Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / O tratamento das doenÃas ocasionadas por biofilmes orais envolve basicamente a remoÃÃo mecÃnica e o uso de antibiÃticos e agentes anti-sÃpticos os quais podem originar cepas resistentes aos antimicrobianos tradicionais. A Terapia FotodinÃmica Antimicrobiana (TFDA) apresenta-se como uma opÃÃo alternativa ao tratamento clÃssico, promovendo a morte bacteriana por meio da fotossensibilizaÃÃo dos componentes microbianos. Este estudo verificou a aÃÃo antimicrobiana da terapia fotodinÃmica sobre biofilmes orais produzidos in vitro e in situ utilizando um diodo emissor de luz (LED) associado ao fotossensibilizador azul de orto-toluidina (TBO). No estudo in vitro, biofilmes de Streptococcus mutans UA159, foram formados sobre discos de hidroxiapatita utilizando um modelo de banhos de cultura e submetidos à TFDA apÃs 5 dias. Para o estudo in situ, vinte e um voluntÃrios foram previamente selecionados para utilizar dispositivos intra-orais palatinos contendo 8 blocos de dentina humana durante 7 dias. SoluÃÃo de sacarose 10% foi gotejada sobre os blocos dentais 8 vezes ao dia. O biofilme formado em um dos lados do dispositivo recebeu tratamento da TFDA, e o lado oposto serviu como grupo controle. O material coletado passou por um processo de disrupÃÃo para a dispersÃo das cÃlulas e diluiÃÃo em sÃrie decimal de 10-1 a 10-4. Em ambos os experimentos, meios de cultura especÃficos para o crescimento de estreptococos totais e estreptococos do grupo mutans foram inoculados e incubados em condiÃÃes ideais para o crescimento desses microrganismos. ReduÃÃes significativas acima de 99,99% (p<0.05) foram observadas na viabilidade das colÃnias de S. mutans UA159 quando expostos ao TBO e LED no estudo in vitro. Entretanto, nos biofilmes formados in situ e submetidos Ãs mesmas condiÃÃes experimentais, nÃo foram verificadas diferenÃas estatisticamente significantes (p&#8805;0.05) da contagem microbiana quando comparadas ao grupo controle. Portanto, podemos concluir que a TFDA foi efetiva na reduÃÃo microbiolÃgica de S. mutans UA159 crescidos em modelo de formaÃÃo de biofilme in vitro, mas, pouco efetiva sobre biofilmes de estreptococos orais formados in situ. / The treatment of diseases caused by oral biofilms involves mechanical removal and use of antibiotics and antiseptics which can lead to problems of bacterial resistance. Photodynamic antimicrobial chemotherapy (PACT) represents an alternative option to conventional treatment, promoting bacterial killing by photo-sensitization of microbial components. This study assessed the antimicrobial action of photodynamic therapy on oral biofilms produced in vitro and in situ using a light emitting diode (LED) associated with the photosensitizer toluidine blue O (TBO). Biofilms of Streptococcus mutans UA159 were grown on hydroxyapatite discs immersed in bathing culture and submitted to PACT after 5 days. For the in situ study, twenty-one volunteers were previously selected to use intra-oral palatal appliances containing eight blocks of human dentin during 7 days. Sucrose solution (10%) was dripped onto the dental blocks 8 times a day. The biofilm formed on one side of the device received treatment (PACT), and the opposite side acted as control. The collected material has gone through a process of disruption to the dispersion of cells and diluted in decimal series (10-1 to 10-4). In both experiments, specific culture media for the growth of total streptococci and mutans streptococci were inoculated and incubated under optimal conditions for growth of these microorganisms. Significant reductions in excess of 99.99% (p<0.05) were observed in the viability of colonies of S. mutans UA159 when exposed to TBO and LED on in vitro study. However, the biofilms formed in situ and subjected to the same experimental conditions showed no statistically significant differences (p&#8805;0.05) in the microbiological counting when compared with control group. Therefore, we conclude that PACT was effective in microbiological reduction of S. mutans UA159 grown in an in vitro biofilm model, but very little effective on oral streptococci biofilms produced in situ.

A imobilização de fotossensibilizadores (FS) em materiais nanométricos tem se mostrado uma excelente alternativa ao seu emprego na forma molecular para terapia fotodinâmica (PDT). O presente trabalho descreve uma série de estratégias sintéticas desenvolvidas para a imobilização de FS em nanoesferas de sílica contendo núcleos magnéticos, bem como o estudo das propriedades foto-químicas, foto-físicas e morfológicas dos nanomateriais obtidos. Os FS utilizados foram o azul de metileno, tionina, azure A, protoporfirina IX, hematoporfirina IX e clorofilina. As metodologias de imobilização empregadas envolveram a adição do FS durante o crescimento da camada de sílica ou a ligação do FS na superfície do nanomaterial já formado. Em alguns casos foi necessário modificar previamente o FS com organossilanos. Os núcleos magnéticos foram preparados por co-precipitação de sais de ferro(II) e ferro(III) em meio básico e o revestimento com sílica foi realizado utilizando-se tetraetilortossilicato em uma microemulsão. Os núcleos magnéticos revestidos com uma camada de sílica mantiveram o comportamento superparamagnético. Para a tionina e o azul de metileno não foi detectada geração de oxigênio singlete após a imobilização. Para o azure A, protoporfirina IX e clorofilina imobilizados, a geração de oxigênio singlete foi detectada e caracterizada por métodos químicos e físicos. O material contendo clorofilina mostrou-se bastante promissor e foi empregado em estudos preliminares com células HeLa, levando à morte celular após irradiação, o que comprova o potencial para PDT. O comportamento do fotossensibilizador imobilizado e a possível proteção extra fornecida pela camada de sílica foram estudados pela supressão de fluorescência com íons brometo e supressão da geração de oxigênio singlete com albumina de soro bovino. Hematoporfirina IX é mais afetada na forma livre do que imobilizada em nanoesferas de sílica, o que indica que o FS imobilizado é capaz de gerar oxigênio singlete e está mais protegido de interferentes. / The immobilization of photosensitizers (PS) in nanometric materials is an excellent alternative to their use in molecular forms for photodynamic therapy (PDT). The present work describes a series of synthetic strategies for the immobilization of PS on silica nanospheres containing magnetic nucleous, as well as the studies of the photochemical, photophysical and morphological properties of the obtained materials. The studied PS were methylene blue, thionin, azure A, protoporphyrin IX, hematoporphyrin IX and chlorophyllin. The immobilization methodologies involve the addition of PS during the growth of the silica shell or the attachment of PS to the material surface. Often, a previous modification of the PS with organosilanes was necessary. The magnetic cores were prepared by co-precipitation of iron(II) and iron(III) salts in basic media and the coating with silica was performed by a microemulsion using TEOS. After coating with silica, the magnetic nanomaterial maintained the superparamagnetic behavior. The generation of singlet oxygen by thionin and methylene blue was not detect after immobilization. The generation of singlet oxygen by immobilized azure A, protoporphyrin IX and chlorophyllin was detected and characterized by chemical and physical methods. The material containing chlorophyllin is promising for PDT and was applied in preliminary studies with HeLa cells, causing cell death after irradiation. The behavior of the immobilized PS and the possible extra protection given by the silica shell were investigated by fluorescence quenching with bromide ions and by suppression of singlet oxygen generation with bovine serum albumin. Hematoporphyrin IX was more affected in the free form than immobilized in silica nanospheres, which indicates that the immobilized FS still generates singlet oxygen and is more protected against interfering species

Foram investigadas através da técnica de absorção eletrônica Uv-Vis e emissão de fluorescência a interação entre uma série de porfirinas e zinco-porfirinas com o polímero de eumelanina sintética obtida pela auto-oxidação da L-DOPA (dihidroxi-fenilalanina). Ocorre a formação de dois complexos na interação entre as porfirinas catiônicas e a eumelanina. Um complexo mais fraco em concentrações mais baixas e intermediárias de eumelanina, e um complexo mais forte em concentrações maiores. Estes complexos podem ser observados pelas variações obtidas nos espectros de absorção em função da concentração da melanina. A eumelanina suprime eficientemente a fluorescência das porfirinas catiônicas, em um processo de supressão por esfera de ação. Este processo ocorre particularmente para os complexos fracos entre a porfirina e a eumelanina, podendo ser inferido pela natureza de polieletrólito da eumelanina que deve apresentar uma ampla superfície de contato carregada negativamente, facilitando a orientação das porfirinas em uma disposição planar em relação ao polímero. A determinação das constantes de supressão estáticas aparentes (KEA) mostra que os substituintes alquílicos influem consideravelmente na formação destes complexos com a eumelanina. As diferentes interações são devidas aos fatores estruturais tais como dimensão da porfirina, polarização dos substituintes e formação de interações de natureza hidrofóbica. As zinco-porfirinas são de forma geral menos suprimidas que as bases livres. Este comportamento pode ser uma evidencia da aproximação planar em relação ao polímero da melanina, uma vez que zinco porfirinas apresentam moléculas de água ligadas axialmente, logo estas moléculas coordenadas axialmente impediriam uma aproximação mais efetiva. Foi realizado um estudo da formação de filmes baseados na técnica de auto-montagem eletrostática entre as porfirinas e a melanina utilizando como substrato o vidro. Observa-se que a adsorção das porfirinas em vidro é diferenciada sendo a TBzPyP a base livre que apresenta a maior adsorção. Pelos espectros de emissão dos filmes de porfirina em vidro foi possível verificar que alguns derivados interagem com uma participação mais efetiva dos piridínios que outros em que a interação é menos direcionada. Nos filmes porfirina melanina também é observada a supressão de fluorescência da porfirina. A diferença na magnitude de supressão nos filmes sugere que estes derivados possam ser utilizados em sistemas de diagnóstico relacionados à detecção de tumores melanóticos. Em um estudo paralelo, foram investigadas as propriedades de fotoisomerização de grupos azobenzênicos coordenados axialmente a uma ftalocianina de Silício(IV). Este sistema apresenta fotoisomerização E-Z e isomerização térmica e sensibilizada para o processo Z-E. As conversões E-Z e Z-E seguem uma cinética de primeira ordem, sendo o processo térmico Z-E cerca de dez vezes mais lento que o fotoquímico E-Z. A intensidade de fluorescência da ftalocianina é dependente do estado de isomerização dos grupos axiais, sendo que a forma E apresenta maior emissão de fluorescência que a forma Z. Portanto este sistema se constitui em um dispositivo molecular do tipo “on-off”, onde a intensidade de fluorescência pode ser controlada por um estímulo externo, no caso o comprimento de onda de excitação para a reação E-Z direta ou Z-E sensibilizada. / In this work we have investigated the interaction between a series of cationic porphyrins and zinc-porphyrins and the synthetic eumelanin polymer obtained by the auto-oxidation of L-DOPA (dihidroxy-phenylalanine) by means of UV-Vis and fluorescence emission techniques. Weak and strong porphyrin-melanin complexes are formed, and the evolution from weak to strong complexes can be monitored by the UV-Vis changes in the pophyrin spectra as the melanin concentration increases. The porphyrin fluorescence emission is quenched efficiently by eumelanin in a “quenching sphere of action” mechanism. This quenching sphere of action process is mainly observed for the weak complexes, and can be rationalized considering the polyelectrolyte nature of the eumelanin polymer which provides a broad contact area coated negatively, that enables a planar approximation of the porphyrins. The apparent static quenching constants (KEA) show that the nature of the alkyl substiutents markedly influences the complex formation. Total porphyrin size, different polarization induced by substituents and specific hydrophobic interactions are probably responsible for the different porphyrin – melanin association, which in turn reflects in the quenching properties of each particular porphyrin - melanin complex. The corresponding Zinc-porphyrins are less efficiently quenched by eumelanin, probably due the fact this molecules are axially coordinated by a water molecule, precluding a more close approximation between zinc-porphyrin and the eumelanin polymer. Electrostatically self-assembled films of porphyrins and eumelanin on glass have been studied. Different porphyrin adsorption on glass were observed, and TBzPyP derivative shown the highest adsorption in the investigated porphyrin series. Fluorescence emission suggests different interaction properties of the adsorbed pophyrin on glass, with specific pyridinium participation for some porphyrin derivatives. Fluorescence quenching of the porphyrins is observed in porphyrin – melanin films. The differences observed in the quenching for each derivative suggest a possible application of these compounds for diagnostic procedures related to melanotic tumors. In a complementary study, have been investigated the photoisomerization properties of a Si(IV)-phthalocyanine axially coordinated by azobenzene groups. Photochemical E-Z and thermal and sensitized Z-E isomerization processes were observed. Both E-Z photochemical and Z-E thermal follow first-order kinetics. Z-E process as expected is considerably slower than E-Z. The pthalocyanine emission is dependent on the isomerization state of the apendent azobenze groups, and the E form is more emissive than the Z form. This system can be considered a molecular device since the emission properties of the phthalocyanine moiety can be modulated by the isomerization state of the axially coordinated azoarenes, creating an on-off (E-Z states) fluorescence signal, and differently from other reported systems the recovery of the initial state does not depend only on the rate of the thermal reaction, since it can be controlled by a sensitized mechanism. This system seems promising taking into account the stability of the axially coordinated moieties, the greater versatility in the achievement of the desired isomerization state and the broad spectral region considering both direct and sensitized excitation.

A terapia fotodinâmica (Photodynamic Therapy - PDT) é um método de tratar neoplasias baseado na interação entre luz, oxigênio molecular e um agente fotossensibilizador. Após a administração do agente, o tumor é iluminado com luz visível, ativando-o e produzindo espécies reativas de oxigênio, altamente citotóxicas, que provocam morte celular e destruição tecidual. Com a destruição do tumor há ativação do sistema imune inato e o subsequente processo inflamatório determina a apresentação de antígenos tumorais aos linfócitos, promovendo uma resposta imunológica adaptativa contra o tecido tumoral. O presente trabalho visou estudar a PDT associando um laser de diodo como fonte de luz e o fotossensibilizante Azul de Metileno (AM) a 1%, avaliando a sua efetividade no tratamento do Tumor de Ehrlich (TE) em sua forma sólida e a resposta imunológica nos animais tratados. Em um primeiro estudo, avaliou-se macro e microscopicamente tumores tratados, determinando a capacidade do protocolo em induzir inflamação e destruição do tecido tumoral. No segundo estudo, a resposta imune foi estudada em camundongos desafiados com um segundo implante de células do tumor de Ehrlich. O primeiro implante tumoral foi tratado com a PDT ou a excisão cirúrgica, comparando-se com um grupo controle sem tratamento. Os parâmetros avaliados após 17 dias foram o crescimento tumoral (p>0,05), peso relativo dos órgãos linfóides [Baço (p<0,05) e Linfonodo poplíteo (p>0,05)], tamanho relativo do linfonodo (p<0,05), presença de metástase em linfonodo poplíteo (p>0,05), contagem de leucócitos sanguíneos (p>0,05) e análise morfométrica quantitativa do tumor secundário [determinação da fração volumétrica de células tumorais (p<0,05), infiltrado inflamatório (p<0,05), necrose (p>0,06) e porcentagem da área tumoral em necrose (p<0,05)]. A PDT com o AM foi capaz de induzir necrose do TE e inflamação, havendo diferenças da resposta imune sistêmica quando comparado aos animais tratados por meio de excisão cirúrgica do tumor de Ehrlich. / Photodynamic therapy (PDT) is a method of treating neoplasms based on the interaction between light, molecular oxygen and a photosensitizing agent. After administration of the photosensitizer, the tumor is illuminated with visible light, activating the agent and producing reactive oxygen species (ROS). This highly cytotoxic ROS cause cell death and tissue destruction. The activation of the innate immune system and the subsequent inflammation induces tumor antigen presentation to lymphocytes, promoting an adaptive immune response against the tumor cells. This work aimed to study the PDT using a diode laser as light source and Methylene Blue (MB) 1% as photosensitizer. It was accessed its effectiveness in treating Ehrlich Solid tumor (ET) and the immune response produced in treated animals. First the treated tumors were evaluated macroscopically and microscopically, determining the ability of the protocol to induce inflammation and tumor tissue destruction. In a second study, the immune response was studied in mice challenged with a second tumor cell implant. The primary tumor was treated with PDT or surgical excision, comparing with a control group without treatment. The parameters evaluated after 17 days were tumor growth (p> 0.05), relative weight of lymphoid organs [spleen (p <0.05) and popliteal lymph node (p> 0.05)], the relative size of the lymph node (p <0, 05), metastasis at lymph node (p>0,05), blood leukocyte count (p> 0.05) and quantitative morphometric analysis of secondary tumor [determining the volume fraction of tumor cells (p <0.05), inflammatory infiltrate (p <0.05), necrosis (p> 0.06) and tumor necrosis area (p <0.05)]. PDT with MB was able to induce necrosis of the ET and inflammation, with differences in the immune response when compared to animals treated surgically to remove the Ehrlich tumor in its solid form.

A terapia fotodinâmica (Photodynamic Therapy - PDT) é um método de tratar neoplasias baseado na interação entre luz, oxigênio molecular e um agente fotossensibilizador. Após a administração do agente, o tumor é iluminado com luz visível, ativando-o e produzindo espécies reativas de oxigênio, altamente citotóxicas, que provocam morte celular e destruição tecidual. Com a destruição do tumor há ativação do sistema imune inato e o subsequente processo inflamatório determina a apresentação de antígenos tumorais aos linfócitos, promovendo uma resposta imunológica adaptativa contra o tecido tumoral. O presente trabalho visou estudar a PDT associando um laser de diodo como fonte de luz e o fotossensibilizante Azul de Metileno (AM) a 1%, avaliando a sua efetividade no tratamento do Tumor de Ehrlich (TE) em sua forma sólida e a resposta imunológica nos animais tratados. Em um primeiro estudo, avaliou-se macro e microscopicamente tumores tratados, determinando a capacidade do protocolo em induzir inflamação e destruição do tecido tumoral. No segundo estudo, a resposta imune foi estudada em camundongos desafiados com um segundo implante de células do tumor de Ehrlich. O primeiro implante tumoral foi tratado com a PDT ou a excisão cirúrgica, comparando-se com um grupo controle sem tratamento. Os parâmetros avaliados após 17 dias foram o crescimento tumoral (p>0,05), peso relativo dos órgãos linfóides [Baço (p<0,05) e Linfonodo poplíteo (p>0,05)], tamanho relativo do linfonodo (p<0,05), presença de metástase em linfonodo poplíteo (p>0,05), contagem de leucócitos sanguíneos (p>0,05) e análise morfométrica quantitativa do tumor secundário [determinação da fração volumétrica de células tumorais (p<0,05), infiltrado inflamatório (p<0,05), necrose (p>0,06) e porcentagem da área tumoral em necrose (p<0,05)]. A PDT com o AM foi capaz de induzir necrose do TE e inflamação, havendo diferenças da resposta imune sistêmica quando comparado aos animais tratados por meio de excisão cirúrgica do tumor de Ehrlich. / Photodynamic therapy (PDT) is a method of treating neoplasms based on the interaction between light, molecular oxygen and a photosensitizing agent. After administration of the photosensitizer, the tumor is illuminated with visible light, activating the agent and producing reactive oxygen species (ROS). This highly cytotoxic ROS cause cell death and tissue destruction. The activation of the innate immune system and the subsequent inflammation induces tumor antigen presentation to lymphocytes, promoting an adaptive immune response against the tumor cells. This work aimed to study the PDT using a diode laser as light source and Methylene Blue (MB) 1% as photosensitizer. It was accessed its effectiveness in treating Ehrlich Solid tumor (ET) and the immune response produced in treated animals. First the treated tumors were evaluated macroscopically and microscopically, determining the ability of the protocol to induce inflammation and tumor tissue destruction. In a second study, the immune response was studied in mice challenged with a second tumor cell implant. The primary tumor was treated with PDT or surgical excision, comparing with a control group without treatment. The parameters evaluated after 17 days were tumor growth (p> 0.05), relative weight of lymphoid organs [spleen (p <0.05) and popliteal lymph node (p> 0.05)], the relative size of the lymph node (p <0, 05), metastasis at lymph node (p>0,05), blood leukocyte count (p> 0.05) and quantitative morphometric analysis of secondary tumor [determining the volume fraction of tumor cells (p <0.05), inflammatory infiltrate (p <0.05), necrosis (p> 0.06) and tumor necrosis area (p <0.05)]. PDT with MB was able to induce necrosis of the ET and inflammation, with differences in the immune response when compared to animals treated surgically to remove the Ehrlich tumor in its solid form.

5-Aminolevulinate synthase (ALAS; EC 2.3.1.37) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the first committed step of heme biosynthesis in animals, the condensation of glycine and succinyl-CoA yielding 5-aminolevuliante (ALA), CoA, and CO2. Murine erythroid-specific ALAS (mALAS2) variants that cause high levels of PPIX accumulation provide a new means of targeted, and potentially enhanced, photosensitization. Transfection of HeLa cells with expression plasmids for mALAS2 variants, specifically for those with mutated mitochondrial presequences and a mutation in the active site loop, caused significant cellular accumulation of PPIX, particularly in the membrane. Light treatment of HeLa cells expressing mALAS2 variants revealed that mALAS2 expression results in an increase in cell death in comparison to aminolevulinic acid (ALA) treatment producing a similar amount of PPIX. Generation of PPIX is a crucial component in the widely used photodynamic therapies (PDT) of cancer and other dysplasias. The delivery of stable and highly active mALAS2 variants has the potential to expand and improve upon current PDT regimes. Mutations in the C-terminus of human ALAS2 (hALAS2) can increase hALAS2 activity and are associated with X-linked erythropoietic protoporphyria (XLEPP), a disease phenotypically characterized by elevated levels or PPIX and zinc protoporphyrin in erythroblasts. This is apparently due to enhanced cellular hALAS2 activity, but the biochemical relationship between these C-terminal mutations and increased hALAS2 activity is not well understood. HALAS2 and three XLEPP variants were studied both in vitro to compare kinetic and structural parameters and ex vivo in HeLa and K562 cells. Two XLEPP variants, delAGTG, and Q548X, exhibited higher catalytic rates and affinity for succinyl-CoA than wild-type hALAS2, had increased transition temperatures, and caused porphyrin accumulation in HeLa and K562 cells. Another XLEPP mutation, delAT, had an increased transition temperature and caused porphyrin accumulation in mammalian cells, but exhibited a reduced catalytic rate at 37[deg]C in comparison to wild-type hALAS2. The XLEPP variants, unlike wild-type hALAS2, were more structurally responsive upon binding of succinyl-CoA, and adopted distinct features in tertiary and PLP cofactor-binding site. These results imply that the C-terminus of hALAS2 is important for regulating its structural integrity, which affects kinetic activity and stability. XLEPP has only recently been identified as a blood disorder, and thus there are no specific treatments. One potential treatment involves the use of the antibiotic isonicotinic acid hydrazide (isoniazid, INH), commonly used to treat tuberculosis. INH can cause sideroblastic anemia as a side-effect and has traditionally been thought to do so by limiting PLP availability to hALAS2 via direct inhibition of pyridoxal kinase, and reacting with pyridoxal to form pyridoxal isonicotinoyl hydrazone. We postulated that in addition to PLP-dependent inhibition of hALAS2, INH directly acts on hALAS2. Using FACS and confocal microscopy, we show here that INH reduces protoporphyrin IX accumulation in HeLa cells expressing either wild-type human hALAS2 or XLEPP variants. In addition, PLP and pyridoxamine 5'-phosphate (PMP) restored cellular hALAS2 activity in the presence of INH. Kinetic analyses with purified hALAS2 demonstrated non-competitive or uncompetitive inhibition with an apparent Ki of 1.5 uM. Circular dichroism studies revealed that INH triggers structural changes in hALAS2 that interfere with the association of hALAS2 with its PLP cofactor. These studies demonstrate that hALAS2 can be directly inhibited by INH, provide insight into the mechanism of inhibition, and support the prospective use of INH in treating patients with XLEPP and potentially other cutaneous porphyrias.

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.

The biosynthesis of tetrapyrolles in eukaryotes and the alpha-subclass of purple photosynthetic bacteria is controlled by the pyridoxal 5?-phosphate (PLP)-dependent enzyme, 5-aminolevulinate synthase (ALAS). Aminolevulinate, the universal building block of these macromolecules, is produced together with Coenzyme A (CoA) and carbon dioxide from the condensation of glycine and succinyl-CoA. The three-dimensional structures of Rhodobacter capsulatus ALAS reveal a conserved active site serine that moves to within hydrogen bonding distance of the phenolic oxygen of the PLP cofactor in the closed, substrate-bound enzyme conformation, and simultaneously to within 3-4 angstroms of the thioester sulfur atom of bound succinyl-CoA. To elucidate the role(s) this residue play(s) in enzyme activity, the equivalent serine in murineerythroid ALAS was mutated to threonine or alanine. The S254A variant was active, but both the KmSCoA and kcat values were increased, by 25- and 2-fold, respectively, suggesting the increase in turnover is independent of succinyl-CoA-binding. In contrast, substitution of S254 with threonine results in a decreased kcat, however the Km for succinyl-CoA is unaltered. Removal of the side chain hydroxyl group in the S254A variant notably changes the spectroscopic properties of the PLP cofactor and the architecture of the PLP-binding site as inferred from circular dichroism spectra. Experiments examining the rates associated with intrinsic protein fluorescence quenching of the variant enzymes in response to ALA binding show that S254 affects product dissociation. Together, the data led us to suggest that succinyl-CoA binding in concert with the hydrogen bonding state of S254 governs enzyme conformational equilibria. As a member of the alpha-oxoamine synthase family, ALAS shares a high degree of structural similarity and reaction chemistry with the other enzymes in the group. Crystallographic studies of the R. capsulatus ALAS structure show that the alkanoate component of succinyl-CoA is bound by a conserved arginine and a threonine. To examine acyl-CoA-binding and substrate discrimination in murine erythroid ALAS, the corresponding residues (R85 and T430) were mutated and a series of CoA substrate analogs were tested. The catalytic efficiency of the R85L variant with octanoyl-CoA was 66-fold higher than that calculated for the wild-type enzyme, suggesting this residue is strategic in substrate binding. Hydrophobic substitutions of the residues that coordinate acyl-CoA-binding produce ligand-induced changes in the CD spectra, indicating that these amino acids affect substrate-mediated changes to the microenvironment of the chromophore. Pre-steady-state kinetic analyses of the R85K variant-catalyzed reaction show that both the rates associated with product-binding and the parameters that define quinonoid intermediate lifetime are dependent on the chemical composition of the acyl-CoA tail. Each of the results in this study emphasizes the importance of the relationship between the bifurcate interaction of the alkanoic acid component of succinyl-CoA and the side chains of R85 and T430. From the X-ray crystal structures of Escherichia coli 8-amino-7-oxonoanoate synthase and R. capsulatus ALAS, it was inferred that a loop covering the active site moved 3-6 Å between the holoenzymic and acyl-CoA-bound conformations. To elucidate the role that the active site lid plays in enzyme function, we shuffled the portion of the murine erythroid ALAS cDNA corresponding to the lid sequence (Y422-R439), and isolated functional variants based on genetic complementation in an ALA-deficient strain. Variants with potentially greater enzymatic activity than the wild-type enzyme were screened for increased porphyrin overproduction. Turnover number and the catalytic efficiency of selected functional variants with both substrates were increased for each of the enzyme variants tested, suggesting that increased activity is linked to alterations of the loop motif. The results of transient kinetics experiments for three isolated variants when compared to wild-type ALAS showed notable differences in the pre-steady-state rates that define the kinetic mechanism, indicating that the rate of ALA release is not rate-limiting for these enzymes. The thermodynamic parameters for a selected variant-catalyzed reaction indicated a reduction in the amount of energy required for catalysis. This finding is consistent with the proposal that, in contrast to the wild-type ALAS reaction, a protein conformational change associated with ALA release no longer limits turnover for this variant enzyme.

Die Photodynamische Therapie (PDT) hat sich zunehmend als vielversprechende Methode zur Behandlung von verschiedenen malignen Neubildungen gezeigt. Die photodynamische Zerstörung der Tumore wird erreicht indem zunächst ein Photosensibilisator entweder lokal oder systemisch appliziert wird und im Anschluss an eine gewisse Inkubationszeit die Tumormasse mittels einer Lichtquelle mit einer spezifischen Wellenlänge durchleuchtet wird. Aufgrund der bevorzugten Anreicherung des Photosensibilisators in Tumorzellen, erlaubt diese Methode eine selektive Abtötung des malignen Tumors, während das umliegende Gewebe weitestgehend verschont wird. Diese Eigenschaften und Anforderungen machen die PDT, insbesondere in den Fällen, wo die chirurgische Enukleation als kurative Option erwogen wird, zu einer attraktiven Therapieoption in der Behandlung von Retinoblastomen (Rb). Die extreme Methode der Enukleation wird noch immer angewendet, wenn die Tumoren nicht ausreichend chemosensibel sind, oder wenn sich die Erkrankung aufgrund von unzureichendem Zugang zu medizinischer Versorgung bereits in einem fortgeschrittenen Stadium befindet. In dieser Studie haben wir zunächst In-Vitro-Untersuchungen mit dem neuen kationischen wasserlöslichen Photosensibilisator Tetrahydroporphyrin-Tetratosylat (THPTS) bezüglich seiner photodynamischen Wirkung auf WERI Rb-1 und Y79-Retinoblastomzellen durchgeführt. Dabei konnten wir zeigen, dass weder die Inkubation mit THPTS ohne anschließende Beleuchtung, noch die alleinige Beleuchtung zu einem signifikanten Effekt auf die Proliferation der Rb-Zellen führte. Die Kombination von THPTS mit anschließender Beleuchtung hingegen führte zu einem maximal zytotoxischen Effekt in den Tumorzellen. Darüber hinaus war die Phototoxizität in normalen Primärzellen des Pigmentepithels der Retina geringer, wodurch ein erhöhter phototoxischer Effekt von THPTS in Krebszellen gegenüber diesem normalen Zelltyp der Retina gezeigt werden konnte. Die vorliegenden Ergebnisse bilden eine ermutigende Grundlage für weiterführende in-vivo-Untersuchungen zum therapeutischen Potential dieses vielversprechenden Photosensibilisators mit der Aussicht auf eine potentiell kurative Therapie des Retinoblastoms unter Erhalt von Augapfel und Visus.

Emerging evidence has shown that the capacity of a tumor to grow and propagate is dependent on a small subset of cells, termed “cancer stem cells“. Conventional treatments, however, may miss the cancer stem cells, which have been shown in several tumor types to be more resistant to standard chemotherapeutic agents, because the cancer stem cell survives and regenerates the neoplasm. The aim of this study was to investigate the applicability of quantum dots in detection and eradication of cancer stem cells. The properties of stem cells were inestigated in breast, pancreatic, ovarian cancer and melanoma cells. The accumulation and distribution of quantum dots was assessed by the means of laser scanning confocal and fluorescence lifetime imaging microscopy. It was shown, that anti-CD44 and quantum dot conjugates are internalized selectively inside CD44+ cells, while anti-CD44 and organic dye FITC conjugates remained attached to cell membrane for at least 24 hours. Moreover, the complex of quantum dot (QD) and photosensitizer chlorin e6 (Ce6) accumulates inside cells and initiate cell death upon QD-directed exposure of 470 nm irradiation. These results show the capability of quantum dots not only to detect and internalize stem-like cells but also to carry the therapeutic agent inside cells without losing its activity. / Pastaruoju metu vis gausėja įrodymų apie vėžyje esančią nedidelę subpopuliaciją, pavadintą „vėžio kamieninėmis ląstelėmis“. Teigiama, jog būtent vėžio kamieninės ląstelės yra atsakingos už naviko formavimą, atsparumą taikomai terapijai bei metastazių formavimą. Šio darbo tyrimo tikslas – ištirti nanodarinių pritaikymo galimybes vėžinių kamieninių ląstelių aptikimui ir sunaikinimui. Darbe buvo ištirtos krūties, kasos, kiaušidžių vėžio ir melanomos ląstelių savybės, pagal literatūroje nurodytą vėžio kamieninių ląstelių aptikimo metodiką. Panaudojant lazerinę skenuojančią konfokalinę ir gyvavimo trukmių vaizdinimo mikroskopiją buvo įvertintas kvantinių taškų, kaip galimų diagnostiką ir terapiją atliekančių nanodarinių kaupimosi ir lokalizacijos ląstelėse tyrimai. Buvo parodyta, jog antikūnais prieš paviršiaus antigeną CD44 padengti kvantiniai taškai selektyviai pateko tik į CD44+ fenotipo ląstelių vidų. Anti-CD44-FITC konjugatai taip pat selektyviai žymėjo norimas ląsteles, tačiau net po 24 valandų nepateko į ląstelių vidų. Taip pat buvo parodyta, jog kvantinio taško (KT) ir sensibilizatoriaus chlorino e6 (Ce6) nanodarinys lokalizuojasi ląstelių viduje pūslelėse ir sukelia ląstelių žūtį tik toms ląstelėms, kurioms pritaikytas apjungtas KT-Ce6 komplekso ir 470 nm spinduliuotės poveikis. Tokie rezultatai parodo, jog galima kvantinį tašką ne tik selektyviai nukreipti į norimas ląsteles, bet taip pat ir įnešti į jų vidų, neprarandant terapinės molekulės aktyvumo.