Reevaluation of analytical methods for photogenerated singlet oxygen

The aim of the present study is to compare different analytical methods for singlet oxygen and to discuss an appropriate way to evaluate the yield of singlet oxygen photogenerated from photosensitizers. Singlet oxygen photogenerated from rose bengal was evaluated by electron spin resonance analysis using sterically hindered amines, spectrophotometric analysis of 1,3-diphenylisobenzofuran oxidation, and analysis of fluorescent probe (Singlet Oxygen Sensor Green®). All of the analytical methods could evaluate the relative yield of singlet oxygen. The sensitivity of the analytical methods was 1,3-diphenylisobenzofuran < electron spin resonance < Singlet Oxygen Sensor Green®. However, Singlet Oxygen Sensor Green® could be used only when the concentration of rose bengal was very low (<1 µM). In addition, since the absorption spectra of 1,3-diphenylisobenzofuran is considerably changed by irradiation of 405 nm laser, photosensitizers which are excited by light with a wavelength of around 400 nm such as hematoporphyrin cannot be used in the 1,3-diphenylisobenzofuran oxidation method. On the other hand, electron spin resonance analysis using a sterically hindered amine, especially 2,2,6,6-tetramethyl-4-piperidinol and 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide, had proper sensitivity and wide detectable range for the yield of photogenerated singlet oxygen. Therefore, in photodynamic therapy, it is suggested that the relative yield of singlet oxygen generated by various photosensitizers can be evaluated properly by electron spin resonance analysis

Bactericidal Action of Photogenerated Singlet Oxygen from Photosensitizers Used in Plaque Disclosing Agents

Photodynamic therapy (PDT) has been suggested as an efficient clinical approach for the treatment of dental plaque in the field of dental care. In PDT, once the photosensitizer is irradiated with light of a specific wavelength, it transfers the excitation energy to molecular oxygen, which gives rise to singlet oxygen., a major causative pathogen of caries, followed by erythrosine and phloxine, both of which showed activity similar to each other. One of the reasons for the discrepancy between the singlet oxygen generating ability and bactericidal activity was the incorporation efficiency of the photosensitizers into the bacterial cells. The incorporation rate of rose bengal was the highest among the three photosensitizers examined in the present study, likely leading to the highest bactericidal activity. Meanwhile, the addition of L-histidine, a singlet oxygen quencher, cancelled the bactericidal activity of any of the three photoactivated photosensitizers, proving that singlet oxygen was responsible for the bactericidal action.It is strongly suggested that rose bengal is a suitable photosensitizer for the plaque disclosing agents as compared to the other two photosensitizers, phloxine and erythrosine, when used for PDT

Incorporation rates of photosensitizers into the bacterial cells.

<p>The bacterial suspension containing 10 μM rose bengal, phloxine, or erythrosine was incubated at room temperature for 3 min. Each value represents the mean of triplicate determinations with standard deviation. Statistically significant differences between the two groups are shown as * (p<0.05).</p

Number of viable <i>S. mutans</i> in the suspension after each treatment.

<p>P(+)L(+), P(+)L(−), P(−)L(+), and P(−)L(−) stand for laser irradiation of each photosensitizer, photosensitizer alone, laser irradiation alone, and no treatment, respectively. Under the conditions of P(+)L(+) and P(+)L(−), the suspension contained 10 μM rose bengal, phloxine, or erythrosine. Each value represents the mean of nonuplicate determinations.</p

A schema showing oxidation of a sterically hindered amine TPC (2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide) by singlet oxygen to give rise to the nitroxide radical, and representative ESR spectra of the laser-irradiated mixtures containing rose bengal and a sterically hindered amine TPC.

<p>The mixture containing 450 mM TPC and 0, 1 or 10 µM rose bengal was irradiated by the laser light with a wavelength of 532 nm for 60 s. Open circle and closed circle indicate Mn²⁺ marker and the nitroxide radical, respectively.</p

Influence of L-histidine, a singlet oxygen quencher, on the bactericidal effects of laser-irradiated photosensitizers.

<p>L-Histidine (25 and 100 mM) was added to the bacterial suspension containing 10 μM rose bengal, phloxine, or erythrosine, followed by laser-irradiation for 60 s. Each vealue represents the mean of nonuplicate determinations with standard deviation. Statistically significant differences from the corresponding P (−)L(−) group are shown as ** (p<0.01).</p