Photodynamic therapy (PDT) is a new form of cancer treatment that uses the localized delivery of light and a photosensitizing drug, which is selectively retained in tumor tissue, to cause photochemically induced cell death. Although PDT mediated by the sensitizer Photofrin (Ph-PDT) is currently in Phase III trials for a number of human cancers, the exact mechanism(s) involved in PDT induced cytotoxicity is not fully understood. Also, Photofrin has a number of drawbacks including extended cutaneous photosensitization and low absorption in the red region of the spectrum. This has lead to the search for improved sensitizers. In vitro, tumor cells resistant to PDT have been developed from PDT sensitive cell lines to examine the mechanism(s) of PDT action. In this work, the sensitivity of RIF-1 murine fibrosarcoma cells and RIF-1 derived Ph-PDT resistant RIF-8A cells was examined following several damaging agents including PDT mediated by the novel Ruthenium phthalocyanine photosensitizer JM2929 (JM2929-PDT), UV, gamma-radiation, and hyperthermia. Gamma-radiation sensitivity of two other RIF-1 derived Ph-PDT resistant variants, CPR-C1 and RIF-P16CL8, was also examined. RIF-8A cells showed cross resistance to UV but increased sensitivity to gamma-rays compared to RIF-1 cells. RIF-1 and RIF-8A cells showed similar sensitivity to JM2929-PDT and hyperthermia. It is possible that Ph-PDT induces a "UV -like" component of damage and/or there is some overlap in the pathways for the repair of UV and Ph-PDT induced damage, but not JM2929-PDT, hyperthermia, and ionizing radiation damage in RIF-1 and RIF-8A cells. A cross resistance to gamma-rays was observed for CPR-C1 but not RIF-P16CL8 cells. Since Ph-PDT resistant CPR-C1 cells, but not RIF-8A cells or RIF-P16CL8 cells, show a cross resistance to gamma radiation, these results suggest that the cellular changes required for RIF-8A, RIF-P16CL8, and CPR-C1 cells to become resistant to Ph-PDT are different. Survival of RIF-1 and RIF-8A cells following gamma-rays in the presence of either Photofrin or JM2929 was also examined. Results suggest sensitization of RIF-1 cells, but not RIF-8A cells, to gamma-radiation in the presence of Photofrin. Gamma-radiation in the presence of JM2929 had no sensitizing effects on the survival of RIF-1 and RIF-8A cells. DNA repair of a UV-damaged reporter gene was also examined in untreated as well as Ph-PDT, JM2929-PDT, UV, cisplatin, and hyperthermia pretreated RIF-1 and RIF-8A cells. Results suggest an increased repair of UV damaged DNA in untreated RIF-1 cells compared to untreated RIF-8A cells. Ph-PDT, JM2929-PDT, and UV pretreatments resulted in an increased reactivation of a UV damaged reporter gene in RIF-1 cells compared to RIF-8A cells. Enhanced reactivation of a UV damaged reporter gene was not observed in either RIF-1 or RIF-8A cells following cisplatin or hyperthermia pretreatment. Enhanced expression of an undamaged reporter gene was greater in RIF-8A cells compared to RIF-1 cells following Ph-PDT pretreatment, but similar to RIF-1 cells following pretreatment with all other agents. These results suggest that the relation between survival, DNA repair of an actively transcribed gene, and transcriptional enhancement of an actively transcribed gene, varies in RIF-1 and RIF-8A cells depending on the damaging agent used. However, decreased reactivation of a UV damaged reporter gene in RIF-8A cells may be related to Ph-PDT and UV resistance seen in RIF-8A cells. / Thesis / Master of Science (MSc)

Yes / Osteoarthritis (OA) is a degenerative disease leading to the breakdown of the hyaline cartilage between a varieties of diarthrodial joints such as the knee joint, carpals of the wrist and etc. When the cartilage is affected by trauma or wear and tear, Osteolysis may occur; broken debris of cartilage found within the synovial fluid may be recognised as a pathogen and therefore, the body’s autoimmune response will directly target the cartilage for destruction. Cytokines are proteins/peptides of glycoproteins that are secreted by cells and are involved in interaction and communication between cells. Transforming Growth Factors Beta 1 (TGF-β1) is one of well-known cytokines and had shown many effects on cellular biology including simulation or inhibition of cell proliferation, differentiation, production of extracellular matrix (ECM), remodelling, and producing both hormones and growth factors. On the other hand, Photonics recently play an important role for treatment of OA. The main aim of this review article is to investigate the effect of TGF-β1 in treatment of OA. Other important aim of this work is to explore the broad applications of optics and photonics in biomedical applications including treatment of OA. Biomedical applications of photonics have broad aspects including laser, carbon nanotubes (CNTs), quantum dots (QDs) and graphene and photodynamic therapy (PDT) which discussed in this review paper.

This thesis describes the study of a series of multimetallic supramolecules containing varied metals and ligands, synthesized by a building block method and characterized by mass spectrometry, electronic absorption spectroscopy, and electrochemistry. Incorporating different functional units into complex systems allowed these multimetallic supramolecules to perform various light activated tasks including DNA cleavage and hydrogen generation from water. The complex [({(bpy)₂Os(dpp)}₂Ru)₂(dpq)](PF₆)₁₂ and [{(bpy)₂M(dpp)}₂Ru(BL)PtCl₂](PF₆)₆ were synthesized (M = Os^II or Ru^II; BL = dpp or dpq; bpy = 2,2^'-bipyridine, dpp = 2,3-bis(2-pyridyl)pyrazine, dpq = 2,3-bis(2-pyridyl)quinoxaline). The building blocks displayed varied electrochemical properties upon complexation. The bridging ligands dpp and dpq display their reduction potentials shifted to less negative values when they changed from monochelating to bischelating. The electronic absorption spectra of the multimetallic systems displayed transitions of each contributing chromophore, with overlapping metal to ligand charge transfer (MLCT) transitions in visible region of spectrum. Spectroelectrochemistry revealed the nature of MLCTs and helped to identify fingerprint features of complex supramolecules. Photophysical measurements include emission spectroscopy with quantum yield measurements and emission lifetime measurements. Photophysical data provided detailed information to aid in developing an understanding of excited state properties of these complexes. Supported by the electrochemical data and spectroelectrochemistry, the hexametallic complex was suggested to have a HOMO localizing in the peripheral Os and a LUMO localizing in the central dpq, separating by a Ru energy barrier. This research systematically investigated photophysical properties of some building blocks and the mixed-metal, mixed-ligand supramolecules constructed by a variety of building blocks coupling light absorbing subunits to a reactive Pt metal center. Preliminary studies suggested [{(bpy)₂Ru(dpp)}₂Ru(dpq)PtCl₂](PF₆)₆ was a photocatalyst for H2 production from water in the presence of a sacrificial electron donor. The complex [{(bpy)₂Ru(dpp)}₂Ru(dpq)PtCl₂](PF₆)₆ had been studied for its catalytic ability in generating hydrogen and was found to have 34 product turnovers after 3 h photolysis. Photolysis and gel electrophoresis revealed that the tetrametallic complexes were able to bind to and then photocleave DNA through an oxygen mediated mechanism. The independence of ionic strength variation when [{(bpy)₂Ru(dpp)}₂Ru(dpp)PtCl₂](PF₆)₆ interacted with DNA, suggested the covalent interaction nature of the complex. These results suggest future work on understanding the excited state properties of supramolecular complexes is suggested. The designs of future photocatalysts for hydrogen production from water and anticancer photodynamic therapy drugs are also proposed. / Ph. D.

The development of therapeutic drugs or drug systems that enhance a cancer patient's quality of life during treatment is a primary goal for many researchers across a wide range of disciplines. Many investigators turn to nanoparticles (~50-200 nm in size) that tend to accumulate in tumor tissues in order to deliver active drug compounds to specific sites in the body. This targeted delivery approach would reduce the total body effects of current cancer drugs that result in unwanted (sometimes painful and even fatal) side effects. One of the main obstacles however, is ensuring that drugs incorporated into the nanoparticles are anchored such that premature drug release is prohibited. Also, while it is important to ensure strong drug-nanocarrier interactions, the nanocarrier must be able to release the drug when it has reached its biological target. We have developed a nanocarrier that provides a platform for drug systems that could achieve this drug release via the use of a light "trigger". Metal-Organic Frameworks (MOFs) are a relatively new class of often highly porous materials that act as "sponges" for the absorption of various small molecules. MOFs are so named because they consist of metal clusters that are linked by organic compounds to form networked solids that are easily tuned based on the choice of metal and organic "linker". We have developed a MOF nanocarrier incorporating benign zirconium (IV) metal clusters bridged by an organic component that changes shape when illuminated with a light source. The resulting material is therefore not stable upon irradiation due to the organic linker shape change that disturbs the MOF structure and causes it to degrade. When loaded with drugs, this photo-enhanced degradation results in the release of the cargo thereby, providing a handle on controlling drug release with the use of a light trigger. We have demonstrated that in the presence of the MOF nanocarrier incorporating 5-fluorouracil (a clinically available cancer drug), very low toxicity to human breast cancer cells is observed in the dark, however, cell death occurs in the presence of a light source. These reports offer a model MOF nanocarrier system that could be used to incorporate various drugs and therefore tune the system to an individual patient's needs. Furthermore, we also developed a material that is capable of providing magnetic resonance imaging (MRI) contrast by changing the metal to manganese (II). MRI contrast agents are compounds that serve to either darken or brighten an MRI image based on the agent used and therefore they aid in clinical diagnosis by making internal abnormalities easier to spot. Currently gadolinium (III) complexes are the most widely used contrast agents; however, the toxicity of gadolinium (III) has been shown to be responsible for the development of nephrogenic systemic fibrosis in some patients. This manganese material has also shown useful for the attachment of fluorescent dyes that can aid in the benchtop optical diagnosis of biopsies. These reports provide a basis for developing ways to offer controlled drug delivery in cancer patients and to aid in cancer diagnosis using MOF materials, in an effort to reach the goals of comfortable cancer treatment. / Ph. D. / The development of therapeutic drugs or drug systems that enhance a cancer patient’s quality of life during treatment is a primary goal for many researchers across a wide range of disciplines. Many investigators turn to nanoparticles (~50-200 nm in size) that tend to accumulate in tumor tissues in order to deliver active drug compounds to specific sites in the body. This targeted delivery approach would reduce the total body effects of current cancer drugs that result in unwanted (sometimes painful and even fatal) side effects. One of the main obstacles however, is ensuring that drugs incorporated into the nanoparticles are anchored such that premature drug release is prohibited. Also, while it is important to ensure strong drug-nanocarrier interactions, the nanocarrier must be able to release the drug when it has reached its biological target. We have developed a nanocarrier that provides a platform for drug systems that could achieve this drug release via the use of a light “trigger”. Metal-Organic Frameworks (MOFs) are a relatively new class of often highly porous materials that act as “sponges” for the absorption of various small molecules. MOFs are so named because they consist of metal clusters that are linked by organic compounds to form networked solids that are easily tuned based on the choice of metal and organic “linker”. We have developed a MOF nanocarrier incorporating benign zirconium (IV) metal clusters bridged by an organic component that changes shape when illuminated with a light source. The resulting material is therefore not stable upon irradiation due to the organic linker shape change that disturbs the MOF structure and causes it to degrade. When loaded with drugs, this photo-enhanced degradation results in the release of the cargo thereby, providing a handle on controlling drug release with the use of a light trigger. We have demonstrated that in the presence of the MOF nanocarrier incorporating 5-fluorouracil (a clinically available cancer drug), very low toxicity to human breast cancer cells is observed in the dark, however, cell death occurs in the presence of a light source. These reports offer a model MOF nanocarrier system that could be used to incorporate various drugs and therefore tune the system to an individual patient’s needs. Furthermore, we also developed a material that is capable of providing magnetic resonance imaging (MRI) contrast by changing the metal to manganese (II). MRI contrast agents are compounds that serve to either darken or brighten an MRI image based on the agent used and therefore they aid in clinical diagnosis by making internal abnormalities easier to spot. Currently gadolinium (III) complexes are the most widely used contrast agents; however, the toxicity of gadolinium (III) has been shown to be responsible for the development of nephrogenic systemic fibrosis in some patients. This manganese material has also shown useful for the attachment of fluorescent dyes that can aid in the benchtop optical diagnosis of biopsies. These reports provide a basis for developing ways to offer controlled drug delivery in cancer patients and to aid in cancer diagnosis using MOF materials, in an effort to reach the goals of comfortable cancer treatment.

Two photon absorption (TPA) is the near simultaneous absorption of two photons of light to achieve an electronically excited state. It has led to huge advances in microscopy and microfabrication due to its quadratic dependence on the local light intensity. This thesis describes the design, synthesis and application of dyes with strong TPA properties, and as such is divided into three chapters. The first introduces the theory and measurement of TPA as well as structure-property relationships known to maximise the efficiency of TPA. The subsequent chapters present explorations of the application of these dyes in biological applications; namely two-photon uncaging and two-photon photodynamic therapy. A recurring theme in my research is the discussion and evaluation of strategies for improving the compatibility of organic macromolecules with biological systems. Uncaging is the use of photolysis to achieve a rapid increase in the local concentration of a physiologically active species via a photoremovable protecting groups. Photoremovable protecting groups are covalently attached to the physiologically active species, thus rendering it inactive. At the desired time and location, a light dose releases the molecule in its active form. There are many compounds known to uncage following photoexcitation, but there are few examples of caging groups which exhibit both strong two-photon absorption properties and highly efficient uncaging. Chapter 2 discusses the rational design of such groups through the development of a new mechanism for uncaging, in which a photoinduced electron transfer (PeT) between a two-photon-excited electron donor and an electron acceptor/release group drives the uncaging event. Photodynamic therapy (PDT) is a treatment for neoplastic disorders such as cancer in which localised cell death is induced through photoexcitation of a sensitiser. Following light absorption, the photosensitiser enters a relatively long-lived excited state which reacts with cellular oxygen to produce its highly cytotoxic singlet form. The main challenges of the field are to achieve deep penetration of light into tissue and to reduce coincident damage to unaffected tissue by light scattering during irradiation. In 2008, the Anderson group reported the development of PDT photosensitisers with highly efficient two-photon absorption as well as high singlet oxygen quantum yields. Chapter 3 discusses strategies for improving the pharmacokinetics and defining the sub-cellular localisation of these photosensitisers.

Ce travail de thèse a été consacré au développement d’un système multicouche constitué de nanoparticules dopées par des ions terres rares (le cœur), entourées d’une première couche cristalline non dopée, permettant de préserver les propriétés optiques du cœur. Une coquille de silice mésoporeuse est ensuite déposée, permettant l’incorporation d’un photosensibilisateur (ZnPc) via les pores de la couche de silice pour une application thérapeutique : la photothérapie dynamique. Différentes matrices ont été étudiées à savoir Y2O3, KY3F10 et NaYF4. Ces matrices ont été codopées Yb3+/Er3+ afin d’obtenir des émissions dans le visible sous l’effet d’une excitation infrarouge (upconversion), le but ultime étant d’exciter le ZnPc in situ. Chacune des matrices a été caractérisée d’un point de vue structural et morphologique dans une première partie, et d’un point de vue spectroscopique dans une deuxième partie. La structure cœur-coquille cristalline renforce l’émission rouge issue du niveau 4F9/2 de l’Er, effet déduit de l’analyse des spectres et de la dynamique de luminescence.La détection de l’oxygène singulet a été réalisée par le protocole de « bleaching » en présence ou pas du ZnPc en évaluant l’intensité de fluorescence de l’ABDA. / This work has been dedicated to the development of a multistep system composed by rare earths doped nanoparticles (core), enclosed by a first undoped crystalline layer (core-shell), serving as protection of the optical properties of the core. Within a shell of mesoporous silica allowing the loading of the photosensitizer (ZnPc) via the pores of the shell of silica for a therapeutic application: the photodynamic therapy. Various lattices were studied namely Y2O3, KY3F10 and NaYF4. These lattices were codoped with Yb3+ and Er3+ ions to obtain emissions in the visible under an infrared excitation (up conversion), the ultimate purpose being to excite in situ ZnPc. Each of these lattices was characterized from a structural and morphological point of view in the first part and, in the second part, spectroscopic studies are developed. The core-shell enhances the red emission stemming from the level 4F9/2 of Er, effect deduced from the analysis of spectra and the dynamics of luminescence. The detection of the singlet oxygen was realized in vitro by the study of the bleaching of ABDA fluorescence. The comparison of the results for nanoparticles loaded with ZnPc and unloaded ones allows demonstrating the generation of singlet oxygen by exciting in the infrared region of the spectra thanks to the efficient upconversion processes occurring in the rare earth doped materials.

Dans le traitement du cancer, l'utilisation de nanoparticules comme vecteurs de molécules thérapeutiques est de plus en plus étudiée dans le but de limiter les effets secondaires toxiques dus à l'administration systémique de molécules thérapeutiques libres. En effet, de par leur taille, les nanoparticules sont capables de s'accumuler de façon plus importante dans les tumeurs que dans les tissus sains. Fonctionnalisées avec des agents de ciblage spécifiques des cellules cancéreuses, leur accumulation dans les tumeurs peut être optimisée. Les nanoparticules de silicium poreux sont particulièrement intéressantes pour des applications biomédicales car elles sont biocompatibles et biodégradables in vivo. Elles possèdent également des propriétés physico-chimiques et photophysiques intéressantes, telles que leurs propriétés texturales, leur photoluminescence intrinsèque et leur capacité à produire de 1O2. L'objectif de cette thèse a été d'étudier le potentiel de nanoparticules de silicium poreux fonctionnalisées pour la thérapie photodynamique, et comme vecteur d'agent de chimiothérapie. Dans un premier temps, la préparation et la caractérisation physico-chimique des nanoparticules de silicium poreux a été réalisée. Les nanoparticules ont ensuite été fonctionnalisées avec un agent de ciblage, le mannose, et des molécules photosensibilisatrices, des porphyrines. Leur utilisation en imagerie et en thérapie photodynamique du cancer avec une excitation mono- ou biphotonique a été montrée in vitro sur des cellules de cancer du sein. Une autre étude a porté sur la vectorisation d'un agent anti-cancéreux hydrophobe, la camptothécine, par des nanoparticules de silicium poreux fonctionnalisées avec des anticorps pour le ciblage de cellules de glioblastome, de neuroblatome et de lymphocytes. Enfin, une étude plus fondamentale de caractérisation de la texture interne du silicium poreux combinant expérience et modélisation moléculaire est présentée. / In cancer therapy, the use of nanoparticles as drug nanovectors is intensively studied in order to overcome the toxic side effects due to the systemic administration of the anti-cancer molecules. Indeed, because of their size, nanoparticles tend to accumulate in tumor cells more importantly than in healthy cells. When functionalized with targeting agents specific to cancer cells, their accumulation in tumors can be optimized. Porous silicon nanoparticles are particularly interesting for biomedical applications because they are biocompatible and biodegradable in vivo. They also have interesting physico-chemical and photochemical properties, such as their textural properties, their intrinsic photoluminescence and their ability to produce 1O2. The goal of this thesis was to study the potential of functionalized porous silicon nanoparticles for photodynamic therapy, and as nanovectors for chemotherapeutic agents. In this purpose, we first studied the preparation and the physico-chemical characterization of the porous silicon nanoparticles. Then, the nanoparticles were functionalized with a targeting agent, mannose, and porphyrin photosensitizers. Their use in imaging and in photodynamic therapy of cancer under 1-photon or 2-photon excitation was shown in vitro on breast cancer cell lines. An other study consisted in the vectorization of a hydrophobic anti-cancer drug, camptothecin, by porous silicon nanoparticles functionalized with antibodies to specifically target glioblastoma, neuroblastoma and lymphocyte cells. Finally, a study of the internal texture of porous silicon combining experiments and molecular modelization is presented.

A protoporfirina IX (PpIX) é uma substância fotodinâmicamente ativa, entretanto, devido a sua alta lipofilia apresenta dificuldades para penetrar nas camadas mais profundas da pele, não sendo administrada topicamente. Assim, o objetivo deste trabalho é estudar a influência de diferentes tipos de dendrímeros de poliamidoamina (PAMAM) na solubilidade, penetração cutânea e penetração celular da PpIX com o intuito de melhorar a terapia fotodinâmica tópica (TFD) com esta substância. Os estudos de solubilidade da PpIX na presença dos dendrímeros PAMAM G4, G4.5 e G4-OH mostraram que o dendrímero PAMAM G4.5, foi o que solubilizou a maior quantidade de PpIX seguido do PAMAM-OH G4> PAMAM G4 > tampão HEPES. A partir dos estudos de solubilidade foi possível identificar que a PpIX na presença dos dendrímeros PAMAM G-4.5 e G4-OH apresentam diagramas de solubilidade tipo BS, e BI quando na presença do PAMAM G4. Os estudos de varredura em espectroscopia UV-VIS e fluorescência indicaram a presença de agregados principalmente nos complexos PpIX-PAMAM G-4.5 obtidos. Os complexos solúveis PpIX-PAMAM G4-OH apresentaram tamanho médio de 13,2 nm, e potencial zeta de -3,41. Já os complexos com o dendrímero PAMAM G-4.5 apresentaram uma população bidispersa, com tamanhos de 31 e 391 nm e um potencial zeta de -17,3. A análise de DSC e espectroscopia de infravermelho com transformada de Fourrier mostraram alterações nas características da PpIX em solução e quando complexada com os dendrímeros. Nos estudos de permeação e retenção cutânea passiva com os complexos contendo 0,006 mg/mL de PpIX, estes não aumentaram a penetração cutânea do fármaco. No entanto, a complexação permitiu que maiores quantidades de PpIX ficassem disponíveis na solução doadora, e um aumento significativo na penetração da PpIX foi observado quando experimentos com 1 mg/mL de PpIX complexada foram conduzidos; ademais notou-se uma possível ação promotora do dendrímero PAMAM G4-OH. Nos estudos de iontoforese verificou-se que a corrente elétrica não aumentou a penetração da PpIX em relação aos experimentos passivos quando o complexo PpIX-PAMAM G4.5 foi estudado, provavelmente devido a maior liberação da PpIX do complexo na presença da corrente elétrica, aumentando os agregados que são grandes o suficientes para não penetrar na pele. Por outro lado, a iontoforese anódica aumentou a penetração do complexo PpIX-PAMAM G4-OH por eletrosmose até as camadas mais profundas da pele, levando a PpIX inclusive até a solução receptora. Os estudos de cultura de células tumorais confirmaram a capacidade dos dendrímeros em aumentar a penetração da PpIX através de membrana. A complexação melhorou a distribuição da PpIX no interior das células e aumentou significativamente a fotocitotoxicidade desta porfirina. / Protoporphyrin IX (PpIX) is a photodynamic active drug that is not topically administered due to its high lipophilicity and consequent low penetration in deep skin layers. The aim of this work is to study the influence of different types of polyamidoamine dendrimers (PAMAM) in PpIX solubility, skin and cell penetration to enhance topical photodynamic therapy (PDT). The solubility studies of PpIX in the presence of PAMAM dendrimers G4, G4.5 and G4-OH demonstrated that the G4.5 solubilized the greatest amount of PpIX, followed by PAMAM G4-OH> PAMAM G4> HEPES buffer. Solubility studies showed that the PpIX in the presence of PAMAM dendrimers G-4.5 and G4-OH presented solubility diagrams of BS type, and BI type in the presence of PAMAM G4. Studies of scanning UV-VIS and fluorescence indicated the presence of aggregates mainly in PpIX-PAMAM G-4.5 solution. The PpIX-PAMAM G4-OH complexes showed a mean size of 13.2 nm and zeta potential of -3.41; the complexes with PAMAM dendrimer G-4.5 had a bi-dispersed population, with sizes of 31 and 391 nm and a zeta potential of -17.3. The analysis of DSC and Fourier transform infrared Fourier showed changes in the characteristics of PpIX when it was complexed with the dendrimers. Passive skin permeation and retention studies with the complexes containing 0.006 mg/ml PpIX had not increased PpIX penetration, however, the complexation allowed greater amounts of PpIX to become available in the donor solution, and experiments with 1 mg/mL of PpIX increased significantly the penetration of the drug. Moreover, dendrimer PAMAM G4-OH seemed to act as a penetration enhancer. Iontophoresis did not increase skin penetration of PpIX compared to passive studies when the PpIX-PAMAM G4.5 was studied, likely because the electric current increased PpIX release from the complexes, forming aggregates large enough to not penetrate through the skin. On the other hand, anodic iontophoresis increased significantly the penetration of the PpIX-PAMAM G4-OH by eletrosmosis, spreading the drug to deep skins layers, also reaching the receiver solution. Studies in cell culture confirmed the ability of dendrimers to increase the penetration of PpIX through the membrane. The complexation improved the distribution of PpIX within the cells and significantly increased photocytotoxic of the porphyrin.

Ftalocianina de alumínio-cloro (AlClPc) é um fotossensibilizador de segunda geração em terapia fotodinâmica (TFD) caracterizado por seu caráter anfifílico e tendência de auto-agregação em meio aquoso, o que prejudica seu potencial de aplicação. O aCHC é um substrato de transportadores de monocarboxilato (MCT) superexpresso em células de MCF-7. Objetivando a solubilização da AlClPc e aumento de internalização em tecidos neoplásicos nos propomos aqui o uso de DSPC e DOPC em diferentes proporções para formar vesículas lipidicas mistas (LV) na presença de aCHC como sistemas veiculadores de fármaco. Lv foi preparado pelo método de injeção etanólica e formou vesículas de dimensões nanométricas (aproximadamente 100 nm) com bom índice de polidispersão, valores negativos de potencial zeta e estáveis em meio aquoso por mais de 50 dias. AlClPc se complexou com o fosfato das LV o que conferiu uma localização interfacial às moléculas de AlClPc como demonstrado pelos resultados de supressão de fluorescência. Medidas de anisotropia, fluorescência estática e resolvida no tempo corroboram com estes resultados e demonstram que a auto-agregação da AlClPc ocorre mesmo em lipossomas. Entretanto, a veiculação da AlClPc por LV em carcinoma de células escamosas oral (OSCC) levou a um processo de desagregação demonstrado por (FLIM). Este incrível comportamento é novo e aumenta o conhecimento científico sobre o mecanismo intracelular de ação de fotossensibilizadores em TFD. Em TFD, ambos os sistemas LVIII+AlClPc e LVIII+AlClPc+aCHC não apresentaram toxicidade no escuro no período de incubação de 3 h com as concentrações de lipídios, AlClPc e aCHC iguais a 0,15 mmol/L, 0,5 umol/L e 10,0 umol/L, respectivamente. De maneira inesperada, o sistema LVIII+AlClPc foi mais eficiente em TFD que o sistema LVIII+AlClPc+aCHC, devido ao caráter antioxidante do aCHC. Estes resultados abrem uma nova perspectiva do potencial uso de LV-AlClPc para o tratamento fotodinâmico. / Aluminum phthalocyanine chloride (AlClPc) is a second-generation photodynamic therapy (PDT) photosensitizer characterized for its high hydrophobicity and self-aggregation tendency in aqueous media, which hampers its potential application. The aCHC is a monocarboxylate transporter substrate (MCT) overexpressed in MCF-7 line cell. Aiming at AlClPc solubilization and uptake increase we proposed here the use of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) at different proportions to form mixed lipid vesicles (LV) in presence of aCHC as a drug delivery system. LV were prepared by ethanol injection method and formed nano-sized vesicles (about 100 nm) with suitable polydispersity index, negative zeta potential, and stable in aqueous medium for at least 50 days. AlClPc strongly interacts with LV (high binding constant values), especially due to aluminum-phosphate specific interactions, which gives a surface localization to AlClPc molecules as demonstrated by fluorescence quenching data. Anisotropy, static and time-resolved fluorescence measurements corroborated with these results and demonstrated that AlClPc self-aggregation occurred even in the liposomes. However, formulation uptake by oral squamous cell carcinoma (OSCC) the AlClPc suffered a disaggregation process demonstrated by fluorescence life-time imaging microscopy. This amazing behavior is new and increases the scientific knowledge about the intracellular mechanism of action of PDT photosensitizers. In PDT, both systems LVIII+AlClPc e LVIII+AlClPc+aCHC did not show toxicity in the dark at 3 h incubation period with lipid, AlClPc, and aCHC concentrations equal at 0,15 mmol/L, 0,5 umol/L, and 10,0 umol/L, respectively. Unexpectedly, the system LVIII+AlClPc was more effective in PDT that the LVIII+AlClPc+aCHC, devido ao caráter antioxidante do aCHC due to the antioxidant character of aCHC. These results open a new perspective to the potential use of ALClPc-LV formulations for photodynamic treatment.

Neste estudo foi proposto um modelo de formação de biofilme fúngico formado por Candida albicans em dois diferentes substratos associado ou não à glicose para o estudo dos efeitos da terapia fotodinâmica (PDT) utilizando o azul de metileno (AM) como fotossensibilizador, avaliando os efeitos de dois comprimentos de onda distintos incluindo a investigação na captação do AM quando sensibilizados previamente com a glicose. Foi avaliada a susceptibilidade da levedura em suspensão de células crescidas por 24 h (início da fase estacionária) com e sem a adição prévia da glicose. Foram investigadas duas linhagens de C. albicans para a padronização do biofilme, ATCC 90028 e ATCC 10231. Com os biofilmes formados, avaliamos a captação de AM para determinarmos a mais eficiente concentração e tempo de pré-irradiação (PIT). Por último, avaliamos sua sensibilidade à PDT em dois comprimentos de onda distintos (λ = 645 nm e λ = 660 nm) em função do tempo de irradiação (potência = 16 mW, taxa de fluência = 127,3 mW/cm2). A linhagem de célula que possibilitou a formação de biofilme foi a ATCC 10231 em discos de hidrogel. A menor concentração que possibilitou uma melhor captação do AM foi de 500 μM com um PIT de 30 min e em contato prévio com a glicose 50 mM por 90 min. O comprimento de onda mais eficiente, que promoveu redução em leveduras e biofilmes foi o de λ = 660 nm, reduzindo melhor no estudo de leveduras quando sensibilizados com glicose. Nos biofilmes, a redução foi iniciada mais precocemente sem a adição de glicose (com 6 min de irradiação e fluência = 46 J/cm2), porém, em 12 min (fluência = 92 J/cm2) o grupo com glicose passou a ter maior eficiência. / In this study we propose a fungal biofilm model of Candida albicans developed on different substrates for photodynamic effect of photodynamic therapy (PDT) study, using the methylene blue (MB) as photosensitizer, evaluating the effects on two wavelengths distinct, including investigating the difference in uptake of MB when sensitized with glucose. Susceptibility was evaluated in the yeast cell suspension with or without the prior addition of glucose to cells grown for 24 h (early stationary phase). Two strains of C. albicans were investigated for the standardization of the biofilm, ATCC 90028 and ATCC 10231. In biofilms, we evaluated the uptake of MB including cell suspension to determine the most efficient concentration and pre-irradiation time (PIT). Finally, we assess its sensitivity to PDT in two distinct wavelengths (λ = 645 nm and λ = 660 nm) as a function of irradiation time (power = 16 mW, fluence rate = 127,3 mW/cm2). The ATCC strain that allowed the biofilm formation was ATCC 10231 on hydrogel disks. The lowest concentration that enabled better uptake of MB was 500 μM with a PIT 30 min and prior contact with 50 mM glucose f or 90 min. The most effective wavelength, which promoted a reduction in biofilms and yeasts was λ = 660 nm, reducing yeast best when primed with glucose and reduction in biofilms started earlier without the addition of glucose (6 min irradiation, fluence = 46 J/cm2), but at 12 min (fluence = 46 J/cm2) the glucose group now has greater efficiency.