Search

Antimicrobial Photodynamic Therapy (aPDT) is an emerging strategy against resistant pathogenic bacteria, a serious global health threat. We describe herein the efficient preparation of photosensitized cellulose nanocrystals (CNC) using trialkoxysilane linkers for covalent incorporation of anionic (Rose Bengal: RB) and cationic (Toluidine blue O: TBO) photosensitizers (PSs), along with a N-alkyl-d-gluconamide ligand to specifically target Escherichia coli, as model nanosystems for aPDT. The synthesized nanomaterials exhibited high PS loading, high singlet oxygen quantum yield comparable to the solution, and good stability in aqueous media with minimal PS release under physiological conditions. Experimental viability tests in bacteria demonstrated their capability for aPDT, mitigating the inherent cytotoxicity of both PSs under dark conditions while retaining high phototoxicity against E. coli bacteria. The presence of gluconamide further enhanced photoactivity, highlighting the importance of surface functionalization with a specific bacterial ligand for improved efficacy. The CNC-supported RB system exhibited sufficient fluorescence for tracking via fluorescence microscopy, making it suitable for theranostics, integrating bioimaging and aPDT. Overall, photosensitized CNCs hold great promise as nanocarriers for combating topical infections caused by Gram-negative bacteria, addressing the urgent need for novel therapeutic strategies in infectious disease management while also mitigating antimicrobial resistance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Photodynamic therapy has always been an antibacterial tool for solving multi-drug resistant bacteria problem, but the side effects and the low efficiency due to the high aggregation and low solubility of photosensitizers limit its application. Due to the anti-inflammatory effect of caffeic acid, two novel photosensitizers (CA-1-TBO, CA-TBO) were synthesized by conjugating caffeic acid with toluidine blue O (TBO). The structures have been characterized by 1 HNMR and high-resolution mass spectrometry. The UV-vis absorption, fluorescence spectra and the octanol-water partition coefficient of two photosensitizers were measured to evaluate their photophysical properties and hydrophilic/hydrophobic properties. Compared with parent TBO, the two modified photosensitizers have shown a higher quantum yield and kinetics constants of singlet oxygen, which has been supported by the simulation results of density functional theory. As drug-resistant representatives of gram-positive and gram-negative bacteria, respectively, S. aureus and P. aeruginosa have been used for in vitro antibacterial experiments. The sterilization efficiencies of the two modified photosensitizers far exceed that of parent TBO. The results of the octanol-water partition coefficient and fluorescence quantification showed that modified photosensitizers CA-1-TBO and CA-TBO could be more accumulated than parent TBO. Based on scanning electron microscopy images, protein gel electrophoresis, and the conductivity of the bacterial solution, the possible mechanism of improved antibacterial photodynamic efficiencies could be induced by membrane permeability due to the caffeic acid effect. The findings demonstrate the significant potential of natural phenolic compounds in the development of photosensitizer molecules with characteristics such as more efficient, biocompatible and less side effects., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Photodynamic therapy (PDT) is an antimicrobial approach that can be used as an adjunctive safe technique for root canal disinfection. In this study, we aimed to evaluate the antibacterial efficacy of PDT with indocyanine green and toluidine blue in combination with sodium hypochlorite (NaOCl) in compared with NaOCl alone against Enterococcus faecalis in infected root canals., Methods: Sixty five root canals of sound human premolars were collected. First, the teeth were sterilized, then 60 root canals were inoculated with the E. faecalis, and 5 samples served as negative control remained uncontaminated. The contaminated root canals were randomly assigned into 4 experimental groups (n=15): (1) NaOCl, (2) NaOCl+ PDT-1 (toluidine blue + 630nm LED), (3) NaOCl+ PDT-2 (indocyanine green + 808nm diode laser), and (4): positive control (no treatment). Then, the colony-forming units (CFU/mL) of E. faecalis were calculated in all the groups. Moreover, 5 samples from each group were examined under a scanning electron microscope. The data were analyzed by independent T-test and the Mann-Whitney U test at the significance level of 0.05., Results: The CFU count was significantly lower in the indocyanine green-PDT group in comparison with other groups (p˂0.05). The mean of CFU in the positive control group was significantly higher than the other groups (p˂0.05). No significant difference was detected between NaOCl and toluidine blue -PDT group (p=1.00)., Conclusion: Indocyanine green- PDT improved the antibacterial effects of NaOCl against E. faecalis, while toluidine blue - mediated PDT had no significant additional effect on NaOCl., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Antimicrobial resistance is an ever-growing global concern, making the development of alternative antimicrobial agents and techniques an urgent priority to protect public health. Antimicrobial photodynamic therapy (aPDT) is one such promising alternative, which harnesses the cytotoxic action of reactive oxygen species (ROS) generated upon irradiation of photosensitisers (PSs) with visible light to destroy microorganisms. In this study we report a convenient and facile method to produce highly photoactive antimicrobial microparticles, exhibiting minimal PS leaching, and examine the effect of particle size on antimicrobial activity. A ball milling technique produced a range of sizes of anionic p(HEMA-co-MAA) microparticles, providing large surface areas available for electrostatic attachment of the cationic PS, Toluidine Blue O (TBO). The TBO-incorporated microparticles showed a size-dependent effect on antimicrobial activity, with a decrease in microparticle size resulting in an increase in the bacterial reductions achieved when irradiated with red light. The >6 log 10 Pseudomonas aeruginosa and Staphylococcus aureus reductions (>99.9999%) achieved within 30 and 60 min, respectively, by TBO-incorporated >90 μm microparticles were attributed to the cytotoxic action of the ROS generated by TBO molecules bound to the microparticles, with no PS leaching from these particles detected over this timeframe. TBO-incorporated microparticles capable of significantly reducing the bioburden of solutions with short durations of low intensity red light irradiation and minimal leaching present an attractive platform for various antimicrobial applications., Competing Interests: Declaration of Competing Interest There are no conflicts of interest to declare., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Streptococcus mutans ( S. mutans ) and Candida albicans ( C. albicans ) are prominent microbes associated with rapid and aggressive caries. In the present study, we investigated the antimicrobial efficacy, cytotoxicity, and mechanism of toluidine blue O (TBO)-mediated antimicrobial photodynamic therapy (aPDT) and potassium iodide (KI). The dependence of KI concentration, TBO concentration and light dose on the antimicrobial effect of aPDT plus KI was determined. The cytotoxicity of TBO-mediated aPDT plus KI was analyzed by cell counting kit-8 (CCK-8) assay. A singlet oxygen ( 1 O 2 ) probe test, time-resolved 1 O 2 detection, and a 1 O 2 quencher experiment were performed to evaluate the role of 1 O 2 during aPDT plus KI. The generation of iodine and hydrogen peroxide (H 2 O 2 ) were analyzed by an iodine starch test and Amplex red assay. The anti-biofilm effect of TBO-mediated aPDT plus KI was also evaluated by counting forming unit (CFU) assay. KI could potentiate TBO-mediated aPDT against S. mutans and C. albicans in planktonic and biofilm states, which was safe for human dental pulp cells. 1 O 2 measurement showed that KI could quench 1 O 2 signals, implicating that 1 O 2 may act as a principal mediator to oxidize excess iodide ions to form iodine and H 2 O 2 . KI could highly potentiate TBO-mediated aPDT in eradicating S. mutans and C. albicans due to the synergistic effect of molecular iodine and H 2 O 2 .

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to conventional antimicrobial therapies, allowing for high morbidity and mortality. Photodynamic antimicrobial chemotherapy (PACT) is one method that combines visible harmless light with the optimum wavelength with photosensitizers or dyes, producing singlet oxygen ( 1 O 2 ) and reactive oxygen strains (ROS), making permanent damages to the target cells. The purpose of this research is to evaluate the suppression efficacy of toluidine blue O (TBO)-mediated PACT on mature MRSA biofilm in vitro., Methods: In this study, the 48 h mature biofilm of the multidrug-resistant Staphylococcus aureus strain MRSA252 was used. The photodynamic therapy (PDT) group was treated with different concentrations of TBO (0.5, 0.75, 1.0 or 1.25 µM) and different doses of red light (635 ± 5 nm wavelength; 30 or 50 J/cm 2 ). The biofilms viability after PDT were evaluated by crystal violet (CV) staining assay and {2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetra-zolium hydroxide} (XTT) assay; meanwhile, the morphological changes were detected by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), separately. Moreover, the biofilms virulence was evaluated by red blood cell (RBC) hemolysis assay and staphylococcal virulence factor enterotoxins A (SEA) detected by enzyme linked immunosorbent assay (ELISA). After PDT, the biofilm was re-cultured for extra 48 h. Its formation viability and virulence were detected again. All data were analyzed by ANOVAs followed by the Games Howell post hoc test (α = 0.05)., Results: The biofilm was inactivated about 2.3 log10 at 1.25 µM with 30 J/cm 2 illumination, and 3.5 log10 with 50 J/cm 2 after PDT (P<0.05). XTT assays demonstrated the viability of mature MRSA biofilms was reduced after PACT. PDT group shows a distinct reduction in RBC hemolysis rate and the concentration of SEA compared to the control groups. The morphological features of the biofilms showed great changes, such as shrinkage, fissure, fragmentation, and rarefaction after being treated by TBO-PDT and observed by SEM. The recovery of the structure and virulence of biofilm were suppressed after PDT., Conclusion: TBO-mediated PDT could destroy the biofilm structure, reduce its virulence and depress its self-recovery., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: The appearance of bacterial resistance to antibiotics a are major health problems. Antimicrobial photodynamic therapy (aPDT) has emerged as an adjunctive treatment for drug-resistant infections. Titanium dioxide (TiO 2 ) and zinc oxide (ZnO) nanoparticles have recently been proposed as effective carriers for photosensitizers. The present study was designed to evaluate the antibacterial activity of ZnO and TiO 2 -nanoparticles accompanying aPDT and antimicrobial photocatalysis approaches against extensively drug-resistant colistin resistant Acinetobacter baumannii (XDR-CO-Ab)., Materials and Methods: A XDR-CO-Ab isolated from burn-wound infection was used in this study. The bacterial suspension was exposed to TBO (Toluidine blue O), TiO 2 , and ZnO-nanoparticles in different groups. Ultraviolet A (UVA) (320 nm) and blue laser light (405 nm) were used to activate the photosensitizer and nanoparticles. The bacterial viability was then determined by counting the number of colonies forming units (CFUs)/mL., Results: Using both aPDT and antimicrobial photocatalysis methods simultaneously, it had an amazing result; so that 99.32 % of XDR-CO-Ab was killed. According to the results, the bactericidal effects of activated TBO using UVA and laser lights was 5.1 % more than TBO was excited with laser light alone., Conclusions: It was found that the combination of photocatalyst (TiO 2 +ZnO-nanoparticles) and TBO as photosensitizer at two-wavelength irradiation with blue laser and UVA lights have been shown to have significant antimicrobial effects., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Antibacterial photodynamic therapy (A-PDT) has been shown to kill oral bacteria in the planktonic culture, dental plaque, and biofilm. This study sought to assess the antimicrobial effect of A-PDT with toluidine blue O (TBO) and diode laser on salivary mutans streptococci in 5-6-year-olds with severe early childhood caries (SECC). This case-control study was conducted on 56 children with SECC divided into four groups, namely 0.1 mg/mL TBO, diode laser (633 nm, 20 mW, 6 J/cm 2 ), combination of the two, and no intervention control group. A-PDT was performed on days 1 and 3. Salivary samples were collected before and after A-PDT on days 1 and 3, and 1 and 2 weeks after the second intervention (day 3). Samples were cultured on mitis salivarius agar, and after incubation, the colonies were counted. Data were subjected to repeated measures ANOVA, ANCOVA, and paired comparisons with least square difference and Tukey's test. Bacterial count significantly decreased on days 1 and 3, and 1 and 2 weeks after the second intervention. Bacterial count also decreased following the use of TBO and laser separately, but these reductions were not significant (P > 0.05). Within the limitations of this study, antimicrobial efficacy of TBO + laser was higher than that of diode laser or TBO alone. Durability of treatment increased with double-dose therapy. This modality may be used to decrease the colony count of salivary mutans streptococci in children with SECC.

Background: During antimicrobial photodynamic therapy (aPDT) in the treatment of endodontic intracanal infection, it is extremely likely that microorganisms would be exposed to sub-lethal doses of PDT (sPDT). Although sPDT would not result in microorganism death, it can considerably influence microbial virulence. This study evaluated the effect of sPDT on gene expression of Enterococci surface protein (esp) as a major virulence factor for biofilm formation in Enterococcus faecalis., Materials and Methods: The lethal and sub-lethal potential of aPDT using indocyanine green (ICG), toluidine blue O (TBO), and methylene blue (MB), as the photosensitizers (PSs) and 660, 635, and 810 nm diode laser against E. faecalis was analyzed using colony-forming unit assays. Considering sub-lethal doses of PSs and photo-irradiation time of the diode laser, the expression of esp was evaluated through quantitative Real-time PCR (qRT-PCR)., Results: ICG at a concentration of 31.2-1000 μg/mL and both TBO and MB at 6.2-100 μg/mL significantly reducedE. faecalis growth. 660, 635, and 810 nm diode laser with energy density of 93.75-140.62, 137.5-206.25, and 31.25-62.5 J/cm 2 , reduced the bacterial count. ICG with a concentration of 15.6 μg/mL and irradiation time of 0.5 min, TBO with a concentration of 3.1 μg/mL and irradiation time of 3 min, and MB with a concentration of 3.1 μg/mL and irradiation time of 3 min were found as a sPDT dose against E. faecalis. The esp expression was significantly reduced at sub-lethal doses of ICG compared to TBO (2 fold) and MB (2.4 fold)., Conclusion: Although all tested PSs showed bacterial reduction, ICG may be considered as the best PS in treating endodontic infection due to its higher efficacy in reduction of esp expression which has a main role in biofilm formation of E. faecalis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Despite the high success rate of endodontic treatment, failure may occur in some cases. In this case, Enterococcus faecalis is the most common species in endodontic treatment failure and post-treatment apical periodontitis. Therefore, a new adjunctive strategy is needed for the prevention of endodontic infections due to E. faecalis. The aim of the present study was to compare the antimicrobial and anti-biofilm activities of different common photosensitizers (PSs) for use in antimicrobial photodynamic therapy (aPDT) against E. faecalis., Materials and Methods: E. faecalis strain ATCC 29212 was used as the tested strain and methylene blue (MB), toluidine blue O (TBO), indocyanine green (ICG), and curcumin (CUR) were used as PSs. Irradiation was carried out using diode laser and light emitting diode (LED) at wavelengths related to the above PSs. Then, antimicrobial and anti-biofilm activities were measured using the microbial viability assay and crystal violet test, respectively., Results: aPDT with using the above PSs significantly decreased the CFU/mL count of E. faecalis compared to the control group (P < 0.05). The killing percentage of E. faecalis via PS mediated aPDT was 99.6%, 98.2%, 85.1%, and 65.0% for CUR, ICG, TBO, and MB, respectively. aPDT using the above PSs significantly decreased the biofilm formation ability of E. faecalis compared to the control group (P < 0.05). The biofilm reduction percentage of the PSs was 68.4%, 62.9%, 59.0%, and 47.6% for CUR, ICG, TBO, and MB, respectively., Conclusion: CUR and ICG mediated aPDT exhibited considerably more antimicrobial activity than other PSs, while TBO and MB demonstrated weaker anti-biofilm effects against E. faecalis compared to other PSs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Several microbial species have been implicated in the pathogenesis of endodontic diseases that colonize the infected root canal system. Since the complete removal of endopathogenic agents is essential in endodontic infection therapy, photo-activated disinfection (PAD) is suggested as an alternative method to traditional antimicrobial therapy. Recent studies reported that the molecular methods with low sensitivity and high efficiency to identify fastidious anaerobic endopathogenic microbiota can be replaced by the cultivation-based approaches. This study aimed to validate the multiplex real-time PCR in order to identify six common microorganisms associated with the endodontic infections before and after the PAD., Materials and Methods: Microbial specimens from the root canals of 50 patients with primary and secondary endodontic infections were collected before PAD treatment using sterile paper points. Toluidine blue O (TBO)-mediated PAD was performed on the root canals, followed by resampling. The prePAD- and postPAD-treatment endodontic samples were transferred to a transport medium and six target microorganisms were then identified from the samples using the microbiological culture techniques and multiplex real-time PCR approach., Results: Multiplex real-time PCR could represent the presence of all target microorganisms in 100% cases before and after the PAD. Before PAD, using the culture method, Enterococcus faecalis (100%) was found to be the most frequent, followed by Veillonella parvula (97.5%), Aggregatibacter actinomycetemcomitans (94.7%), Porphyromonas gingivalis (84.3%), Lactobacillus rhamnosus (84.3%), and Actinomyces naeslundii (66.6%); whereas, after PAD these microbial frequencies changed to 80%, 83.3%, 66.6%, 80%, 66.6%, and 33.3%, respectively. The sensitivity and negative predictive value of the multiplex real-time PCR were 100% before and after the PAD, whereas the highest and the lowest specificities were 100% and 82% before PAD, and 97% and 89% after PAD for E. faecalis and P. gingivalis, respectively. The highest (100%) and the lowest (66%) positive predictive values were for V. parvula and A. naeslundii before and after the PAD, respectively., Conclusion: As observed from the results, multiplex real-time PCR demonstrated high sensitivity and specificity when compared to the culture technique. Therefore, it can prove to be a highly sensitive technique to detect the endodontic infections microflora., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: It is essential to identify the root canal microbial diversity and count. This is due to the polymicrobial nature of the primary endodontic infection that is associated with the microbial diversity and increased resistance to the antimicrobial agents. Photo-activated disinfection (PAD), also known as antimicrobial photodynamic therapy, is a new promising non-antibiotic approach, studied to prevent microbial resistance and treatment failure., Materials and Methods: In this study, we investigated the effect of PAD on reduction of microbial diversity and count, related with primary endodontic infections. Microbial specimens were collected before PAD from patients infected with the primary endodontic infection. PAD with toluidine blue O (TBO), in combination with diode laser, was performed on infected root canals. Resampling was carried out on the root canal after PAD, and microorganisms were identified by classical microbiological tests using biochemical and analytical profile index (API ® 20A) assays and nucleic acid approaches., Results: From the 36 subjects studied before TBO-PAD, 187 cultivable isolates from 14 different genera and 19 various microbial species were retrieved. Of the bacterial isolates, 45.4% were strict anaerobes including Veillonella parvula, Porphyromonas gingivalis, Propionibacterium acnes, Lactobacillus acidophilus, Campylobacter rectus, and Slackia exigua, in order of their frequency; 45.4% were facultative anaerobes; and 9.2% were microaerophilic bacteria (Aggregatibacter actinomycetemcomitans). This in vivo study revealed significant decrease in the microbial diversity and count of the infected root canal after TBO-PAD (P < 0.05); whereas P. gingivalis strains, the most resistance microorganisms, were recovered in 34% of the samples after TBO-mediated PAD (P > 0.05)., Conclusions: TBO-mediated PAD is an effective in exhibiting efficient antimicrobial activity due to the substantial reduction of the microbial diversity and count in the primary endodontic infections., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: The infected root canal system harbors one of the highest accumulations of polymicrobial infections. Since the eradication of endopathogenic microbiota is a major goal in endodontic infection therapy, photo-activated disinfection (PAD) can be used as an alternative therapeutic method in endodontic treatment. Compared to cultivation-based approaches, molecular techniques are more reliable for identifying microbial agents associated with endodontic infections. The purpose of this study was to evaluate the ability of designed multiplex real-time PCR protocol for the rapid detection and quantification of six common microorganisms involved in endodontic infection before and after the PAD., Materials and Methods: Samples were taken from the root canals of 50 patients with primary and secondary/persistent endodontic infections using sterile paper points. PAD with toluidine blue O (TBO) plus diode laser was performed on root canals. Resampling was then performed, and the samples were transferred to transport medium. Then, six target microorganisms were detected using multiplex real-time PCR before and after the PAD., Results: Veillonella parvula was found using multiplex real-time PCR to have the highest frequency among samples collected before the PAD (29.4%), followed by Porphyromonas gingivalis (23.1%), Aggregatibacter actinomycetemcomitans (13.6%), Actinomyces naeslundii (13.0%), Enterococcus faecalis (11.5%), and Lactobacillus rhamnosus (9.4%). After TBO-mediated PAD, P. gingivalis strains, the most resistance microorganisms, were recovered in 41.7% of the samples using molecular approach (P > 0.05)., Conclusion: As the results shown, multiplex real-time PCR as an accurate detection approach with high-throughput and TBO-mediated PAD as an efficient antimicrobial strategy due to the significant reduction of the endopathogenic count can be used for detection and treatment of microbiota involved in infected root canals, respectively., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Photodynamic therapy has been studied as a method for inactivating bacterial growth. Workers have used planktonic bacterial as well as biofilm bacterial cultures to evaluate the potential of photodynamic therapy in inactivating bacteria. However, almost all the studies use a photosensitiser in aqueous solution, which could be detrimental to the efficiency of photodynamic therapy., Methods: In this study, the photodynamic killing effect of toluidine blue O (TBO) has been investigated on Staphylococcal biofilms in-vitro. The sensitivity of the in-vitro biofilms to photodynamic killing action was compared using different formulations of TBO, different dosages of photosensitiser and different light irradiation strengths. Effect of TBO formulations on bacterial quorum sensing system was evaluated using a colorimetric assay. Finally, dual staining using hoechst and propidium iodide stains was carried out on the photodynamically treated biofilms to visualise and compare the effects of photodynamic therapy. Scanning electron microscope imagery was also carried out to evaluate the photodynamic killing effect on the in-vitro biofilms., Results: The sensitivity of biofilms to the photodynamic killing effect increased proportionally with the photosensitiser dosage and the light irradiation duration. TBO encapsulated in microemulsion was more effective in killing the biofilm bacteria than only TBO in water. The combination of TBO in microemulsion with EDTA was another effective way of increasing the photodynamic killing effect on the bacterial biofilms. Effect of encapsulated TBO on the quorum sensing system of bacteria was greater than the effect of aqueous solution of TBO. The in-vitro Staphylococcal biofilms could thus be inhibited by the photodynamic effect, and TBO encapsulated in microemulsion was much more effective than only TBO in water., Conclusions: The encapsulation of a photosensitiser is an effective way of increasing the likelihood of the complete and successful inactivation of the biofilm growth. The encapsulated photosensitiser achieves higher inactivation of the bacterial biofilm than that of the aqueous solution of a photosensitiser., (Copyright © 2017. Published by Elsevier B.V.)

Background: The endodontic disinfection therapy majorly aims to eradicate microbial pathogens. Photo-activated disinfection (PAD), also known as antimicrobial photodynamic therapy, is an alternative antimicrobial modality used to control the microorganisms causing endodontic infections. Notably, microorganisms may be exposed to sub-lethal doses of PAD (sPAD), influencing microbial virulence. The present study assessed the effects of sPAD on expression profiling of the gene associated with the biofilm formation being the most essential virulence factor in Porphyromonas gingivalis strain cells that survive the photodynamic reatment in vivo., Materialas and Methods: Sixteen clinical strains of PAD resistant P. gingivalis that were isolated in vivo, were further photosensitized in vitro with toluidine blue O (TBO), methylene blue (MB), and indocyanine green (ICG) as the photosensitizing agents, which were excited with specific wavelength of light based on the photosensitizer. After evaluating sPAD, its effects on the fimA gene expression were assessed using real-time quantitative reverse transcription PCR (qRT-PCR)., Results: In this study, maximum values of sPAD against P. gingivalis were 6.25μg/mL TBO at a fluency of 171.87J/cm 2 , 15.6μg/mL ICG at fluency of 15.6J/cm 2 , and 25μg/mL MB at fluency of 93.75J/cm 2 . MB-, TBO-, and ICG-sPAD could cause about 4.6-, 14.4-, and 17.3-fold suppression of fimA expression, respectively. P. gingivalis strains expressed less virulence in cells surviving PAD., Conclusions: In conclusion, the gene expression profiling was reduced in the bacterial cells, wherein greater reduction was observed for ICG-sPAD than TBO- and MB-sPAD., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Photodynamic therapy (PDT) has been successfully employed in the treatment of oral cancer. Toluidine blue O (TBO) is a photosensitizer (PS) that has exhibited remarkable photocytotoxicity in a variety of tumour cells; however, its physicochemical properties, as well as the physicochemical properties of oral mucosa, prevent the drug from reaching the target site at a therapeutic concentration. The aim of this study was to evaluate the influence of Tween 80 ® (TW), which has shown potential as a penetration enhancer, on the mucosal retention of TBO for the PDT of oral cancer. 4% Chitosan-based mucoadhesive gels (CH gels) containing or not 5%TW were prepared (both containing 1%TBO), and their physicochemical properties (pH, rheology and mucoadhesion), TBO in vitro release profiles and TBO in vitro mucosal retention were evaluated. In vivo mucosal penetration studies of TBO followed by laser exposition were also carried out. The results showed that 4%CH gels containing 5%TW and 1%TBO have adequate mucoadhesive and rheological properties for oral mucosa use, although they present a slightly acid pH. TBO release studies showed that TW reduces TBO release, but it prolongs TBO release and increases TBO retention in the mucosa. In vivo studies showed that 4%CH gels containing 5%TW and 1%TBO cause an increase in the number of apoptotic cell, after laser exposition. In summary, 4%CH gels containing 5%TW may be a promising vehicle to optimize the penetration of TBO in oral mucosa and to improve the PDT response for the treatment of oral cancer., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Widespread methicillin resistant Staphylococcus aureus (MRSA) and absence of effective antimicrobial agents has led to limited therapeutic options for treating MRSA infection. We aimed to evaluate the effect of antimicrobial photodynamic therapy (aPDT) on the expression of novel identified methicillin resistance markers (NIMRMs) in S. aureus using complementary DNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) approaches to address the therapeutic alternatives for MRSA infections., Materials and Methods: We used cDNA-AFLP to compare MRSA and methicillin susceptible S. aureus (MSSA) for identification of target genes implicated in methicillin resistance. To determine the sub-lethal aPDT (sPDT), MRSA and MSSA clinical isolates photosensitized with toluidine blue O (TBO), and then were irradiated with diode laser. After sPDT, the colony forming units/mL was quantified. Antimicrobial susceptibility against methicillin was assessed for cell-surviving aPDT. Effects of sPDT on the expression of NIMRMs were evaluated by real-time quantitative reverse transcription PCR., Results: According to our results, serine hydrolase family protein (Shfp) encoding gene and a gene encoding a conserved hypothetical protein (Chp) were implicated in methicillin resistance in MRSA. sPDT reduced the minimum inhibitory concentrations of methicillin by 3-fold in MRSA. sPDT could lead to about 10- and 6.2- fold suppression of expression of the Chp and Shfp encoding genes, respectively., Conclusion: sPDT would lead to reduction in resistance to methicillin of MRSA in surviving cells by suppressing the expression of the Shfp and Chp encoding genes associated with methicillin resistance. This may have potential implications of aPDT for the treatment of MRSA infections., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Secondary/persistent endodontic infection can be the outcome of failure of endodontic treatment. Photo-activated disinfection (PAD) can be a useful adjunct to mechanical and antimicrobial agents in eliminating endopathogenic microorganisms. In this study, we evaluated the effect of PAD on diversity and count of microbiota related to secondary/persistent endodontic infections., Materials and Methods: Root canal samples were taken using sterile paper points from the root canals of 14 patients with secondary/persistent endodontic infections after removing the root-canal filling materials. PAD was performed on teeth with toluidine blue O (TBO) in combination with diode laser. Then re-sampling was conducted from the canal root using sterile paper points and transferred to transport medium. The samples were plated and pure cultures of the target microorganisms were then isolated and identified by analytical profile index (API ® 20A) assays and 16S ribosomal RNA gene sequencing., Results: Before TBO-PAD, a total of 31 cultivable isolates could be retrieved; 25.8% of the isolated species were obligate anaerobic or microphilics including Veillonella parvula, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Propionibacterium acnes, and Lactobacillus acidophilus, and 74.2% of the isolated species were facultative anaerobic such as Enterococcus faecalis, Actinomyces naeslundii, L. rhamnosus, L. casei, Streptococcus sanguinis, S. mitis, and Candida albicans. According to this in vivo study, the diversity and count of microbiota in root canal-treated teeth were decreased after TBO-PAD, so that E. faecalis, V. parvula, and C. albicans were the microorganisms that recovered after PAD., Conclusion: TBO-PAD is an effective approach that exhibited anti-microbial potential activity against microbiota involved in secondary/persistent endodontic infection., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Antimicrobial photodynamic therapy (aPDT) is a novel technique for reduction of pathogenic microorganisms in dentistry. The aim of this study was to evaluate the effects of aPDT on Streptococcus mutans reduction in children with severe early childhood caries., Methods: Twenty-two children with severe early childhood caries aged 3-6 years were treated with toluoidine blue O (TBO) for 1min and irradiated by a Light Emitting Diode (LED; FotoSan, CMS Dental, Denmark) with the exposure time of 150s. Saliva samples were collected at baseline, 1h and 7 days after treatment. S. mutans counts were determined using the Dentocult SM Strip mutans., Results: The counts of S. mutans in saliva decreased significantly after 1h (P<0.001). However, the difference in reduction of S. mutans counts in saliva was not significant between the baseline and 7 days after treatment (P>0.05)., Conclusion: aPDT seems to be efficient to reduce salivary S. mutans immediately after treatment in children with severe early childhood caries. However, further research is needed to evaluate different doses and frequency of irradiation in combination with restoring carious teeth to find more durable results., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Porphyromonas gingivalis is one of the etiological agents in the initiation of combined periodontal-endodontic (perio-endo) lesions. Successful treatment of perio-endo lesions with photo-activated disinfection (PAD) as a novel therapeutic approach depends on the selection of an appropriate target site, specific photosensitizer (PS), and wavelength of light. Since the fimbrillin (FimA) is the most important pathogenic agent in P. gingivalis it was evaluated as a target site against PAD with toluidine blue O (TBO) as a PS., Materials and Methods: We evaluated the potential of FimA using a number of bioinformatic tools and computer simulation molecular modeling. In silico analysis was done to predict the hierarchical nature of structure, topology, backbone, physicochemical properties, and functional characterization of FimA., Results: The predicted structure of FimA exhibited that it is an anionic protein with negative charge (-7.00) in pH7 and random coil which dominates other secondary structures located outside the cell. It has three motifs with seven ligand binding site residues and one active site residue which can be a site of interaction for cationic TBO., Conclusion: Based on the results of this study cationic TBO tends to interact with FimA during PAD as a major target to increase the efficiency of treatment of perio-endo lesions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Microorganisms treated by photo-activated disinfection (PAD) in combined periodontal-endodontic (perio-endo) lesions would be exposed to sub-lethal doses of PAD (sPAD). This study evaluated the effect of sPAD using toluidine blue O (TBO) in combination with diode laser irradiation on the growth and biofilm-formation ability of Porphyromonas gingivalis as an endo-periodontal pathogen., Methods: The antibacterial and antibiofilm potential of sPAD against P. gingivalis was analyzed at sub-lethal doses of TBO and irradiation time of diode laser on a colony-forming unit and crystal violet assays, respectively., Results: TBO-mediated PAD, using 6.25-100μg/mL at a fluency of 171.87J/cm 2 and 12.5-100μg/mL at a fluency of 137.5J/cm 2 , showed a significant dose-dependent reduction in P. gingivalis growth when compared to the control. TBO-mediated PAD showed a significantly inhibitory effect on biofilm formation in P. gingivalis than TBO-PAD at sub-lethal levels., Conclusion: High doses of sPAD revealed antibacterial and antibiofilm potential activity, whereas lower doses of sPAD had conflicting results. Therefore, when PAD is prescribed in combined perio-endo lesions treatment, the dose of PAD used in vivo should be taken into account., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: During photodynamic therapy (PDT) in the treatment of a primary endodontic infection, it is extremely likely that microorganisms would be exposed to sub-lethal doses of PDT (sPDT). Although sPDT cannot kill microorganisms, it can considerably influence microbial virulence. This study was conducted to characterize the effect of sPDT using toluidine blue O (TBO), methylene blue (MB), and indocyanine green (ICG) on biofilm formation ability and metabolic activity of Enterococcus faecalis., Methods: The antimetabolic and antibiofilm potential of ICG-, TBO-, and MB-sPDT against E. faecalis was analyzed at sub-lethal doses (1/2-1/64 minimum inhibitory concentration) using the XTT reduction assay, crystal violet assay, and scanning electron microscopy., Results: Higher doses of sPDT adversely affected biofilm formation ability and metabolic activity. ICG-, TBO-, and MB-PDT at a maximum sub-lethal dose markedly reduced the formation of biofilm up to 42.8%, 22.6%, and 19.5%, respectively. ICG-, TBO-, and MB-sPDT showed a marked reduction in bacterial metabolic activity by 98%, 94%, and 82%, respectively. ICG-PDT showed a stronger inhibitory effect on biofilm formation in E. faecalis than MB- and TBO-PDT at sub-lethal levels. Interestingly, a gradual increase in metabolic activity and biofilm formation upon exposure to a lower dose of test sPDT were observed., Conclusion: sPDT showed dual effect on biofilm formation ability and metabolic activity of E. faecalis. High doses revealed antimetabolic and antibiofilm potential activity, whereas lower doses had conflicting results. Hence, when PDT is prescribed in clinical settings, the dose of PDT used in vivo should be taken into consideration., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Photodynamic therapy (PDT) is an emerging treatment that has demonstrated potential for the clinical treatment of buccal cancer. It is based on the photoactivation of a photosensitizer (PS) when irradiated by light at a specific wavelength. The light-excited PS generates reactive oxygen species that cause the destruction of tumor cells by apoptosis or necrosis. Toluidine Blue O (TBO) is a PS that has shown potential for PDT in cancer treatment. However, saliva and mechanical activities quickly remove the PS from the surface of the buccal mucosa. Therefore, the bioavailability of PS at the surface of target tissues is reduced. The aim of this study was to evaluate the potential of chitosan (CH) gels in TBO delivery to buccal tissue., Methods: CH gels were obtained at different concentrations and their physico-chemical properties (pH and rheology), mucoadhesion, in vitro release profile, in vivo retention and in vivo efficacy by the ability to induce cell apoptosis were evaluated., Results: CH-based mucoadhesive gels optimized the release and adherence of preparations at the target site. Specifically, 4% (w/w) CH gel showed adequate properties for buccal use, such as pH value, mucoadhesion, pseudoplastic behavior, extended release, minimal permeation and higher TBO retention by the mucosa. In vivo studies showed the potential of the gel to enhance TBO retention and induce cell apoptosis after laser irradiation., Conclusion: 4% (w/w) CH based mucoadhesive gel can be explored as a TBO delivery system in the PDT of oral cancer., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Aim:   Light-emitting diodes have been investigated as new light activators for photodynamic therapy. We investigated the bactericidal effects of high-power, red light-emitting diodes on two periodontopathic bacteria in vitro., Methods:   A light-emitting diode (intensity: 1100 mW/cm(2) , peak wavelength: 650 nm) was used to irradiate a bacterial solution for either 10 or 20 s. Bacterial solutions (Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans) at a concentration of 2.5 × 10(6) c.f.u./mL were mixed with an equal volume of either methylene blue or toluidine blue O (0-20 μg/mL) and added to titer plate wells. The plate wells were irradiated with red light-emitting diode light from a distance of 22 or 40 mm. The contents were diluted, and 50 μL was smeared onto blood agar plates. After 1 week of culturing, bacterial c.f.u. were counted., Results:   The light-emitting diode energy density was estimated to be approximately 4 and 8 J/cm(2) after 10 and 20 s of irradiation, respectively. Red light-emitting diode irradiation for 10 s from a distance of 22 mm, combined with methylene blue at concentrations >10 μg/mL, completely killed Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans., Conclusion:   High-power, red light-emitting diode irradiation with a low concentration of dye showed effective bactericidal effects against two periodontopathic bacteria., (© 2011 Blackwell Publishing Asia Pty Ltd.)

Background Oral care is important for oral and systemic health, especially for elderly institutionalized individuals and compromised patients. However, conventional mechanical plaque control is often difficult for these patients because of the pain or the risk of aspiration. Despite that antimicrobial photodynamic therapy (aPDT), which is considered an alternative or adjunct to mechanical means, has potential application as a less stressful method of daily plaque control, no clinical application has been reported. Methods We chose the combination of toluidine blue O (TBO), and a red light-emitting diode (LED), and investigated the associated inhibitory effect on dental plaque formation in healthy volunteers. For determining the concentration of TBO, preliminary experiments were performed in vitro to evaluate the bactericidal effect of aPDT on Streptococcus oralis, and to clarify the safety in fibroblast cells. In order to survey the mechanism of TBO-mediated aPDT, the quality and quantity of reactive oxygen species (ROS) generated during aPDT were also examined using electron spin resonance (ESR) spectroscopy. Following the in vitro experiments, the inhibitory effect of the aPDT on dental plaque formation was investigated in eleven subjects as a clinical pilot study. The right or left side mandible premolars were randomly assigned to the treatment (with aPDT) or control (without aPDT) groups. In total, aPDT was applied six times (twice per day) to the test group teeth over a period of four days. On the fourth day, the study concluded and the analyses were performed. Results The results indicated that the combination of 500 or 1000 µg/ml TBO and LED irradiation for 20 s significantly decreased the number of colony forming unit of Streptococcus oralis. The cytotoxicity of aPDT was comparable to that of standard antiseptics used in oral cavity. Hydroxyl radicals were detected by ESR analysis, but not singlet oxygen. The randomized controlled trial demonstrated that aPDT with 1000 µg/ml TBO and red LED irradiation significantly suppressed dental plaque formation without harmful events to teeth or surrounding tissues. Conclusions aPDT has the potential to be a promising novel technical modality for dental plaque control. [ABSTRACT FROM AUTHOR]

Abstract: The synthesis of chromophoric di- and trimethyl derivatives of azure A has been carried out along with the physicochemical profiling of the resulting derivatives. Structure–function relationships of the derivatives are proposed with a view to the development of novel photosensitisers with antimicrobial applications. All derivatives exhibited similar magnitudes of photosensitising efficacy in vitro to the standard dyes, azure A and toluidine blue O, but were generally more lipophilic in nature. Chromophoric methylation furnished more active photoantibacterials compared to the lead compounds, both against Gram-positive and Gram-negative organisms. [Copyright &y& Elsevier]

Abstract: Mucocutaneous oropharyngeal candidiasis is predominately caused by Candida albicans. The overall incidence of oral candidiasis in young adults has increased dramatically with the spread of HIV/AIDS. Conventional treatments have been shown to have a fungistatic rather than a fungicidal effect, resulting in an inadequate treatment outcome for patients. In addition, increasing resistance of C. albicans to antifungal agents has made effective treatment more difficult. Accordingly, interest has arisen in development of new prophylaxis/treatment regimens. One such alternative treatment is photodynamic antimicrobial chemotherapy (PACT), in which a combination of a photosensitising drug and visible light cause selective destruction of microbial cells. Due to the highly coloured nature of photosensitisers and the potential for staining of teeth, lips and buccal mucosa, administration of photosensitisers to humans as a liquid mouthwash is undesirable. Targeted delivery of the photosensitiser directly to the site of infection should be the aim. The current study, therefore, reports on a mucoadhesive patch containing toluidine blue O (TBO), as a potential delivery system for use in PACT of oropharyngeal candidiasis. Patches prepared from aqueous blends of poly(methyl vinyl ether/maleic anhydride) and tripropyleneglycol methyl ether possessed suitable properties for use as mucoadhesive drug delivery systems and were capable of resisting dissolution when immersed in artificial saliva. When releasing directly into an aqueous sink, patches containing 50 and 100mg TBO cm−2 both generated receiver compartment concentrations exceeding the concentration (2.0–5.0mgml−1) required to produce high levels of kill (>90%) of both planktonic and biofilm-grown C. albicans upon illumination. However, the concentrations of TBO in the receiver compartments separated from patches by membranes intended to mimic biofilm structures were an order of magnitude below those inducing high levels of kill, even after 6h release. Therefore, short application times of TBO-containing mucoadhesive patches should allow treatment of recently-acquired oropharyngeal candidiasis, caused solely by planktonic cells. Longer patch application times may be required for persistent disease where biofilms are implicated. [Copyright &y& Elsevier]

Abstract: It is known that Staphylococcus aureus is susceptible to photodynamic inactivation in general, but the significant variation among particular strains in the response to the treatment exists. However, factors that determine the observed phenomenon remain unclear. This study was aimed to explore the PDI effect of two sensitizers (protoporphyrin diarginate and toluidine blue O) against clinical as well as reference strains of S. aureus. Obtained results indicate that the same isolate could be characterized as highly resistant or highly sensitive to PDI according to a sensitizer used. Moreover, the same sensitizing agent could be successfully used for total eradication of some isolates and could be non-effective in the case of other strains. Additionally, changing the photosensitizer, we are able to reverse the PDI “resistant” phenotype into “sensitive” one. Thus, one could conclude that photoinactivation involving several sensitizing agents and several isolates of the same bacterial species should be undertaken to make antimicrobial photodynamic inactivation reliable. [Copyright &y& Elsevier]