Thesis (Ph.D.) -- McMaster University, 2001. / Includes bibliographical references. Also available via World Wide Web.

Made available in DSpace on 2014-08-13T14:50:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-05-08Bitstream added on 2014-08-13T18:01:09Z : No. of bitstreams: 1 000737321_20150508.pdf: 458900 bytes, checksum: 37734b9c708dcabe02230f14146dcf69 (MD5) Bitstreams deleted on 2015-05-11T13:13:20Z: 000737321_20150508.pdf,Bitstream added on 2015-05-11T13:13:54Z : No. of bitstreams: 1 000737321.pdf: 921005 bytes, checksum: a5bf7910d36287fe4d0b08c8e8ad1e65 (MD5) / A Terapia Fotodinâmica (TFD) tem sido proposta como terapia auxiliar no tratamento da periodontite, onde um corante não-tóxico (fotossensibilizador, FS) reage com o oxigênio dos tecidos e que, na presença de luz, é capaz de eliminar ou reduzir os microorganismos presentes nos sítios da doença. A hipericina (HY) é um pigmento fotoativo natural muito potente. O objetivo deste estudo foi avaliar os efeitos da TFD com HY no tratamento da doença periodontal in vivo. Para isso, 84 ratos foram submetidos à indução de peridontite por inserção de ligaduras em torno dos 2os molares superiores. Após 7 dias, as ligaduras foram removidas e os animais foram randomicamente distribuídos em 4 grupos e submetidos aos seguintes procedimentos: Grupo I - Controle, doença sem tratamento; Grupo II - RAR, raspagem e alisamento radicular manual com curetas periodontais; Grupo III - TFD, emprego de HY (10μg/ml) por 5 minutos na bolsa periodontal seguido de irradiação com LED 630nm por 4 minutos/dente (35,15J/cm2); Grupo IV- RAR+TFD, associação dos tratamentos dos grupos II e III. Sete, 15 e 30 dias após os procedimentos, os animais foram sacrificados. Avaliação tridimensional por microCT e análise histométrica revelou diferenças significativas entre o grupo controle e os demais grupos aos 7 e 15 dias pós-tratamento. Análise imunohistoquímica por TRAP e RANKL apresentou marcação semelhante nos grupoI, III e IV. Dentre os limites deste estudo, podemos concluir que a TFD com hipericina foi capaz de reduzir a progressão da periodontite induzida in vivo de modo similar ao tratamento mecânico. / Photodynamic therapy (PDT) has been proposed as an auxiliary therapy on the treatment of peridontitis. This technique uses a non-toxic dye (photosensitizer, PS) that reacts with oxygen of the tissues in the presence of light, and is able to eliminate or reduce microorganisms on the sites of the disease. Hypericin (HY) is a very potent photoactive natural pigment. The aim of this study was evaluate the effects of HY-PDT in the treatment of periodontal disease induced in vivo. Thus, 84 rats were submitted to peridontitis induction by placing ligatures around superior 2nd molars. After 7 days, ligatures were removed and animals were randomically distributed in 4 groups and submitted to the following procedures: Group I – Control, disease without treatment; Group II – SRP, scaling and root planning with manual periodontal curettes; Group III – PDT, application of HY (10μg/ml) for 5 minutes in the periodontal pocket followed by 4 minutes of 630 nm irradiation per tooth (35,15J/cm2); Group IV - SRP+PDT, association of the treatments of groups II and III. Seven, 15 and 30 days post-treatment the animals were sacrificed. MicroCT tridimensional evaluation and histometric analysis showed statistical differences between Control and treatments groups at 7 and 15-days post treatment. TRAP and RANKL immunohistochemistry analysis showed similar marking on groups I, III and IV. Therefore, HY-PDT was able to reduce periodontitis progression in vivo in a similar way to mechanical treatment.

The synthesis of symmetrically and unsymmetrically substituted zinc phthalocyanines (ZnPc) derivatives is done according to reported procedures. The unsymmetrical ZnPc derivatives are synthesized by ring expansion of sub-phthalocyanine complexes. Ring substitution is effected with tert-butyl phenol, naphthol, and hydroxybenzoic acid. Comparison of the redox potentials for the complexes substituted with varying numbers of tert-butyl phenol: 1, 2, 3, 6 and 8 show that the complex with the highest number of substituents are more difficult to oxidize and easier to reduce. Water soluble sulphonated ZnPc (ZnPcSn) was prepared. The possibility of using axial ligation to increase the solubility and the photochemical activity of sulphotnated ZnPc in aqueous solutions was investigated. Pyridine, aminopyridyl and bipyridyl were used as axial ligands. When bipyridyl was used as the axial ligand, solubility of the ZnPcSn increased, shown by the increase in the Q-band of the monomer species in solution and the singlet oxygen quantum yields was relatively higher than that of the unligated ZnPcSn. The singlet oxygen quantum yields by the various complexes in DMF using diphenylisobenzofuran as a chemical quencher for organic solvent were determined. Singlet oxygen quantum yields for the unsymmetrically ring substituted complexes range from 0.22 to 0.68. Photobleaching quantum yields are in the order of 10-5, which means that the complexes are relatively photostable.

Substituted zinc and magnesium phthalocyanine and porphyrazine derivatives were synthesized according to the reported procedures. The magnesium and zinc phthalocyanine and porphyrazine derivatives were synthesized by ring enlargement of subphthalocyanine and statistical condensation of the two phthalonitrile derivatives. Characterization of the complexes involved the use of infrared spectroscopy, nuclear magnetic resonance spectroscopy, ultraviolet and visible spectroscopy, and Maldi-TOF spectroscopy (for selected compounds) and elemental analysis. Photochemical and photophysical properties of the complexes in non-aqueous solution was then investigated. Photobleaching quantum yields are in order of 10⁻⁵ indicating their relative photostability. Complexes containing more electron-donating substituents were more easily oxidized. For complexes 66 and 69 (as these complexes have the same number of substituents but differ in the metal center) photobleaching quantum yield for the ZincPc complex 69 was slightly less than that of the MgPc complex 66. Singlet oxygen quantum yields of the various complexes in DMSO using diphenylisobenzofuran (DPBF) as a quencher in organic solvents were determined. Singlet oxygen quantum yields of the complexes range from 0.23 to 0.67. High values of Φ[subscript]Δ ZnPc complexes was observed compared to the corresponding MgPc, complexes. This was evidenced by complexes 66 and 69 with Φ[subscript]Δ values of Φ[subscript]Δ = 0.26 and 0.40, respectively. Varying number of phenoxy substituents, complex 71 gave significantly large value of Φ[subscript]Δ compared to 70 (that is, the presence of more electron-donating substituted group, gave higher singlet oxygen quantum yields (0 .67 and 0.25 for 71 and 70 repectively). The triplet quantum yields and triplet lifetimes were determined by laser flash photolysis for selected compounds. The triplet quantum yields increase as the number of substituents increases e.g 68 > 67 > 66. Comparing porphyrazine complexes (63, 64 and 65), 63 with benzene attached to the ring, has higher triplet state lifetime (420 μs) compared to 64 and 65 containing long alkyl chain and tertbutyl substituents, 350 and 360 μs,respectively). The observed Φ[subscript]f values for 68 and 63 were quiet suprising, since low values are observed compared to the rest of the complexes (e.g 0.03 and 0.02 respectively). Although these values seem so low, they are sufficient for fluorescence imaging applications. The Φ[subscript]f values for the complexes under study are within the range reported for complexes currently used for PDT. / KMBT_363 / Adobe Acrobat 9.54 Paper Capture Plug-in

A detailed photophysicochemical study of some non-transition metal (AI, Zn, Si, Ge, and Sn) metallophthalocyanine (MPc) derivatives is presented. The effects of substituents, central metal ions and solvents on the photophysical and photochemical properties are investigated and rationalized accordingly. The presence of peripheral substituents on the macrocycle enhances the yield of the triplet state. Near infra-red absorptions of the solvents reveal that solvents which absorb around 1100 nm and around 1270 nm, quench the triplet state of the MPc derivative and singlet oxygen, respectively. Although water has a high singlet oxygen quenching effect, the singlet oxygen quantum yield (Φ∆) value for sulphonated zinc phthalocyanine in water is still reasonably high at 0.48, which may provide an explanation for the efficient photosensitization by this molecule in photodynamic studies. The lowering of Φ∆ following protonation of the MPc macrocycle is attributed to the lowering of triplet energy to the level where energy transfer to ground state oxygen is no longer favourable. MPc inclusion complexes with cyclodextrins showed larger Φ∆ values when compared to the complexes before inclusion. Job's plots show that 2:1 and 4:1 (CD:MPc) complexes may be formed. Fluorescence quenching by electron donors and acceptors were analysed by StemVolmer relationship and the results used in determining fluorescence lifetimes of the complexes. Qualitative and quantitative interpretations of the interaction of sulphonated MPcs with bovine serum albumin (BSA) are provided in this thesis. 1:1 adducts were formed with BSA, but the binding feasibilities varied markedly. Spectral, photophysical and photochemical properties of the complexes are altered in the presence of BSA.

The synthesis, photophysicochemcial properties, in vitro dark toxicity and photodynamic therapy (PDT) activities of different derivatives of zinc phthalocyanine (ZnPc) conjugates with biomolecules (folic acid, bovine serum albumin (BSA), ascorbic acid, uridine or spermine) and single-walled carbon nanotubes (SWCNTs) are presented in this work. The fluorescence quantum yields (ΦF) (Subscript F) of the ZnPc derivatives or ZnPc-biomolecule conjugates remained relatively the same as compared to the precursor Pcs. Slight increases were observed in the ΦF (Subscript F) values of conjugates containing substituents such as pyrene, folic acid or BSA with intrinsic fluorescence properties. The triplet quantum yield (ΦT ) (Subscript T) values for some ZnPc conjugates increases compared to the precursor ZnPcs due to extended π conjugation (for the conjugate with pyrene) and the presence of phenyl ring that support spin-orbit charge transfer intersystem crossing to triplet state. While some conjugates showed decreases in the ΦT (Subscript T) values compared to precursor ZnPcs due to the presence of substituents that could quench photo-excited state properties. The singlet oxygen quantum yield (ΦΔ ) values follow the trends of the triplet quantum yield values. The conjugates containing BSA also show increases in the ΦΔ values without corresponding increases in ΦT (Subscript T) values due to the ability of BSA to generate free radicals including singlet oxygen. The presence of SWCNTs decreases the photophysicochemcial properties of some ZnPc-SWCNT conjugates compared to the precursor ZnPcs due to photo-induced electron transfer from an excited Pc complex (electron donor) to SWCNTs (electron acceptor). However, increases were observed in some ZnPc-SWCNT conjugates as a result of fast charge recombination process due to highly short-lived radical ion pair produced. These phenomena affected the ΦF (Suscript F) values, ΦT (Suscript T) values, and the ΦΔ values. Increases or decreases in ΦT (Suscript T) values resulted in corresponding increases or decreases in ΦΔ values

Various axially substituted Silicon octaphenoxyphthalocyanines were synthesised as potential photosensitisers in the photodynamic therapy of cancer. Conventional reflux reactions were used for synthesis as well as new microwave irradiation reactions, wherein the reaction times were decreased tenfold with a marginal increase in reaction yield and product purity. An interesting series of oligomeric (dimer to a nonamer) silicon octaphenoxyphthalocyanines were also successfully synthesised in a reaction similar to polymerisation reactions. These compounds were found to undergo an axial ligand transformation upon irradiation with red light (> 600 nm) in dimethylsulphoxide solution. All the ligands were transformed into the dihydroxy silicon octaphenoxyphthalocyanine with varying degrees of phototransformation quantum yields ranging in order from 10⁻³ to 10⁻⁵ depending on the axial ligand involved. During and after axial ligand transformations a photodegredation of the dihydroxy silicon octaphenoxy phthalocyanine was observed upon continued irradiation. The oligomers were found to undergo the same axial ligand transformation process with a phototransformation quantum yield of 10⁻⁵ The singlet oxygen quantum yields of the unaggregated monomeric silicon octaphenoxy phthalocyanines were all found to be approximately 0.2 with the exception of a compound with two (trihexyl)siloxy axial substituents that had a singlet oxygen quantum yield of approximately 0.4 in dimethylsulphoxide solutions. The oligomers showed a surprising trend of an increase in singlet oxygen quantum yield with an increase in phthalocyanine ring number up to the pentamer and then a dramatic decrease to the nonamer. The triplet quantum yield and triplet lifetime were determined by laser flash photolysis for selected compounds and no correlation was observed with any of these properties and the singlet oxygen quantum yields. These selected compounds all fluoresce and a very good correlation was found between the fluorescence lifetimes determined experimentally by laser photolysis and the Strickler-Berg equation for the non-aggregated compounds. Electrochemical measurements also indicate the importance of the axial ligands upon the behaviour of the phthalocyanines as cyclic voltammetric behaviour was determined by the nature of the axial ligand.

Orientador: Rosemary Adrianna Chierici Marcantonio / Banca: Alessandra Nara de Souza Rastelli / Banca: Joni Augusto Cirelli / Banca: Priscila Fenanda de Campos Menezes / Banca: Giuseppe Alexandre Romito / Resumo: A Terapia Fotodinâmica (TFD) tem sido proposta como terapia auxiliar no tratamento da periodontite, onde um corante não-tóxico (fotossensibilizador, FS) reage com o oxigênio dos tecidos e que, na presença de luz, é capaz de eliminar ou reduzir os microorganismos presentes nos sítios da doença. A hipericina (HY) é um pigmento fotoativo natural muito potente. O objetivo deste estudo foi avaliar os efeitos da TFD com HY no tratamento da doença periodontal in vivo. Para isso, 84 ratos foram submetidos à indução de peridontite por inserção de ligaduras em torno dos 2os molares superiores. Após 7 dias, as ligaduras foram removidas e os animais foram randomicamente distribuídos em 4 grupos e submetidos aos seguintes procedimentos: Grupo I - Controle, doença sem tratamento; Grupo II - RAR, raspagem e alisamento radicular manual com curetas periodontais; Grupo III - TFD, emprego de HY (10μg/ml) por 5 minutos na bolsa periodontal seguido de irradiação com LED 630nm por 4 minutos/dente (35,15J/cm2); Grupo IV- RAR+TFD, associação dos tratamentos dos grupos II e III. Sete, 15 e 30 dias após os procedimentos, os animais foram sacrificados. Avaliação tridimensional por microCT e análise histométrica revelou diferenças significativas entre o grupo controle e os demais grupos aos 7 e 15 dias pós-tratamento. Análise imunohistoquímica por TRAP e RANKL apresentou marcação semelhante nos grupoI, III e IV. Dentre os limites deste estudo, podemos concluir que a TFD com hipericina foi capaz de reduzir a progressão da periodontite induzida in vivo de modo similar ao tratamento mecânico. / Abstract: Photodynamic therapy (PDT) has been proposed as an auxiliary therapy on the treatment of peridontitis. This technique uses a non-toxic dye (photosensitizer, PS) that reacts with oxygen of the tissues in the presence of light, and is able to eliminate or reduce microorganisms on the sites of the disease. Hypericin (HY) is a very potent photoactive natural pigment. The aim of this study was evaluate the effects of HY-PDT in the treatment of periodontal disease induced in vivo. Thus, 84 rats were submitted to peridontitis induction by placing ligatures around superior 2nd molars. After 7 days, ligatures were removed and animals were randomically distributed in 4 groups and submitted to the following procedures: Group I - Control, disease without treatment; Group II - SRP, scaling and root planning with manual periodontal curettes; Group III - PDT, application of HY (10μg/ml) for 5 minutes in the periodontal pocket followed by 4 minutes of 630 nm irradiation per tooth (35,15J/cm2); Group IV - SRP+PDT, association of the treatments of groups II and III. Seven, 15 and 30 days post-treatment the animals were sacrificed. MicroCT tridimensional evaluation and histometric analysis showed statistical differences between Control and treatments groups at 7 and 15-days post treatment. TRAP and RANKL immunohistochemistry analysis showed similar marking on groups I, III and IV. Therefore, HY-PDT was able to reduce periodontitis progression in vivo in a similar way to mechanical treatment. / Doutor

Orientador: Ana Claudia Pavarina / Banca: Eunice Teresinha Giampaolo / Banca: Carlos Eduardo Vergani / Banca: Maria Aparecida de Andrade Moreira Machado / Banca: Karin Hermana Neppelenbroek / Resumo: Esta investigação teve como objetivo avaliar: 1. a viabilidade de utilização da curcumina como agente fotossensibilizador (PS) em Terapia Fotodinâmica (PDT) para inativação de uma cepa padrão de Candida albicans e seu efeito tóxico sobre culturas celulares de macrófagos; 2. a efetividade da PDT mediada pela curcumina para inativação de suspensões planctônicas e biofilmes formados por diferentes isolados clínicos de C. albicans, Candida tropicalis e Candida glabrata; 3. a fotoinativação de C. albicans, presente em candidose induzida em camundongos, por meio da utilização de diferentes concentrações de curcumina. No primeiro estudo, suspensões planctônicas de C. albicans (ATCC 90028) foram expostas a nove concentrações de curcumina (0,005; 0,01; 0,05; 0,1; 0,5; 1, 5, 10 e 20μM) e oito doses de luz (1,32; 2,64; 3,96; 5,28; 6,60; 13,20 e 26,4J/cm2). O efeito de diferentes tempos de pré-irradiação (PIT), a possível captação de curcumina pelas células fúngicas e participação do oxigênio singlete na fotoinativação também foram avaliados. Adicionalmente, a PDT mediada pela curcumina foi avaliada em biofilmes formados pela cepa de referência de C. albicans e sobre culturas celulares de macrófagos. Além disso, as características ópticas da solução de curcumina foram investigadas em função da dose de luz utilizada. Os resultados foram submetidos à análise descritiva, Análise de Variância (ANOVA) e Kruskal-Wallis (α= 5%). As suspensões planctônicas de C. albicans foram completamente inativadas com a utilização de 20μM de curcumina e 5,28J/cm2. Além disso, foi observada redução significativa na viabilidade celular dos biofilmes de C. albicans. O efeito fotodinâmico foi acentuado pela presença da curcumina durante a iluminação... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this investigation was to evaluate: 1. the Photodynamic Therapy (PDT) mediated by curcumin for in vitro inactivation of a reference strain of Candida albicans and its citotoxic effects against a macrofage cell line; 2. the effectiveness of PDT mediated by curcumin to inactivate clinical isolates of C. albicans, Candida tropicalis and Candida glabrata, both in planktonic and biofilm phases; 3. the photoinactivation of C. albicans in a murine model of oral candidiasis using different curcumin concentrations. In the first study, suspensions of C. albicans were treated with nine curcumin concentrations (0.005; 0.01; 0.05; 0.1; 0.5; 1, 5, 10 and 20μM) and exposed to LED light at different fluences (1.32; 2.64; 3.96; 5.28; 6.60; 13.20 and 26.4J/cm2). The effect of the pre-irradiation time (PIT) on PDT effectiveness, the uptake of curcumin by C. albicans cells and the possible involvement of singlet oxygen in the photodynamic action was also evaluated. In addition, curcumin-mediated PDT was assessed against biofilms. Similar protocols were tested on a macrophage cell line and the effect was evaluated by MTT and SEM analysis. The optical properties of curcumin were investigated as a function of illumination fluence. Data were analyzed with Analysis of Variance (ANOVA), and Kruskal-Wallis test (α= 5%). The results showed a statistically significant reduction in C. albicans viability after PDT (p<0.05), for both planktonic and biofilm cultures. Photodynamic effect was greatly increased with the presence of curcumin in the surrounding media and the PIT of 20 minutes improved PDT effectiveness. Although PDT was phototoxic to macrophages, the therapy was more effective in inactivating the yeast cell than the defense cell. The spectral changes showed a high photobleaching rate of curcumin. In the second study, planktonic... (Complete abstract click electronic access below) / Doutor

M. Tech. / Photodynamic therapy (PDT) is a type of phototherapy which is based on the interaction of photosensitizer with visible light in the presence of oxygen resulting in the production of reactive oxygen species (Pfaffel-Schubart et al., 2008). PDT has rapidly matured in the past 6 years and is an accepted standard treatment for various cancerous diseases. The main advantages of PDT include significant drug selectivity in tumour cells, absence of toxicity in the dark, possibility to treat only cancer cells and the ability to retreat a tumour in order to improve the prognosis (Pfaffel-Schubart et al., 2008). Photofrins are the most studied photosensitizers and their disadvantages is the inability of these photosensitizers to localize specifically in tumour cells and are retained in normal cells for prolonged periods (Nowis et al., 2005). This factor has stimulated the development of second generation photosensitizers with improved physical, chemical and spectral properties (Sharman et al., 1999). Phthalocyanine compounds are second generation photosensitizers which have a potential as photosensitizers in PDT treatment of many cancers. Traditional treatments of cancer show limited efficiency and other treatment modalities need to be explored. PDT has shown promising results in the treatment of many cancers using phthalocyanine compounds as photosensitizers but little is known about Zn sulfophthalocyanine on lung cancer cells. This study investigated the effects of Zn sulfophthalocyanine photosensitizer and laser irradiation on a lung cancer cell line (A549). Lung cancer cells were cultured in RPMI medium 1640 supplemented with 10% foetal bovine serum and antibiotics and incubated at 37 °C with 5% CO2 and 85% humidity. Cells were divided into 4 groups. Group 1 was an unirradiated control not treated with a photosensitizer. Group 2 was photosensitized at a concentration of 15.8 μM but not irradiated. Group 3 was irradiated but not photosensitized while Group 4 was irradiated and photosensitized at a concentration of 15.8 μM. Laser irradiations were done using a diode laser emitting 636 nm with an output of 110 mW at 5.3 J/cm2. Cell morphology was evaluated using the light inverted microscope. Cell viability was assessed using adenosine triphosphate (ATP) luminescence assay and Trypan blue exclusion test while cell proliferation was measured using the alamarBlue assay. Cytotoxicity was evaluated by assessing membrane permeability for lactate dehydrogenase and DNA damage was evaluated by comet assay. The mode of cell death was assessed by annexin V-FITC apoptosis detection kit using flow cytometry. In addition, expression of Bcl-2/Bax was monitored by western blot analysis and to determine the levels of induced Heat shock protein 70, Hsp 70 ELISA was used. Cells treated with photosensitizer at 15.8 μM and irradiated with 5.3 J/cm2 showed changes in cell morphology, decrease in cellular viability, proliferation and Hsp 70 expression and an increase in cytotoxicity and DNA damage. This indicated that the metalated phthalocyanine was effective in inducing cell death and the analysis of cell death by annexin FITC-V revealed an apoptotic cell death pattern. Furthermore, loss of the antiapoptotic Bcl-2 protein suggested that there was no inhibition of apoptosis while the absence of proapoptotic Bax suggested that other proapoptotic protein might have played a role in the induction of apoptosis. Untreated cells, irradiated cells and cells treated with photosentitizer alone showed no changes in morphology, increase in cellular viability, proliferation, expression of Bcl2/Bax, and Hsp 70 and a decrease in DNA damage and cell membrane damage. However, treatment of cell with photosensitizer alone caused DNA damage. These results indicate that irradiation or photosensitizer alone has no effect on the A549 cells but photosensitizer alone induces DNA damage.