This study evaluated Curcuma longa, Citrus lemon, Hamamelis virginiana and Hypericum perforatum available on the market as photosensitizers (FS) in Antimicrobial Photodynamic Therapy (aPDT). Each FS were analyzed in a spectrophotometer between 350 and 750 nm, to determine the ideal light source. Once the absorption bands were determined, 3 light sources were selected to determine the concentration of use, the compounds were verified at different concentrations on bovine dentin samples to evaluate the risk of staining. Once the concentration was determined, FS were evaluated for cytotoxicity on fibroblast culture. Each compound was then irradiated with each light source and evaluated for indirect reactive oxygen species (ROS) production. The bacterial reduction was tested in culture of E. faecalis in planktonic form and in biofilm using energy of 10 J and Energy Density of 26 J/cm2. The tested compounds had light absorption in three bands of the visible spectrum: violet (405 nm), blue (470 nm) and red (660 nm). At 1:6 concentration, none of the compounds promoted tooth staining, as none of the compounds showed significant toxicity in cells or bacterial suspension. In addition, when the compounds were irradiated at each wavelength, significant ROS production was observed. When the aPDT was performed on the planktonic and biofilm bacteria, significant microbial reduction was observed for both cases, reaching up to 5Logs of reduction. In conclusion, extracts of Curcuma longa, Citrus lemon, Hamamelis virginiana and Hypericum perforatum, showed potential for use in aPDT as photosensitizing agents.
Cryo-electron tomography (cryo-ET) is an emerging technology that enables thin samples, including small intact prokaryotic cells, to be imaged in three dimensions in a near-native 'frozenhydrated' state to a resolution sufficient to recognize very large macromolecular complexes in situ. This methodology has been fundamental to provide information about cellular ultrastructure. This study used cryo-ET to evaluate the photodynamic effect on the viability and envelope architecture of a Gram-negative bacteria. Bacterial suspension of E. coli minicells were submitted to photodynamic treatment with methylene blue solution (100μM) and a 100mW low power diode laser emitting at 660nm with 6 and 18J of energy. As a control group, a suspension of minicells were submitted to 462 IU/mL penicillin G for 60 min at 30 °C, to compare the damage in cell wall structure. After treatment bacteria were immediately plunge-frozen across EM grids and standard cryo-ET tilt series were collected, 3D images reconstructions were calculated and recorded. The imagens showed detachment of the bacterial cell walls and mesosome-like structures. In addition, some sites showed interrupted stretches in both inner and outer membranes and cell wall degradation, indicating bacterial envelope damage. Cryo-electron tomography revealed that the effects of photodynamic therapy on Gram negative bacteria was based on damage to the outer membrane, cell wall and inner membrane and occurs in an energy-dependent manner.
Photodynamic antimicrobial therapy (PAT) may become a useful clinical tool to treat microbial infections, overcoming
microbial resistance that is a major problem nowadays. The aim of our work was to verify the damage caused by
photosensitization over a Escherichia col) via atomic force microscopy (AFM), looking for structural changes that might
occur in cells after PAT. Cells culture were grown until a stationary phase to reach a concentration of approximately 108
cells/mL allowing the production of extracellular slime in a
biofilm-like structure. The cells including the extracellular
matrix were put in a slide and its structure was observed using AFM; subsequently a water solution of methylene blue at
60μM was applied over the cells and a pre-irradiation time of 3 minutes was waited and followed by illumination with a
diode laser (λ=660nm, power 40mW, 3min, fluence 180J/cm2, beam diameter 0.04cm2). The same cells were observed
and the images stored. A second set of experiments was performed with a smaller number of cells/area and without
extracellular slime, using the parameters abovementioned. The results showed alterations on cellular scaffold markedly
dependent on the number of cells and the presence of extracellular slime. The slime is targeted by the photosensitizer,
and after irradiation a destruction of the matrix was observed; when fewer cells were evaluated the destruction is much
more evident. The images suggested rupture of the cellular membrane and cellular fragments were observed. Our
findings indicate that AFM seems is a useful tool to investigate parameters linked with photodestruction of
microorganisms.
This study compares the antimicrobial effect of photodynamic therapy (PDT) combined to endodontic treatment
with conventional endodontic treatment alone in patients with necrotic pulp and has a 6-month radiographic follow up
comparing the healing of periapical lesions. Fifteen patients with periapical lesion and requiring root canal treatment
were selected. Microbiological samples were taken after accessing the root canal, conventional manual endodontic
therapy (group 1 n=5) and after accessing the canal, endodontic therapy and PDT (group 2 n=10). All the root canals
were filled with a calcium hydroxide paste for 1 week. Radiographs were taken after obturation and following 6 months.
Endodontic therapy alone presented an 87% reduction in microorganisms while the combination with PDT had a 95%
reduction. Radiographic follow up showed 32% higher reduction in the lesion area in PDT group. Results suggest that
the use of PDT added to conventional endodontic treatment leads to a further major reduction of microbial load. PDT is
an efficient alternative to chemical antimicrobial agents. It is a non-cumulative local treatment, which may be an
appropriate approach for the treatment of infections in the oral cavity.
Photodymanic antimicrobial therapy (PAT) has been proposed to treat oral infections and the phenothiazinic compound
Methylene Blue (MB) has been considered as a suitable photosensitizer for this application. MB is a known
methachromatic compound and the dimerization process may lead to different photochemical reactions, into the oral
cavity, where the complete isolation of the saliva may not be possible. The aim of this study is to monitor the
dimerization process of MB in the presence of human saliva through absorption spectroscopy. Absorption spectra of
30μM MB solutions in water and in human saliva were recorded in the wavelengths ranging from λ=400nm to λ=700nm.
The spectra were recorded immediately after mixture and 1 min, and 5 minutes after blending. The results were evaluated
by spectral analyses and through the calculus of the dimer/monomer ratio. The results demonstrated that immediately
after mixture a hypochromic effect characterized by the diminishing on the total absorption in the visible range of the
spectrum (λ= 400nm - 700nm) is observed, but the aggregation process is not detectable. After 1 minute the ratio between
dimer and monomer absorption increase and this increase became higher upon increasing the contact time. The results
indicate that the addition of saliva into the mixture leads to and hypochromic effect follow by the dye aggregation.
Aggregation is probably an important variable to be analyzed when choosing the pre-irradiation time in oral cavity
application, because it may lead to different photochemical routes.
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