access to three new arylporhycenes: tetra(p-methoxyphenyl)porphycene, tetra(m-isoproxyphenyl)porphycene and tetra(m-hydroxyphenyl)porphycene. The second family of compounds studied is characterized by the N - and O – functionalization of position 9 of the 2,7,12,17-tetraphenylporphycene (TPPo). Thus, 9-nitro-2,7,12,17-tetraphenylporphycene was obtained and reduced to 9-amino-2,7,12,17-tetraphenylporphycene. The 9-acetoxy-2,7,12,17-tetraphenylporphycene was also obtained from 2,7,12,17-tetraphenylporphycene (TPPo). Finally, the more relevant photophysical properties of these new porphycenes were characterized in order to determine its applicability in the photodynamic therapy. This work has resulted in several publications that are included as an annex.

Photodynamic therapy (PDT), which utilizes the combination of light and a photosensitizing drug to cause tissue damages, has emerged as a novel clinical approach for the treatment of numerous cancers, as well as some other non-malignant conditions. Although a few photosensitizers have been approved for clinical uses, the mechanism of drug action, especially the initial photochemical reactions that lead to the formation of the reactive oxygen species (ROS), is still not well understood. Moreover, the PDT efficiency of currently used drugs is limited due to the strong attenuation of light by tissues in the wavelength range of 630-690 nm, where these drugs are photo-activated. Photosensitizers which are sensitive to near infrared (NIR) light are believed to be able to overcome this limitation. In this thesis work, the molecular mechanism of action of indocyanine green (ICG), a potential NIR PDT drug, was investigated using our femtosecond time-resolved laser spectroscopy. Femtosecond time-resolved fluorescence decay profiles of ICG in water were obtained using the fluorescence up-conversion technique. The lifetime of ICG excited singlet state was determined to be about 150 ps, directly from the fluorescence decay kinetic traces. The excited triplet-state yield of ICG in water was found to be extremely low, according to the result of the ground-state bleaching recovery measurement. This observation is contrary to the conventional understanding that the ROS would be generated mainly from the excited triplet state of the photosensitizer and, therefore, suggests the existence of a new reaction pathway. Pump-probe transient absorption spectroscopy was applied to study the reaction between ICG and oxygen in more details. The results reveal that the formation of ICG and oxygen ground-state complexes ([ICG]_m:[O₂]_n) is a key step in the generation of the ROS. Electron transfer from the excited singlet state of ICG to oxygen has been proposed to be a possible pathway for the generation of ROS.

Photodynamic therapy (PDT), which utilizes the combination of light and a photosensitizing drug to cause tissue damages, has emerged as a novel clinical approach for the treatment of numerous cancers, as well as some other non-malignant conditions. Although a few photosensitizers have been approved for clinical uses, the mechanism of drug action, especially the initial photochemical reactions that lead to the formation of the reactive oxygen species (ROS), is still not well understood. Moreover, the PDT efficiency of currently used drugs is limited due to the strong attenuation of light by tissues in the wavelength range of 630-690 nm, where these drugs are photo-activated. Photosensitizers which are sensitive to near infrared (NIR) light are believed to be able to overcome this limitation. In this thesis work, the molecular mechanism of action of indocyanine green (ICG), a potential NIR PDT drug, was investigated using our femtosecond time-resolved laser spectroscopy. Femtosecond time-resolved fluorescence decay profiles of ICG in water were obtained using the fluorescence up-conversion technique. The lifetime of ICG excited singlet state was determined to be about 150 ps, directly from the fluorescence decay kinetic traces. The excited triplet-state yield of ICG in water was found to be extremely low, according to the result of the ground-state bleaching recovery measurement. This observation is contrary to the conventional understanding that the ROS would be generated mainly from the excited triplet state of the photosensitizer and, therefore, suggests the existence of a new reaction pathway. Pump-probe transient absorption spectroscopy was applied to study the reaction between ICG and oxygen in more details. The results reveal that the formation of ICG and oxygen ground-state complexes ([ICG]_m:[O₂]_n) is a key step in the generation of the ROS. Electron transfer from the excited singlet state of ICG to oxygen has been proposed to be a possible pathway for the generation of ROS.

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 / #8217 / 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.