Cationic galactoporphyrins on microorganisms photoinactivation

• Autor(a) principal: Gomes, Maria Clara Ferreira de Almeida Cardia
• Data de Publicação: 2010
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/8908
• topic: Microbiologia; Terapia fotodinâmica; Porfirinas

Antimicrobial photodynamic therapy (aPDT) is becoming a promising alternative to inactivate microbial pathogens. This therapy combines three nontoxic components, a photosensitizer (PS), light and oxygen, that when combined leads to the formation of highly cytotoxic reactive oxygen species, mainly singlet oxygen (1O2). This specie can oxidize many types of biological molecules, such as proteins, nucleic acids and lipids. The combination of positively charged groups and carbohydrate moieties with porphyrin derivatives results in an increased cell recognition and water solubility, which improves cell membrane penetration and accumulation in sub-cellular compartments. The aim of this work was to synthesize new meso-substituted glycosyl porphyrins derivatives and evaluated the efficacy of these compounds as PS in the photoinactivation of two environmental gram positive bacteria, Brevibacterium sp. and Micrococcus sp., and one gram negative bacteria, bioluminescent Escherichia coli. Brevibacterium sp. and Micrococcus sp were chosen for these studies because they were, respectively, representative of very sensitive and very resistant types to UV-B irradiation experiments. It was also evaluated the effect of 1O2 at the lipid and protein oxidation level, generated during the aPDT assay, on the two gram positive bacteria. The derivatives of meso-tetrapyridyl porphyrin were cationized by methyl iodide or by carbohydrate moieties. All synthesized compounds were characterized by proton and fluor nuclear magnetic resonance and by mass spectrometry. Two of the compounds synthesized 5,10,15,20-tetrakis(Nmethylpyridinium- 4-yl)porphyrin tetra-iodide (PS 1) and 5-[N-(Isopropylidene-6- deoxy-galactopyranos-6-yl)pyridinium-4-yl]-10,15,20-tris(N-methylpyridinium-4- yl)porphyrin tetra-iodide (PS 2) were used as PS in the aPDT assays. For the aPDT assays pure bacterial suspensions were irradiated after pre-incubation in the dark, at concentrations of 0.5, 1 and 5 μmol dm-3 of PS in the case of gram positive bacteria, and 5 μmol dm-3 in the case of gram negative bacteria. The kinetics of irradiation was evaluated by the quantification of colony forming units in aliquots collected during 15 minutes of irradiation, under 150 mW cm-2. Light and dark controls were included in all experiments. Photophysical testes (photostability and 1O2 genereation studies) were also performed. Lipid oxidation was assessed by Tiobarbituric acid (TBA) assay and results were expressed in terms of Malondialdehyde (MDA) (nmol dm-3). Protein oxidation was evaluated by 2,4-dinitrophenylhydrazine (DNPH) assay and results were expressed in terms of Protein carbonyl concentration (nmol cm-3). aPDT assays revealed that PS 2 was more effective (3.0 log of reduction) than the PS 1 (2.0 log) with 5 μmol dm-3 against E. coli. In the case of gram positive bacteria, both PS showed the same photoinactivation effect, presenting complete inactivation after 2 minutes of irradiation. However, with lower concentration PS 1 showed to be more effective than PS 2 in both gram positive bacteria. Photophysical studies showed that both PS are photostable and good 1O2 producers. ! Lipid peroxidation assays displayed different results for both environmental bacteria. In Micrococcus no lipid oxidation was observed with PS 1 while with PS 2 was observed around 31% of lipid oxidation (0.083 nmol dm-3). In Brevibacterium PS 1 caused 28% (0.063 nmol dm-3) and PS 2 50% (0.093 nmol dm-3) of lipid peroxidation. According to the protein oxidation results, Micrococcus showed around 2.1 nmol mL-1 and 6.2 nmol mL-1 of protein carbonyls with PS 1 and 2, respectively. In the case of Brevibacterium 5.0 nmol mL-1 and 4.8 nmol mL-1 were observed with PS 1 and 2, respectively. Both porphyrins showed good photoinactivation results on gram negative and gram positive bacteria. Susceptibility of Brevibacterium sp. and Micrococcus sp. to aPDT were different to those showed in UV-B irradiation by these same bacteria. Lipid oxidation assays allowed to conclude that, in Brevibacterium, both PS act in plasma membrane while in Micrococcus this only happens with the PS with a carbohydrate moiety. Protein oxidation led to the conclusion that protein damage may have occurred due to lipid oxidation or direct interaction of 1O2 with proteins.