The combined use of a photosensitizing agent and a low power laser directed at the access cavity was bactericidal to S. intermedius biofilms in root canals but was unable to achieve total kill, unlike 3% NaOCl.

The aims of this study were to (1) determine the effect of dosimetric and physiological factors on the lethal photosensitization of Porphyromonas gingivalis using toluidine blue O (TBO) and light from a helium/neon (HeNe) laser; (2) determine the influence of sensitizer concentration, preirradiation time, serum and growth phase on sensitizer uptake by P. gingivalis. The dosimetric factors studied were concentration of TBO, light dose and preirradiation time. The physiological factors were presence of serum, pH and bacterial growth phase. Sensitizer uptake by P. gingivalis under various conditions was determined using tritiated TBO (3H-TBO). In the presence of TBO, a light dose-dependent increase in kill was attained (100% kill at 4.4 J). There was no significant effect on the numbers killed when TBO was increased from 12.5 to 50 micrograms/mL. An increase in preirradiation time gave slightly increased kills. High kills were achieved at all three pH (6.8-8.0). Although kills were substantial in the presence of serum, they were significantly less than those obtained in the presence of saline. Cells in all three growth phases were susceptible to lethal photosensitization, although stationary phase cells were slightly less susceptible. Maximum uptake of TBO occurred within 60 s and uptake in serum was less than in saline. The uptake by the log phase cells was greater at lower concentrations of sensitizer (50 micrograms/mL), compared to the other two phases.

The purpose of the study was to determine the distribution of the photosensitizer toluidine blue O (TBO) within Porphyromonas gingivalis and the possible mechanism(s) involved in the lethal photosensitization of this organism. The distribution of TBO was determined by incubating P. gingivalis with tritiated TBO (3H-TBO) and fractionating the cells into outer membrane (OM), plasma membrane (PM), cytoplasmic proteins, other cytoplasmic constituents and DNA. The percentage of TBO in each of the fractions was found to be, 86.7, 5.4, 1.9, 5.7 and 0.3%, respectively. The involvement of cytotoxic species in the lethal photosensitization induced by light from a heliumneon (HeNe) laser and TBO was investigated by using deuterium oxide (D2O), which prolongs the lifetime of singlet oxygen, and the free radical and signlet oxygen scavenger L-tryptophan. There were 9.0 log10 and 2 log10 reductions in the presence of D2O and H2O (saline solutions), respectively, at a light dose of 0.44 J (energy density = 0.22 J/cm2), suggesting the involvement of singlet oxygen. Decreased kills were attained in the presence of increasing concentrations of L-tryptophan. The effect of lethal photosensitization on whole cell proteins was determined by measuring tryptophan fluorescence, which decreased by 30% using 4.3 J (energy density = 4.3 J/cm2) of light. Effects on the OM and PM proteins were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. There was evidence of change in the molecular masses of several PM proteins and OM proteins compared to controls. There was evidence of damage to the DNA obtained from irradiated cells. Scanning electron microscopic studies showed that there was coaggregation of P. gingivalis cells when sensitized and then exposed to laser light. These results suggest that lethal photosensitization of P. gingivalis may involve changes in OM and/or PM proteins and DNA damage mediated by singlet oxygen.

Current photodynamic therapy (PDT) of cancer is limited by inefficiencies involved in specifically targeting photosensitizers to tumors. Although antibodies are being explored as targeting vehicles, they present significant challenges, particularly in terms of pharmacokinetics and drug-coupling. We describe here a novel and effective system to covalently attach multiple photosensitizer molecules (both preclinical, pyropheophorbide-a and clinically approved, verteporfin photosensitizers) to single-chain Fvs. Further, we demonstrate that not only do the resulting photoimmunoconjugates retain photophysical functionality, they are more potent than either free photosensitizer, effectively killing tumor cells in vitro and in vivo. For example, treatment of human breast cancer xenografts with a photoimmunoconjugate comprising an anti-HER-2 scFv linked to 8-10 molecules of pyropheophorbide-a leads to significant tumor regression. These results give an insight into the important features that make scFvs good carriers for PDT drugs and provide proof of concept of our unique approach to targeted photodynamic therapy (tPDT). This promises to significantly improve on current photodynamic therapies for the treatment of cancer. ' 2007 Wiley-Liss, Inc.Key words: photodynamic therapy; single chain Fv; pyropheophorbide-a; verteporfin Photodynamic therapy (PDT) is a minimally invasive procedure used in a range of conditions where superficially localized lesions such as age-related macular degeneration (AMD) or tumors need to be treated. 1 PDT is typically a 2-step process that involves the administration of a photosensitizer (PS) leading to marginal accumulation in the tumor. Following this, the PS is activated by exposure to light of an appropriate wavelength. This ultimately leads to the conversion of molecular oxygen into reactive oxygen species (ROS), primarily singlet oxygen, leading to tumor cell death via irreversible damage to cellular components such as proteins, lipids and DNA. 2 Current clinical use of PDT achieves efficacies similar to conventional therapies but with lower morbidity, simplicity of use and improved functional and cosmetic outcome. 3,4 PDT has mainly been used where conventional approaches have failed or were unsuitable. These include premalignant dysplastic lesions and noninvasive cancers, which are commonly found in the mucosa of the aerodigestive 5 and urinary tracts. 6 Success in treating these types of cancers has been achieved using Photofrin 1 , 7 Levulan 18 and Foscan 1 . 9 The most successful application of PDT has been for wet age-related macular degeneration (AMD) for which the photosensitizer verteporfin (Visudyne 1 ) has been used to destroy ocular neovasculature. 10 PDT has also had great successes in dermatology because of its impressive cosmetic outcome. Methyl 5-aminolaevulinate (Metvix 1 ) has been used to treat actinic keratosis with up to 90% cure. 11 Although PSs accumulate in cancer cells, the tumor specificity ratios are low and their inherent hydrophobicity, causes them to persist...

We report the synthesis, spectroscopic properties and intracellular imaging of recombinant antibody single chain fragment (scFv) conjugates with photosensitizers used for photodynamic therapy of cancer (PDT). Two widely-studied photosensitizers have been selected: preclinical pyropheophorbide-a (PPa) and verteporfin (VP), which has been clinically approved for the treatment of acute macular degeneration (Visudyne). Pyropheophorbide-a and verteporfin have been conjugated to an anti-HER2 scFv containing on average ten photosensitizer molecules per scFv with a small contribution (

We have previously demonstrated that Porphyromonas gingivalis is susceptible to killing by toluidine blue O (TBO) when irradiated with light from a helium-neon (HeNe) laser. The aim of this study was to determine whether a TBO-antibody conjugate (Ab-TBO) could be used to specifically target P. gingivalis to lethal photosensitization in the presence of Streptococcus sanguis or human gingival fibroblasts (HGFs). When a mixture of P. gingivalis and S. sanguis was exposed to 4 g of TBO/ml and irradiated with HeNe laser light, there were 1.5-and 4.0-log 10 -unit reductions in the viable counts, respectively. In contrast, when TBO was conjugated with a murine monoclonal antibody against P. gingivalis lipopolysaccharide, the reductions in viable counts of P. gingivalis and S. sanguis amounted to 5.0 and 0.1 log 10 units, respectively. Lethal photosensitization of P. gingivalis in the presence of HGFs using unconjugated TBO resulted in a 0.7-log 10 -unit reduction in P. gingivalis viable counts and a 99% reduction in the incorporation of tritiated thymidine ([ 3 H]Tdr) by the HGFs. In contrast, when the Ab-TBO conjugate was used, there was a 100% reduction in P. gingivalis viable counts but no significant reduction in the incorporation of [ 3 H]Tdr by HGFs. These results demonstrate that specific targeting of P. gingivalis can be achieved using TBO conjugated to a monoclonal antibody raised against a cell surface component of this organism.

The purpose of the study was to determine the distribution of the photosensitizer toluidine blue O (TBO) within Porphyromonas gingivalis and the possible mechanism(s) involved in the lethal photosensitization of this organism. The distribution of TBO was determined by incubating P. gingivalis with tritiated TBO (3H-TBO) and fractionating the cells into outer membrane (OM), plasma membrane (PM), cytoplasmic proteins, other cytoplasmic constituents and DNA. The percentage of TBO in each of the fractions was found to be, 86.7, 5.4, 1.9, 5.7 and 0.3%, respectively. The involvement of cytotoxic species in the lethal photosensitization induced by light from a heliumneon (HeNe) laser and TBO was investigated by using deuterium oxide (D2O), which prolongs the lifetime of singlet oxygen, and the free radical and signlet oxygen scavenger L-tryptophan. There were 9.0 log10 and 2 log10 reductions in the presence of D2O and H2O (saline solutions), respectively, at a light dose of 0.44 J (energy density = 0.22 J/cm2), suggesting the involvement of singlet oxygen. Decreased kills were attained in the presence of increasing concentrations of L-tryptophan. The effect of lethal photosensitization on whole cell proteins was determined by measuring tryptophan fluorescence, which decreased by 30% using 4.3 J (energy density = 4.3 J/cm2) of light. Effects on the OM and PM proteins were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. There was evidence of change in the molecular masses of several PM proteins and OM proteins compared to controls. There was evidence of damage to the DNA obtained from irradiated cells. Scanning electron microscopic studies showed that there was coaggregation of P. gingivalis cells when sensitized and then exposed to laser light. These results suggest that lethal photosensitization of P. gingivalis may involve changes in OM and/or PM proteins and DNA damage mediated by singlet oxygen.

Previous studies have shown one of the causative agents of periodontitis, Porphyromonas gingivalis, can be killed by red light in the presence of the light-activated antimicrobial agent toluidine blue O (TBO). The purpose of this study was to determine the effects of irradiating the organism with red light in the presence of TBO on its proteolytic enzyme activity.Suspensions of P. gingivalis were exposed to light with a wavelength of 633 nm in the presence of various concentrations of TBO. Samples were taken at various times and their proteolytic activity determined by assay of azocasein hydrolysis. On exposure to 126 J of red light in the presence of 12.5 µg/ml of TBO the proteolytic enzyme activity was reduced by 100%.The results of this study have shown that the main virulence factor of P. gingivalis, its proteolytic activity, can be inactivated by red light in the presence of TBO. This, together with the known bactericidal activity of the TBO/light combination, suggests that photodynamic therapy may prove important in reducing the effectiveness of P. gingivalis as a periodontopathogen in vivo.