Evaluation of photo-activated disinfection effectiveness with methylene blue against Porphyromonas gingivalis involved in endodontic infection: An in vitro study

BackgroundEradication or suppression of microbial pathogens is a major goal in endodontic infection therapy. Sub-lethal doses of photo-activated disinfection (sPAD) as a new treatment method might be able to control the microorganisms involved in endodontic infections normally treated with PAD. This study evaluated the effect of sPAD using methylene blue (MB) in combination with diode laser irradiation on the growth and biofilm formation ability of Porphyromonas gingivalis as an endodontic pathogen.Materials and methodsThe anti-microbial and anti-biofilm potential of sPAD against P. gingivalis were assessed at sub-lethal doses of MB and irradiation by diode laser on colony forming unit and crystal violet assays, respectively.ResultsMB-sPAD using 25 μg/mL at a fluency of 117.18 J/cm² and 50–100 μg/mL at a fluency of 93.75 J/cm² significantly P. gingivalis growth when compared to the control. MB at 100 μg/mL at a fluency of 117.18 J/cm² in MB-mediated PAD showed a significant inhibitory effect on biofilm formation in P. gingivalis compared with MB-sPAD.ConclusionHigh doses of MB-mediated sPAD exhibited anti-microbial and anti-biofilm potential activity, whereas lower doses of MB-mediated sPAD did not display this ability. Therefore, the dose of PAD used in vivo should be taken into account for endodontic treatment.     

Monitoring gene expression of rcpA from Aggregatibacter actinomycetemcomitans versus antimicrobial photodynamic therapy by relative quantitative real-time PCR

BackgroundAggregatibacter actinomycetemcomitans is an important pathogen that is frequently found in various infections, particularly aggressive periodontitis. In this study, we described the outcome of the expression level of A. actinomycetemcomitans virulence factor following treatment by antimicrobial photodynamic therapy (aPDT) with indocyanine green (ICG) as a photosensitizing agent.Materials and methodsTo determine the aPDT effect on the cell-surviving assay and expression ratio of the rcpA gene in A. actinomycetemcomitans by a colony-forming unit and relative quantitative (q) real-time PCR (qRT-PCR) assays, respectively, the proper dosing of sub-lethal aPDT was specified.ResultsThe results of the current study showed that ICG-mediated aPDT, using 250–1000 μg/mL, showed a significant reduction in A. actinomycetemcomitans growth when compared to the control group (P < 0.05). Also, a sub-lethal dose of aPDT against A. actinomycetemcomitans was 125 μg/mL ICG, with a 30 s diode laser irradiation time at fluency of 15.6 J/cm² that could reduce the expression of rcpA gene approximately 6-fold.DiscussionaPDT with ICG could reduce the cell survival and the virulence agent of A. actinomycetemcomitans. Thus, use of the appropriate aPDT dosage can be used for the successful treatment of periodontitis in vivo.     

The in vitro effect of antimicrobial photodynamic therapy with indocyanine green on Enterococcus faecalis: Influence of a washing vs non-washing procedure

IntroductionThe purpose of this study was to evaluate the in vitro effect of washing and non-washing of indocyanine green (ICG) as photosensitizer (PS) on bacterial count, biofilm formation, development and degradation of Enterococcus faecalis.MethodsThe anti-bacterial, anti-biofilm formation, anti-biofilm development and biofilm degradation of anti-microbial photodynamic therapy (aPDT) against E. faecalis was determined at concentrations of 3 to 2000 μg/mL of ICG, subject to 18 J/cm² dose of diode laser (808 nm) in washing and non-washing producers. Bacterial viability measurements and biofilm assays were evaluated by broth microdilution method and crystal violet assays, respectively.ResultsICG-mediated aPDT, using 25 to 2000 μg/mL and 50 to 2000 μg/mL showed significant reduction in E. faecalis growth when compared to the control in non-washing and washing producers, respectively (P < 0.05). Also, ICG-mediated aPDT showed a significantly inhibitory effect on biofilm formation of E. faecalis in concentration of 6 to 2000 μg/mL and 100 to 2000 μg/mL in non-washing and washing groups (P < 0.05). The biofilm development was inhibited by concentrations of 12 to 2000 μg/mL and 100 to 2000 μg/mL in non-washing and washing groups. The biofilm degradation increased from concentrations of 12 to 2000 μg/mL and 250 to 2000 μg/mL in non-washing and washing groups, respectively.ConclusionThis study shows that the application of ICG should be accompanied by laser irradiation without being washed out to achieve better result for bacterial count reduction and anti-biofilm effects.     

The effect of indocyanine green loaded on a novel nano-graphene oxide for high performance of photodynamic therapy against Enterococcus faecalis

BackgroundRecently developed photodynamic therapy (PDT) has gained attention for achieving effective root canal disinfection. Using an optimized nontoxic photosensitizer (PS), such as indocyanine green (ICG), is an imperative part of this technique. Therefore, the objective of the current study was to improve ICG photodynamic properties through incorporation of ICG into nano-graphene oxide (NGO) in order to produce NGO-ICG as a new PS and also to assess the antimicrobial effects of NGO-ICG against Enterococcus faecalis after photodynamic therapy.Materials and methodsNGO-ICG was synthesized based on oxidation of graphite flakes and direct loading of ICG onto NGO. NGO-ICG formation was confirmed using the Fourier Transform Infrared Spectroscopy (FT-IR), Scanning Electron Microscopy (SEM), and UV–vis spectrometry. The antimicrobial and anti-biofilm potential of NGO-ICG-PDT against E. faecalis was assessed via colony forming unit and crystal violet assays, respectively.ResultsFT-IR, SEM and UV–vis spectrometry confirmed successful synthesis of NGO-ICG containing 200 μg/mL of ICG. NGO-ICG-PDT at an energy density of 31.2 J/cm² showed a significant reduction (2.81 log) in the count of E. faecalis (P < 0.05). NGO-ICG-PDT significantly reduced the biofilm formation ability of E. faecalis up to 99.4% (P < 0.05). The overall antimicrobial and anti-biofilm potential of NGO-ICG-PDT was higher than PDT based on ICG (1000 μg/mL) (47% and 21%, respectively).ConclusionBecause NGO-ICG-PDT showed a significant reduction in the number and biofilm formation ability of E. faecalis at low ICG concentrations (200 μg/mL), it could be a new approach to adjuvant treatment of endodontic infections.     

In vitro evaluation of adhesion/proliferation of human gingival fibroblasts on demineralized root surfaces by toluidine blue O in antimicrobial photodynamic therapy

BackgroundAntimicrobial photodynamic therapy (aPDT) in Dentistry has important effects as bacterial destruction in areas with periodontal disease. Some dyes applied in aPDT could present low pH and, consequently, result in tooth demineralization. This study evaluated demineralization produced by aPDT with toluidine blue O (TBO) at low pH and analyzed adhesion/proliferation of human gingival fibroblasts (HGF).MethodsIn the 1st phase, bovine enamel and root dentin fragments received 2 treatments: PDT4 group (TBO–100 μg/ml—pH 4–60 s) plus laser (660 nm, 45 J/cm², 1.08 J, 30 mW, 30 s, spot 0.024 cm², 1.25 W/cm², sweeping, non-contact) and CA group (citric acid plus tetracycline—pH 1–180 s). Surface hardness loss and tooth wear were statistically analyzed (Student’s t test, ANOVA/Tukey, p < 0.05). In the 2nd phase, human dentin fragments were divided in C (control group—scaling and root planing), PDT4 and CA. HGF (10⁴, 5th passage) were cultured on these fragments for 24, 48 and 72 h and counted in scanning electron microscopy photographs. Number of HGF was analyzed using repeated-measures ANOVA and Tukey (p < 0.05).ResultsPercentage of surface hardness loss was similar in dentin for PDT4 (71.5%) and CA (76.1%) (p > 0.05) and higher in enamel for CA (68.0%) compared to PDT4 (34.1%) (p < 0.05). In respect to wear, no difference was found between PDT4 (dentin: 12.58 μm, enamel: 12.19 μm respectively) and CA (dentin: 11.74 μm and enamel: 11.03 μm) (p > 0.05). Number of HGF was higher after 72 h in CA group (2.66, p < 0.05) compared to PDT4 (2.2) and C (1.33).ConclusionPDT4 is not as aggressive as CA for enamel. However, dentin demineralized promoted by PDT4 does not stimulate HGF adhesion and proliferation as CA.     

Sub-additive effects of photodynamic therapy combined with erlotinib for the treatment of epidermoid carcinoma: An in vitro study

BackgroundPhotodynamic therapy (PDT) is an antitumour treatment that employs the combination of a photosensitive compound, oxygen and visible light. To improve the antitumour activity of PDT, the present study used the strategy of combining PDT with erlotinib (ERL), a drug frequently used in the treatment of epidermoid carcinoma.MethodsAn MTT cell viability assay was used to evaluate the cytotoxicity of PDT combined with ERL on A431 epidermoid carcinoma cells in vitro. This study evaluated the cytotoxicity of the following treatments: red laser irradiation (660 nm) at different power densities (1.25–180 J/cm²), the photosensitizer methylene blue (MB) at concentrations of 0.39–100 μM, PDT (12.5 μM MB and laser power densities from 1.25 to 180 J/cm²), and PDT (12.5 μM MB and a laser density of 120 J/cm²) plus ERL (1 μM).ResultsThe laser power densities that were tested showed no cytotoxicity in A431 cells. MB showed a dose-dependent cytotoxicity. In PDT, an increase in the dose of light resulted in an increase in the cytotoxicity of MB. In addition, there was a sub-additive effect between PDT and ERL compared to the effect of each therapy alone.ConclusionsThe sub-additive effect between PDT and ERL suggests that their combination may be an important strategy in the treatment of epidermoid carcinoma.     

Efficacy of erythrosine and cyanidin-3-glucoside mediated photodynamic therapy on Porphyromonas gingivalis biofilms using green light laser

BackgroundThe purpose of this in vitro study was to evaluate the efficacy of erythrosine and cyanidin-3-glucoside as photosensitizers in PDT for the elimination of Porphyromonas gingivalis (P. gingivalis) biofilms.MethodsP. gingivalis biofilms were prepared from a chronic periodontitis subject. Erythrosine and cyanidin-3-glucoside were prepared and randomly allocated as follows: 110, 220, 330, and 440 μM erythrosine; 101, 202, 303, and 404 μM anthocyanin; and 440 μM erythrosine + 404 μM cyanidin-3-glucoside. There were 18 PDT experimental groups (non-irradiated/irradiated with a 532-nm green light diode laser at 1.29 J/cm² for 60 s). The 3 controls were grouped as follows: biofilms exposed to the photosensitizers alone, biofilms exposed to the laser alone, and biofilms exposed to 0.12% chlorhexidine. All sample groups were cultured at 1, 3 and 6 h after PDT and incubated in an anaerobic chamber at 37 °C for 4 days. The surviving fraction was calculated from the log₁₀ CFU/ml. The 330 and 440 μM erythrosine and the 440 μM erythrosine + 404 μM cyanidin-3-glucoside were mixed with spin traps (TEMPO, DMPO), and the electron spin resonance spectra were evaluated.ResultsThe log₁₀ CFU/ml measurements showed that the PDT groups with 330 μM or 440 μM erythrosine and 440 μM erythrosine + 404 μM cyanidin-3-glucoside had statistically significant differences from the other groups (one-way ANOVA and Bonferroni’s multiple comparison test, p- value ≤ 0.05).ConclusionsPDT using 330 μM erythrosine, 440 μM erythrosine or 440 μM erythrosine + 404 μM cyanidin-3-glucoside irradiated with the laser more effectively inhibited P. gingivalis in biofilms.     

Photodynamic therapy effect on cell growth inhibition induced by Radachlorin and toluidine blue O on Staphylococcus aureus and Escherichia coli: An in vitro study

IntroductionPhotodynamic therapy is an innovative treatment modality, which is appropriate for tumor detection and for the treatment of cancer as well as nontumoral diseases, such as psoriasis (2), bacterial and viral eradication.Material and methodEffect of two photosensitizer (toluidine blue O (TBO) and Radachlorin was investigated on Staphylococcus Aureus ATCC 25923 (American Type Culture Collection) and Escherichia coli (ATCC 25922).ResultsPDI by TBO caused S. aureus 5.83 log10 killing (P.Value < 0.0001) and reduce 0.08 log 10 in E. coli (P.Value = 0.321). PDI by Radachlorin^® reduce 0.17 log 10 in E. coli (P.Value < 0.0001) and S. aureus showed 6.1 log 10 colony count reduction.ConclusionWithin the limitation of this in vitro study, we can conclude that both PS have the same effect on S. aureus and E. coli with good inhibition effect on S. aureus and partial inhibition effect E. coli.     

Antimicrobial photodynamic therapy on Streptococcus mutans is altered by glucose in the presence of methylene blue and red LED

BackgroundDental caries are a multifactorial disease that progressively produces tooth destruction as a result of bacterial colonization of enamel surface, especially Streptococcus mutans. The objective of this work was to investigate the role of glucose in antimicrobial photodynamic therapy (aPDT) on S. mutans.MethodsS. mutans ATCC 25175 were cultured on microaerophilia at 37 °C for 48 h, and we tested aPDT in the presence of 50 mM glucose. Bacterial suspension was used to investigate aPDT with 100 μM methylene blue (MB) under LED emitting radiation at ʎ = 660 nm and parameters as following (P = 473 mW; I = 166.8 mW/cm², and doses of 5, 10 and 20 J/cm²). A seventy-two hours biofilm was grown on 96 flat buttoned well-plate and irradiation was performed from 10 to 80 J/cm² at similar conditions.ResultsThere was no dark toxicity nor bacterial death regarding LED irradiation on suspension and on biofilm. Nevertheless, aPDT presented expressive bacterial inactivation following 1 and 2 min of irradiation on cell suspension. On the other hand, there was no inactivation in the presence of glucose under the same conditions. Biofilm was completely inactivated by MB-mediated aPDT after 6 min of irradiation. However, the presence of glucose delayed the complete inactivation of the biofilm.ConclusionThe presence of glucose in the suspension drastically delayed the effect of aPDT on S. mutans and this effect is more pronounced in bacterial suspension than on biofilm.     

The comparison of penetration depth of two different photosensitizers in root canals with and without smear layer: An in vitro study

BackgroundThe main objective of this study is to evaluate the penetration depth of suggested photosensitizers in the lateral wall of the human root canal.Materials & methodsForty extracted single-rooted human teeth with straight canals that extracted for periodontal reasons were collected and stored in the sterile saline until employment in the experiment. Teeth were decoronated to a standard 12 mm root segment using diamond disc. After instrumentation of specimens, the external root surface was sealed with two layers of nail polish to avoid environmental contamination. The apical foramen was subsequently closed with composite material. Teeth were divided randomly in two major groups consist of indocyanine green solution (ICG) and tolonium chloride solution (TCH) with and without EDTA in their subgroups. Specimens in all groups grooved longitudinally with a diamond disc and split in two halves with a stainless steel chisel. The measurements were done by the stereo microscope under 20× magnification in three zones of each specimen and the penetration depth of dye was measured.ResultsThe results of this study showed that the mean of lateral penetration depth of ICG (224.04 μm) was significantly (P < 0.05) higher than TCH (70.15 μm). Regarding to the influence of EDTA, in ICG group without consideration to the different regions, the usage of EDTA improved the mean of lateral penetration depth of ICG, but this improvement was not statistically significant (P > 0.05).ConclusionFurther to the findings of this study, it could be assumed that ICG could penetrate in deeper regions of the root canal wall.     

Methylene blue,curcumin and ion pairing nanoparticles effects on photodynamic therapy of MDA-MB-231 breast cancer cell

PurposeThe aim of current study was to use methylene blue-curcumin ion pair nanoparticles and single dyes as photosensitizer for comparison of photodynamic therapy (PDT) efficacy on MDA-MB-231 cancer cells, also various light sources effect on activation of photosensitizer (PS) was considered.MethodIon pair nanoparticles were synthesized using opposite charge ions precipitation and lyophilized. The PDT experiments were designed and the effect of PSs and light sources (Red LED (630 nm; power density: 30 mW cm⁻²) and blue LED (465 nm; power density: 34 mW cm⁻²)) on the human breast cancer cell line were examined. The effect of PS concentration (0–75 μg. mL⁻¹), incubation time, irradiation time and light sources, and priority in irradiation of blue or red lights were determined.ResultsThe results show that the ion pairing of methylene blue and curcumin enhance the photodynamic activity of both dyes and the cytotoxicity of ion pair nanoparticles on the MDA-231 breast cancer cell line. Blue and red LED light sources were used for photo activation of photosensitizers. The results demonstrated that both dyes can activate using red light LED better than blue light LED for singlet oxygen producing.ConclusionNano scale ion pair precipitating of methylene blue-curcumin enhanced the cell penetrating and subsequently cytotoxicity of both dyes together.     

Photodynamic therapy with high-power LED mediated by erythrosine eliminates Enterococcus faecalis in planktonic forms

BackgroundThe failure of endodontic treatment is linked to the presence of microorganisms, particularly Enterococcus faecalis, in the root canals. This study evaluated the effectiveness of photodynamic therapy (PDT) using erythrosine irradiated by a high-power curing light on a planktonic suspension culture of E. faecalis.MethodsBacterial suspensions of E. faecalis were adjusted and then mixed in a 1:1 proportion, in triplicate, in treatment groups by varying the length of irradiation time (120 and 240 s) and the molarity of the erythrosine (5 and 10 μM). In order to verify the post-treatment bactericidal effect, a count of the viable bacteria was performed (CFU mL⁻¹) and transformed into Log10 CFU. The one-way ANOVA with Tukey post-hoc test was applied to check for differences between the groups.ResultsThe bacteria were completely eradicated in the groups that used PDT with 5 μM 240 s, 10 μM 120 s and 10 μM 240 s (p ≪ 0.001). The effect of the PDT 5 μM 120 s group was significant (p ≪ 0.05) in comparison with the groups using only light or only erythrosine. Positive control (exposure to 2.5% NaClO for 120 and 240 s) completely eradicated E. faecalis. The negative control (PBS) did not alter the quantities of E. faecalis CFU with 9.605 Log10 CFU at 120 s and 9.621 Log10 CFU at 240 s.ConclusionPDT with erythrosine in a concentration of 10 μM and high-power LED is capable of totally eliminating E. faecalis in planktonic suspension.     

Periopathogens differ in terms of the susceptibility to toluidine blue O-mediated photodynamic inactivation

BackgroundThe main goal of periodontal therapy is to eliminate the infection spreading in periodontium. Antimicrobial photodynamic therapy may be applied in order to eradicate pathogens remaining in periodontal tissues after conventional mechanical debridement, to improve the treatment results. The aim of this in vitro study was to evaluate the susceptibility of selected key periopathogens to toluidine blue O-mediated photodynamic inactivation and the influence of photosensitizer’s concentration and light dose on the effectiveness of this process.MethodsFollowing bacterial strains were used in the experiments: Porphyromonas gingivalis ATCC 33277, Aggregatibacter actinomyctemecomitans ATCC 33384, Fusobacterium nucleatum ATCC 10953. Toluidine blue O (TBO) was used in concentration ranging from 0.004 to 0.5 mg/mL. Irradiation was performed by a non-laser red light source.ResultsComplete eradication of P. gingivalis was obtained upon the application of TBO in the concentration of 0.1 mg/mL and the highest light dose. A, actinomycetemcomitans was, in turn, not susceptible to photodynamic inactivation regardless of the dosimetric parameters applied. High viability reductions were also obtained for F. nucleatum, however no complete eradication. The effectiveness of photodynamic inactivation of susceptible periopathogens was dependent on the light dose and photosensitizer’s concentration.ConclusionsPeriopathogens differ in terms of their susceptibility to photodynamic inactivation. Antimicrobial PDT may be valuable in the treatment of those cases of periodontal disease, in which P. gingivalis is a dominating pathogen. Microbiological examination prior to clinical application of aPDT may be recommended.     

Effect of methylene blue-induced photodynamic therapy on a Streptococcus mutans biofilm model

BackgroundSeveral studies have reported the use of antimicrobial photodynamic therapy (aPDT) to control biofilm but its efficacy depends on several factors, such as biofilm model used. This study aims to examine whether exposure to diode laser combined with methylene blue affects the bacterial viability and polysaccharide content in a Streptococcus mutans cariogenic biofilm model, which simulated ‘feast-famine’ episodes of exposure to sucrose that occur in the oral cavity.Materials and methodsS. mutans biofilms were formed on acrylic resin discs and exposed to a 10% sucrose solution for 1 min, eight times/day. After growing for 48 h, the biofilms were submitted to the following treatments, twice daily (n = 4): (i) 0.9% NaCl (NaCl) as the negative control; (ii) 0.12% chlorhexidine digluconate (CHX) as the positive antibacterial control; (iii) diode laser combined with methylene blue, using an energy density of 320 J/cm² (aPDT). After 120 h of growth, the biofilm formed on each disc was collected to determine the viable bacterial counts and concentration of insoluble exopolysaccharides (IEPS) and intracellular polysaccharides (IPS).ResultsBacterial counts in the biofilms formed differed among the treatments. Compared with NaCl, aPDT significantly destabilized biofilm (p < 0.0001). aPDT and CHX equally lowered the concentration of IEPS and IPS in biofilms.ConclusionUnder the experimental conditions assessed, our findings indicate that a twice-daily treatment with diode laser combined with methylene blue effectively decreased bacterial viability and the intra- and extracellular polysaccharide concentration in biofilms of S. mutans, a cariogenic bacterium.     

Evaluation of antimicrobial photodynamic therapy with indocyanine green and curcumin on human gingival fibroblast cells: An in vitro photocytotoxicity investigation

Recent investigations have suggested that antimicrobial photodynamic therapy (aPDT) can be an alternative treatment for the management of periodontal infections. However, currently there is very limited data regarding the photocytotoxicity of this method on human gingival fibroblast (HuGu) cells.AimThe in vitro optimal concentrations of indocyanine green (ICG) and curcumin as photosensitizers (PSs) and the irradiation time of diode laser emission were evaluated by assessing the photocytotoxicity of the treatment on HuGu cells.Materials and methodMonolayers of HuGu cells were incubated with various final concentrations of ICG (500, 750, 1000, 1250, 1500, 1750, and 2000 μg/ml) and curcumin (3, 4, 5, 10, and 20 mM). Three exposure times of the diode laser (30 s, 60 s, and 2 × 30 s irradiation with an interval of 1 min between each) and one of exposure time of 5 min for LED were tested; cell viability was determined using neutral red assay. Chlorhexidine (CHX) as a gold standard antimicrobial agent for periodontal disease was considered as a control group.ResultsICG and curcumin significantly reduced HuGu cell viability at concentrations below 1000 μg/ml and 10 mM, respectively (P < 0.01). Cytotoxicity was higher when the cells were treated for 2 × 30 s irradiation with an interval of 1 min and then again exposed to the laser for 30 s (2% and 0.1%). CHX demonstrated no significant reduction in HuGu cell survival.ConclusionPhotocytotoxicity is influenced by PS concentration, exposure time of PS, and time of irradiation. High doses of ICG and curcumin with lowest exposure time of light source and without cytotoxic effects may be an effective strategy for aPDT as an alternative treatment for periodontal disease.     

Comparison of photoinactivation of T. rubrum by new methylene blue (NMB) and indocyanine green (EmunDo®)

BackgroundSuperficial mycotic skin infections which are predominantly caused by Trichophyton rubrum, poorly responsd to conventional therapies. A great amount of attention has focused on finding more effective treatments. The current work is aimed to compare the effectiveness of phoinactivation of Trichophyton rubrum by two relatively new photosensitizers: a phenothiazinium dye(New methylene blue) and Indocyanine green (EmunDo^®).Materials and methodsA Final inoculum of T. rubrum which corresponded to 10⁶ colony forming unit per milliliter (CFU ml⁻¹) was prepared. Antimicrobial Photodynamic treatment (aPDT) of T. rubrum was carried out by either EmunDo^® (1 mg/ml, Infra-red laser (IRL, λ = 810 nm, Energy Density 55 J/cm²)) or NMB (10 μM, Red laser (RL), λ = 630 nm, Energy Density of 5 J/cm²). The suspensions thereafter were subcultured on Sabouraud dextrose agar (SDA) and were counted on due time. based on colony-forming unit per milliliter (CFU/ml).ResultsaPDT with either EmunDo^® (E) or NMB (N) considerably diminished the viability of inoculated T. rubrum with respective reduction of 0.64 log and 0.4 log compared to the control group (P < 0.001). No significant difference was found between two laser only groups (P = 0.79) and two aPDT groups (P = 0.73), however significant reduction of T. rubrum in red laser only group (P = 0.04) and EmunDo^® only group (P = 0.04) was found as compared to the control group (P < 0.05).ConclusionThe study provides evidence regarding satisfactory photodynamic inactivation of T. rubrum with EmunDo^® or NMB as photosensitizers. Irradiation by only red laser source was found superior to only infra-red laser source. Dark toxicity of EmunDo^® was more successful than new methylene blue dye.     

Effects of photodynamic therapy with talaporfin sodium on squamous cell carcinoma and sarcoma cells

BackgroundPhotodynamic therapy (PDT) uses a photosensitizer and light to destroy abnormal cells. Talaporfin sodium (NPe6) is a second-generation photosensitizer.MethodsWe evaluated the toxic effects of different combinations of laser and NPe6 doses on squamous cell carcinoma (KLN205) and sarcoma (Meth A) cell lines. The cells were incubated with 0, 5, 10, or 30 μg/mL NPe6 for 24 h. The cells were then irradiated with 0, 5, 15, or 30 mW/cm² of laser power, and 0, 1, 5, 10, or 20 J/cm² of laser energy. Cell viability was evaluated after 24 h. We also evaluated the cytotoxic effects of continuous wave or square-wave modulated laser irradiations (2, 5, or 10 Hz, 50% duty) on Meth A cells.ResultsThe median lethal doses of NPe6 against the KLN205 and Meth A cells after treatment at a fluence rate of 15 mW/cm² and a light dose of 20 J/cm² were 18.6 and 5.0 μg/mL, respectively. Meth A cells were more sensitive to PDT than KLN205 cells. There was no significant difference between the effects of continuous wave and square-wave modulated lasers on Meth A cell viability.ConclusionsNPe6 PDT induced cell death in a dose-dependent manner in KLN205 and Meth A cells. More work is required to evaluate the cytotoxic effects of square-wave modulated laser therapy at low light doses.     

Streptococcus mutans photoinactivation using a combination of a high potency photopolymerizer and rose bengal

BackgroundLong treatment periods to reach a substantial inactivation of microorganisms are one of the critical challenges in the photodynamic therapy field.MethodsPlanktonic suspensions of Streptococcus mutans were treated in different groups: presence of rose bengal (RB at 2 μM) and light exposure by a new high potency photopolymerizer (L at wavelength = 440–480 nm; dosimetry = 96 J/cm² – 40 s of irradiation; potency density = 1200 mW/cm²; dosage = 48 J) – RB+L+ (PDT), just with dye – RB+L−, just with light – RB−L+ and absence of light and dye RB−L− (control group). Aliquots of each group were transferred to Petri dishes to colony counting (CFU/mL) with the data transformed in log₁₀. The results were submitted to ANOVA and Tukey test at 5%.ResultsPDT group presented total eradication of microorganisms showing statistical difference with all the other groups (5.82 log reduction ≅ 99%).ConclusionThe high potency photopolymerizer in pulsed emission at an extra short irradiation and low concentration of rose bengal could be considered as a progressive alternative to the control of S. mutans suspensions.     

Irradiance dependence of the conduction block of an in vitro cardiomyocyte wire

BackgroundTo obtain therapeutic condition precisely by in vitro experiment, we studied the irradiance dependence of the electrical conduction blockage caused by a photodynamic reaction using a high extracellular concentration of talaporfin sodium on a novel in vitro cardiomyocyte electrical conduction wire.MethodsThe cardiomyocyte wires were constructed on patterned cultivation cover glass, which had cultivation areas 60 μm in width, and a maximum length of 10 mm. The talaporfin sodium concentration was set to 20 μg/mL. The photodynamic reaction with a high extracellular photosensitizer concentration was performed with a short time interval (approximately 15 min) between photosensitizer exposure and irradiation. A 663-nm laser was applied to the cardiomyocyte wire, and the irradiance was varied between 3 and 120 mW/cm². The cardiomyocyte electrical conduction was evaluated using the cross-correlation function of intracellular Ca²⁺ probe fluorescence brightness from an upper and lower section outside the laser irradiation area of a wire every 10 s, which lasted up to 600 s.ResultsThe onset of electrical conduction blockage was defined by an 85% decrease in the cross-correlation function, compared with its initial value. The time for the electrical conduction blockage decreased from 600 to 300 s as the irradiance was increased. Also, the probability of electrical conduction blockage was found to increase with increasing irradiance.ConclusionsWe found a strong dependence on the irradiance for the time and probability of electrical conduction blockage.