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Background: Sonodynamic antimicrobial chemotherapy (SACT) is an effective antimicrobial treatment that can avoid the production of drug-resistant bacteria. Design and development of new high-efficiency sonosensitizers play a key role in the practical application of SACT., Methods: The bacteriostatic effects of two phenothiazine compounds, toluidine blue (TB) and azure A (AA) combined with ultrasonic (US) on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were studied, and the sonodynamic antibacterial activities of TB and AA were compared. The reactive oxygen species (ROS) and the types of ROS produced in the sonodynamic system were detected and the sonodynamic mechanisms of TB and AA were proposed., Results: The sonodynamic bacteriostasis mediated by TB and AA increased with the increasing concentration of sonosensitizer, the extension of sonication time and the increase of reaction temperature. The production of ROS was the main reason that TB and AA had excellent sonodynamic antibacterial performance. Singlet oxygen ( 1 O 2 ) and hydroxyl radical (•OH) were the main ROS types in the sonodynamic antibacterial system. The ROS produced by the combined action of AA and US was higher than that of TB., Conclusion: Both TB and AA displayed excellent sonodynamic antibacterial activities. Moreover, AA had a higher sonodynamic activity than TB. The electron donation effect and steric hindrance effect of the methyl group of phenothiazine parent nucleus of TB might be the cause of the decrease of its sonodynamic activity. These results would provide a valuable reference for the further study of phenothiazines sonosensitizers and their clinical application in SACT., 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 Elsevier B.V. All rights reserved.)

Background: Streptococcus mutans is a major contributor to dental caries due to its ability to produce acid and survive in biofilms. Microbial resistance towards common antimicrobial agents like chlorhexidine and triclosan has shifted the research towards antimicrobial Photodynamic therapy (PDT). In this context, Toluidine Blue O (TBO) is being explored for its photosensitizing properties against Streptococcus mutans. There is a huge variation in the effective concentration of TBO among the current studies owing to the differences in source of and delivery system TBO as well as the time, power and energy densities of light., Objective: The primary objectives of this study are to encapsulate improved Toluidine Blue O (ITBO) in nanoliposomes (NLITBO), characterize it, and evaluate its antibacterial photosensitizing potential against Streptococcus mutans suspensions in vitro., Method: ITBO was synthesised as per Indian patent (number -543908). NLITBO was prepared using the thin-film hydration method. Dynamic light scattering experiment determined the vesicle size, polydispersity index (PDI), and zeta potential. Surface features were characterized by Scanning and Transmission Electron microscopy. ITBO release from NLITBO was assessed using the extrapolation method. The antibacterial activity of the NLITBO was determined by evaluating the zone of inhibition (ZOI) in the Streptococcus mutans culture and comparing with 2% chlorhexidine gluconate. The minimum inhibitory concentration (MIC) of NLITBO as a photosensitizer with red light (wavelength 650nm, power density 0.1 W/cm2, energy density 9-9.1 J/ cm2, 90seconds time) was evaluated against Streptococcus mutans cells by colorimetric method in 96 well plate., Results: Percentage drug loading, loading efficiency, yield percentage, vesicle size, PDI, Zeta potential of NLTBO was reported as 9.3±0.4%, 84.4±7.6%, 73.5%, 123.52 nm, 0.57, -39.54mV respectively. Clusters of uni-lamellar nanovesicles with smooth non-perforated surfaces were observed in SEM and TEM. The size of the vesicle was within 100 nm. At 24 hours, a cumulative 79.81% of ITBO was released from NLITBO. Mean ZOI and MIC of NLITBO (1 μg /ml) were found to be 0.7±0.2 mm, 0.6μg/ml respectively., Conclusion: We have synthesized and encapsulated improved Toluidine Blue O (ITBO) in nanoliposomes (NLITBO) and thoroughly characterized the formulation. The antibacterial efficacy of NLITBO without light was demonstrated by ZOI which is similar to 2% chlorhexidine gluconate. MIC of NLITBO as a photosensitiser along with the optimal light parameter was also proposed in this study. These findings suggested that NLITBO could serve as an effective alternative to conventional antibacterial treatments in managing Streptococcus mutans rich biofilms. It can have potential pharmaceutical application in oral health care., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Panda et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: The co-existence of S. mutans and C. albicans is frequently detected in root caries and early child caries and is reported to be associated with recurrent caries. The aim of this study was to investigate the effects of potassium iodide (KI) in combination with toluidine blue O-mediated antimicrobial photodynamic therapy (aPDT) on S. mutans and C. albicans mixed-species biofilm, as well as the antibiofilm mechanisms involved., Methods: Mixed-species biofilm was constructed of S. mutans and C. albicans on dentin blocks. The antibiofilm efficacy, cytotoxicity and antibiofilm mechanism of KI in combination with aPDT were determined and evaluated., Results: KI+TBO-aPDT treatment caused reduction in microorganism counts, metabolic activity, and biofilm biomass of mixed-species biofilm without inducing cytotoxicity to hDPCs (human dental pulp cells). Observations such increased ROS (reactive oxygen species) levels, impaired cell membrane function, cell apoptosis and reduced expression in several genes seem to be artifacts of reduced growth and general killing by KI+TBO-aPDT treatment., Discussion: These data suggested that KI in combination with aPDT as an innovative approach to combat S. mutans and C. albicans biofilm, and thus as an optional treatment for caries., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Huang, Du, Wang, Cai and Huang.)

Objective: Evaluation of the effect of phycocyanin (PC) and toluidine blue (TBO) along with sodium fluoride varnish (FV) or titanium tetrafluoride (TiF 4 ) under the conditions of antimicrobial photodynamic therapy (PDT) on a dual-species cariogenic biofilm and on remineralization process., Design: After the development of Streptococcus mutans and Lactobacillus acidophilus dual-species biofilms on the human enamel disks, they were divided into 11 groups (n = 9): Control (0.9 % saline), PC, TBO, FV, and TiF 4 alone, PC and TBO in combination with a 635 nm diode laser (PDT treatment), PC-PDT + (PC + FV or TiF 4 + 635 nm diode laser), and TBO-PDT + (TBO + FV or TiF 4 + 635 nm diode laser). After the treatment, crystal violet assay was performed to determine the reduction of cariogenic biofilms. Enamel remineralization changes were analyzed using energy dispersive X-ray spectroscopy (EDX) and field emission scanning electron microscopy (FESEM) for the calcium and phosphorus (Ca/P) ratio., Results: Only TBO-PDT + showed superior antibiofilm activity when TiF 4 was applied. Furthermore, the highest Ca/P ratio was found after treatment of enamel surfaces with TiF 4 -TBO-PDT + . The FESEM images showed that the enamel disks treated with TiF 4 plus TBO-mediated PDT exhibited surface coating. However, TiF 4 plus PC-mediated PDT cannot repair demineralized enamel., Conclusions: These data suggest that TBO-PDT along with TiF 4 can effectively reduce cariogenic biofilms and significantly remineralize enamel disks, opening new avenues in caries prevention., 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 Author(s). Published by Elsevier B.V. All rights reserved.)

Objectives: This study compared the effects of conventional, ultrasonic, and laser-activated irrigation (LAI) on penetration depth of three photosensitizers (PSs) in the root canal system., Materials and Methods: In this in vitro, experimental study, 120 extracted anterior teeth were decoronated such that the remaining root length was standardized at 12 mm. After root canal instrumentation with the ProTaper rotary system and irrigation with 5.25% NaOCl, the roots were assigned to 12 groups for the application of toluidine blue (TB), curcumin, and phycocyanin PSs combined with the LAI using erbium laser with 0.4 mm and 0.6 mm tips, ultrasonic activation, and conventional irrigation. The specimens were sectioned apicocoronally, and the dye penetration depth was quantified in the coronal, middle, and apical thirds under a stereomicroscope at x20 magnification. Data were analyzed by the Kruskal-Wallis and Dunn test (alpha=0.05)., Results: The effects of irrigation technique, PS type, and their interaction on dye penetration depth were significant at the apical, middle, and coronal thirds (P < 0.0001). TB + LAI with 0.4- and 0.6-mm laser tips showed the highest penetration depth while phycocyanin + LAI or conventional irrigation showed the lowest penetration depth at all areas. Dye penetration depth was the highest in the coronal, and the lowest in the apical third., Conclusion: The LAI technique with erbium laser (0.4- and 0.6-mm tips) enhanced the penetration depth of TB. The tested irrigation techniques had no significant efficacy for enhancement of the penetration depth of curcumin and phycocyanin., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Photodynamic therapy (PDT) is an effective method for bacterial infection control in root canals of teeth with a broad-spectrum antibacterial activity. However, its application in root canal treatment is limited due to its inefficiency under hypoxic conditions and dentin staining. Triton X-100 (TX) shows great potential in enhancing the efficiency of antimicrobial agents through improving bacterial membrane permeability. The present study employed a combination of toluidine blue O (TB)-mediated PDT with TX to target the Enterococcus faecalis (E. faecalis), a bacterium with strong resistance to various antibacterial agents and mostly detected in infected root canals. PDT combined with TX showed enhanced antibacterial efficiency against both planktonic cells and biofilms of E. faecalis. At the same time, TX enhanced the antibacterial effect in dentinal tubules and reduced the incubation time. Mechanism studies revealed that TX improved reactive oxygen species (ROS) production through increasing the proportion of TB monomers. Additionally, increased membrane permeability and wettability were also observed. The findings demonstrated the PDT combined with TX could be used as a highly effective method for the root canal disinfection of teeth., 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 Elsevier B.V. All rights reserved.)

Objective: To study the efficacy of PAD TM Plus-based photoactivated disinfection (PAD) for treating denture stomatitis (DS) in diabetic rats by establishing a diabetic rat DS model., Methods: The diabetic rat DS model was developed by randomly selecting 2-month-old male Sprague-Dawley rats and dividing them into four groups. The palate and denture surfaces of rats in the PAD groups were incubated with 1 mg/mL toluidine blue O for 1 min each, followed by a 1-min exposure to 750-mW light-emitting diode light. The PAD-1 group received one radiation treatment, and the PAD-2 group received three radiation treatments over 5 days with a 1-day interval. The nystatin (NYS) group received treatment for 5 days with a suspension of NYS of 100,000 IU. The infection group did not receive any treatment. In each group, assessments included an inflammation score of the palate, tests for fungal load, histological evaluation, and immunohistochemical detection of interleukin-17 (IL-17) and tumor necrosis factor (TNF-α) conducted 1 and 7 days following the conclusion of treatment., Results: One day after treatment, the fungal load on the palate and dentures, as well as the mean optical density values of IL-17 and TNF-α, were found to be greater in the infection group than in the other three treatment groups ( P < 0.05). On the 7th day after treatment, these values were significantly higher in the infection group than in the PAD-2 and NYS groups ( P < 0.05). Importantly, there were no differences between the infection and PAD-1 groups nor between the PAD-2 and NYS groups ( P > 0.05)., Conclusions: PAD effectively reduced the fungal load and the expressions of IL-17 and TNF-α in the palate and denture of diabetic DS rats. The efficacy of multiple-light treatments was superior to that of single-light treatments and similar to that of NYS., Competing Interests: The authors declare that they have no competing interests., (© 2024 Zhang et al.)

Background: Eradication of endodontic biofilms from the infected root canal system is still the main concern in endodontics. In this study, the role of the power density parameter in the efficacy of antimicrobial photodynamic therapy (PDT) with toluidine blue O (TBO) and phycocyanin (PC) activated by a 635 nm diode laser (DL) against Enterococcus faecalis biofilm in the root canal model was investigated., Materials and Methods: The E. faecalis biofilm in the root canal was treated with TBO and PC with different power densities (636, 954, 1273, and 1592 W/cm 2 ). The untreated biofilm represented the control group. After the treatments, the biofilms were analyzed based on the number of colonies per milliliter., Results: TBO and PC activated with 635 nm DL with a power density of 1592 W/cm 2 were more efficient in removing E. faecalis biofilms within the root canals than those with a power density of 636 W/cm 2 (p = 0.00)., Conclusion: The light power density optimized the bacterial reduction of E. faecalis biofilms in the root canal spaces. These results provide information on the decisive parameters for performing PDT on intracanal biofilms., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Background: The aim of this study was to investigate the effects of antimicrobial photodynamic therapy (PDT) using 635 nm diode laser irradiation with an energy density of 6 to 30 J/cm 2 and toluidine blue O (TBO) as a photosensitizer on the viability of Aggregatibacter actinomycetemcomitans attached to the surface of titanium implants., Materials and Methods: Titanium implants contaminated with A. actinomycetemcomitans were treated with TBO alone or in combination with different exposure parameters (light doses of 6 - 30 J/cm 2 at 635 nm) and 0.2 % chlorhexidine (CHX). After treatment, colony forming units (CFUs)/ml were determined to assess PDT efficacy. The structure of the biofilm of A. actinomycetemcomitans was analyzed by field emission scanning electron microscopy (FESEM)., Results: Under optimal conditions, the colony count was reduced by ∼90 %. Treatment with CHX was somewhat more effective (colony formation was reduced by ∼95 %), but this agent has adverse effects that can be avoided with PDT., Conclusion: This study confirms the efficacy of PDT against A. actinomycetemcomitans depending on the light dose. Treatment with TBO + 635 nm diode laser has an effect that may be equivalent to that of CHX, but perhaps with fewer adverse effects., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. 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.)

Coronaviruses (CoVs) belong to the group of enveloped positive-sense single-strand RNA viruses and are causative agents of respiratory, gastro-intestinal, and central nervous systems diseases in many host species, i.e., birds, mammals, and humans. Beta-CoVs revealed a great potential to cross the barrier between species by causing three epidemics/pandemics among humans in the 21st century. Considering the urgent need for powerful antiviral agents for decontamination, prevention, and treatment of BCoV infections, we turned our attention to the possibility of photodynamic inactivation with photosensitizers in combination with light irradiation. In the present study, we evaluated, for the first time, the antiviral activity of toluidine blue O (TBO) against Beta-coronavirus 1 (BCoV) in comparison to methylene blue (MB). First, we determined the in vitro cytotoxicity of MB and TBO on the Madin-Darby bovine kidney (MDBK) cell line with ISO10993-5/Annex C. Thereafter, BCoV was propagated in MDBK cells, and the virus titer was measured with digital droplet PCR, TCID 50 assay and plaque assay. The antiviral activity of non-toxic concentrations of TBO was estimated using the direct inactivation approach. All effects were calculated in MAPLE 15 ® mathematical software by developing programs for non-linear modeling and response surface analysis. The median inhibitory concentration ( IC 50 ) of TBO after 72 h of incubation in MDBK cells was 0.85 µM. The antiviral activity of TBO after the direct inactivation of BCoV ( MOI = 1) was significantly stronger than that of MB. The median effective concentration (EC 50 ) of TBO was 0.005 µM. The cytopathic effect decreased in a concentration-dependent manner, from 0.0025 to 0.01 µM, and disappeared fully at concentrations between 0.02 and 0.3 µM of TBO. The number of virus particles also decreased, depending on the concentration applied, as proven by ddPCR analysis. In conclusion, TBO exhibits significant potential for direct inactivation of BCoV in vitro, with a very high selectivity index, and should be subjected to further investigation, aiming at its application in veterinary and/or human medical practice.

This study evaluated the photobiocidal performance of four widely distributed visible-light-activated (VLA) dyes against two bacteria (Staphylococcus epidermidis and Escherichia coli) and two bacteriophages (phages MS2 and phi 6): rose bengal (RB), crystal violet, methylene blue, and toluidine blue O (TBO). The photobiocidal performance of each dye depended on the relationship between the type of dye and microorganism. Gram-negative E. coli and the non-enveloped structure of phage MS2 showed more resistance to the photobiocidal reaction than Gram-positive S. epidermidis and the enveloped structure of phage phi 6. RB had the highest potential to yield reactive oxygen species. However, the photobiocidal performance of RB was dependent on the magnitude of the surface charge of the microorganisms; for example, anionic RB induced a negative surface charge and thus electrical repulsion. On the other hand, the photobiocidal performance of TBO was observed to be less affected by the microorganism type. The comparative results presented in our study have significant implications for selecting photodynamic antimicrobial chemotherapy (PACT) dyes suitable for specific situations and purposes. Furthermore, they contribute to the advancement of PACT-related technologies by enhancing their applicability and scalability., 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 Inc. 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.)

Background: Although Candida albicans is the most frequent etiological agent of candidiasis, it has been reported a sizable number of infections related to the non-albicans Candida (NAC) species, Candida krusei. In addition, dual biofilms (biofilms composed by two species) may easily occur in vivo, becoming even more challenging the treatment of an infection. The fungicide effect of Photodynamic Therapy (PDT), using toluidine blue O (TBO) on both C. albicans and C. krusei development has been demonstrated. Thus, the objective of this study was to investigate the effects of PDT on dual-species biofilms of Candida albicans and Candida krusei., Methods: The effect of PDT was observed on the metabolic activity of mature dual-species biofilms of Candida albicans and Candida krusei by a metabolic assay based on the reduction of XTT (2,3-bis(2‑methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt) assay and the identification of Candida albicans and Candida krusei was performed on CHROMagar Candida medium., Results: it was observed a reduction of ∼30% in the metabolic activity of a mature biofilm treated with PDT, using 0.05 mg·mL -1 TBO and during biofilm formation a predominance of C. albicans on C. krusei was observed. The inhibition observed was related to reduction in the number of Colony Forming Units (CFU) of Candida albicans from 31.33 ± 3.7 to 17.0 ± 1.5. The number of CFU of C. krusei was not significantly modified., Conclusions: These results demonstrated the efficiency of PDT in inhibiting the dual-species biofilms of Candida albicans and Candida krusei by reducing C. albicans development., Competing Interests: Declaration of Competing Interest The authors have no financial, personal, or other conflicts of interest related to this work., (Copyright © 2023. Published by Elsevier B.V.)

Background: Novel combination of Toluidine Blue O (TBO) embedded silicone catheter with domestic/household LED bulb has a potential in clinical infection such as prevention of multi drug resistant catheter-associated urinary tract infections (CAUTIs) through photodynamic therapy., Material and Methods: Preliminarily, TBO was entrapped into silicone catheter by swell-encapsulation-shrink method. Further, in vitro study was carried out to check the antimicrobial photodynamic efficacy of TBO with domestic/household LED light. Antibiofilm activity was evaluated by scanning electron microscopy., Results: The results showed that these modified TBO embedded silicone catheters showed significant antimicrobial and antibiofilm activity against vancomycin resistant Staphylococcus aureus VRSA. Small piece (1 cm) of TBO-embedded silicone catheter (700 µM) showed 6 log 10 reduction in the viable count when exposed for only 5 min of domestic/household LED bulb, while 1 cm piece of 500 µM and 700 µM concentration of TBO-embedded catheter eradicated all bacterial load when exposed to 15 min of light. Segment of medical grade TBO-embedded silicone catheters were used to carry out investigation of reactive oxygen species generation mainly singlet oxygen that contributes to type II phototoxicity., Conclusion: These modified catheter provides cost effective, easy to manage and less time consuming therapy to eliminate CAUTIs., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2023. Published by Elsevier B.V.)

Background/aims: Alzheimer's disease is a progressive neurological disorder characterized by the intracellular accumulation of Tau protein aggregates. In the present work, we studied the effect of Toluidine Blue and photo-excited Toluidine Blue on the aggregation of repeat Tau using in vitro assays., Methods: The in vitro experiments were carried out on recombinant repeat Tau which was purified by cation exchange chromatography. The ThS fluorescence analysis was used to study the aggregation kinetics of Tau. CD spectroscopy and electron microscopy were used to study the secondary structure and morphology of Tau respectively. The actin cytoskeleton modulation was studied in Neuro2a cells with help of immunofluorescent microscopy., Results: Results showed that Toluidine Blue efficiently inhibited the formation of higher-order aggregates, which was evidenced by Thioflavin S fluorescence assay, SDS-PAGE, and TEM. Immunofluorescence studies on the cytoskeleton of Neuro2a cells showed that Toluidine Blue and photo-excited Toluidine Blue treatment at a non-toxic concentration of 0.5 µM stimulated the formation of actin-rich lamellipodia and filopodia structures. Tubulin networks were also differentially modulated after the treatment of Toluidine Blue and photo-excited Toluidine Blue. End-binding protein 1 (EB1) levels were observed to increase after Toluidine Blue and photo-excited Toluidine Blue treatment indicating accelerated microtubule polymerization., Conclusion: The overall study suggested that Toluidine Blue inhibited the aggregation of soluble Tau and photo-excited Toluidine Blue disaggregated the pre-formed Tau filaments. In our study, TB and PE-TB were observed to be potent against Tau aggregation. We observed a distinctive modulation of actin, tubulin networks, and EB1 levels after TB and PE-TB treatment, which suggested that TB and PE-TB have potency against cytoskeleton deformities., Competing Interests: The authors declare that they have no competing interest., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

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.)

Purpose: This study aimed to evaluate the antimicrobial effect of photodynamic therapy (PDT) against Staphylococcus aureus biofilm on a titanium surface and to compare the differences in the effect of PDT using toluidine blue O (TBO) and methylene blue (MB) as a photosensitizer., Methods: The bacterial strain S. aureus ATCC 25,923 was used. Sandblasted and acid-etched (SLA) disks were divided into the following six groups: phosphate buffer saline (PBS), TBO, MB, PBS with laser (PBS + L), TBO with laser (TBO + L), and MB with laser (MB + L). The laser group samples were irradiated by a cold diode laser for 60 s. After treatment, the number of surviving bacteria was calculated by counting the colony-forming units (CFUs) and confocal laser scanning microscopy (CLSM) was applied to observe the bacteria on the disk surface., Results: The TBO + L and MB + L groups showed significantly lower CFU/ml than the other groups (p < 0.01). The TBO + L group showed significantly lower CFU/ml than the MB + L group (p = 0.032). There was no significant difference between the PBS, TBO, MB, and PBS + L groups. Within the limitations of this in vitro study, PDT with TBO and MB can effectively reduce S. aureus biofilm on SLA titanium surfaces. TBO is more effective than MB as a photosensitizer. PDT with TBO may be applied to the treatment of peri‑implant disease in the future., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest in this study., (Copyright © 2023 Elsevier B.V. All rights reserved.)

To investigate the effect and mechanism of simvastatin on cell components of tendon-bone healing interface. The tendon-bone healing model was established by inserting the end of the Achilles tendon into the tibial tunnel on 24 rats, and simvastatin was used locally at the tendon-bone interface. Healing was evaluated at 8 weeks by mechanical testing, micro-CT, and qualitative histology including H&E, Toluidine blue, and immunohistochemical staining. In vitro, bone marrow stromal cells (BMSCs) and tendon-derived mesenchymal stem cells (TDSCs) underwent osteogenic and chondrogenic differentiation respectively by plate co-culture. An analysis was performed on days 7 and 14 of cell differentiation. Biomechanical testing demonstrated a significant increase in maximum stiffness in the simvastatin-treated group. Micro-CT analysis showed that the bone tunnels in the simvastatin group were smaller in diameter and had higher bone density. H&E and Toluidine blue staining demonstrated that tendon-bone healing was significantly greater with better tissue arrangement and more extracellular matrix in the simvastatin-treated group than that in the control group, and immunohistochemical staining showed the expression of VEGF in simvastatin group was significantly higher. Histological staining and RT-PCR confirmed that simvastatin could promote the differentiation of co-cultured BMSCs and TDSCs into osteoblasts and chondroblasts, respectively. The effect of promoting osteogenic differentiation was more tremendous at 14 days, while its effect on promoting chondroblast differentiation was more evident on the 7th day of differentiation. In conclusion, local administration of simvastatin can promote the tendon-bone healing by enhancing neovascularization, chondrogenesis, and osteogenesis in different stages of the tendon-bone healing process., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Toluidine blue O (TBO) is a phenothiazine dye that, due to its photochemical characteristics and high affinity for biomembranes, has been revealed as a new photosensitizer (PS) option for antimicrobial photodynamic therapy (PDT). This points to a possible association with membranous organelles like mitochondrion. Therefore, here we investigated its effects on mitochondrial bioenergetic functions both in the dark and under photostimulation. Two experimental systems were utilized: (a) isolated rat liver mitochondria and (b) isolated perfused rat liver. Our data revealed that, independently of photostimulation, TBO presented affinity for mitochondria. Under photostimulation, TBO increased the protein carbonylation and lipid peroxidation levels (up to 109.40 and 119.87%, respectively) and decreased the reduced glutathione levels (59.72%) in mitochondria. TBO also uncoupled oxidative phosphorylation and photoinactivated the respiratory chain complexes I, II, and IV, as well as the F o F 1 -ATP synthase complex. Without photostimulation, TBO caused uncoupling of oxidative phosphorylation and loss of inner mitochondrial membrane integrity and inhibited very strongly succinate oxidase activity. TBO's uncoupling effect was clearly seen in intact livers where it stimulated oxygen consumption at concentrations of 20 and 40 μM. Additionally, TBO (40 μM) reduced cellular ATP levels (52.46%) and ATP/ADP (45.98%) and ATP/AMP (74.17%) ratios. Consequently, TBO inhibited gluconeogenesis and ureagenesis whereas it stimulated glycogenolysis and glycolysis. In conclusion, we have revealed for the first time that the efficiency of TBO as a PS may be linked to its ability to photodynamically inhibit oxidative phosphorylation. In contrast, TBO is harmful to mitochondrial energy metabolism even without photostimulation, which may lead to adverse effects when used in PDT., (© 2022. The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology.)

Background: The application of neuroprotective agents in combination with stem cells is considered a potential effective treatment for multiple sclerosis (MS). Therefore, the effects of lithium chloride as a neuroprotective agent and a GSK3-β inhibitor were evaluated in combination with human adipose derived stem cells on re-myelination, oligodendrocyte differentiation, and functional recovery., Methods: After inducing a mouse model of MS and proving it by the hanging wire test, the mice were randomly assigned to five experimental groups: Cup, Sham, Li, hADSC, and Li + hADSC. Additionally, a control group with normal feeding was considered. Finally, toluidine blue staining was carried out to estimate the level of myelination. Furthermore, immunofluorescent staining was used to evaluate the mean of OLIG2 and MOG positive cells. The mRNA levels of β-Catenin, myelin and oligodendrocyte specific genes were determined via the Real-Time PCR., Results: The results of the hanging wire test and toluidine blue staining showed a significant increase in myelin density and improvements in motor function in groups, which received lithium and stem cells, particularly in the Li + hADSC group compared with the untreated groups (P < 0.01). Moreover, immunostaining results indicated that the mean percentages of MOG and OLIG2 positive cells were significantly higher in the Li + hADSC group than in the other groups (P < 0.01). Finally, gene expression studies indicated that the use of lithium could increase the expression of β-Catenin, myelin and oligodendrocyte specific genes., Conclusion: The use of Lithium Chloride can increase stem cells differentiation into oligodendrocytes and improve re-myelination in MS., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Iron homeostasis plays a positive role in articular cartilage health. Excessive iron or iron overload can induce oxidative stress damage in chondrocytes and ferroptosis cell death, advancing knee osteoarthritis (KOA). However, up to date, few effective agents treat iron overload-induced KOA (IOKOA). Chinese herbal medicine (CHM) provides abundant resources for drug selection to manage bone metabolic conditions, including osteoporosis. Biochanin A (BCA) is a novel bioactive multifunctional natural compound isolated from Huangqi, which has protective effects on bone loss. Nevertheless, the function and mechanism of BCA in treating IOKOA are still elusive., Purpose: This study seeks to uncover the potential therapeutic targets and mechanisms of BCA in the management of KOA with iron accumulation., Methods: Iron dextrin (500 mg/kg) was intraperitoneally injected into mice to establish the iron overloaded mice model. OA was induced through surgery, and the progression was evaluated eight weeks following surgery. OA severity was evaluated with micro-CT and Safranin-O/Fast green staining in vivo. Iron deposition in the knee joint and synovium was assessed using Perl's Prussian blue staining. Ferric ammonium citrate (FAC) was then administered to primary chondrocytes to evaluate iron regulators mediated iron homeostasis. Toluidine blue staining was utilized to identify chondrocytes in vitro. The vitality of the cells was assessed using the CCK-8 test. The apoptosis rate of cells was measured using Annexin V-FITC/PI assay. The intracellular iron level was detected utilizing the calcein-AM test. Reactive oxygen species (ROS), lipid-ROS, and mitochondrial membrane potentiality were reflected via fluorescence density. Utilizing RT-qPCR and western blotting, the expression level was determined., Results: Micro-CT and histological staining of knee joints showed greater cartilage degradation and higher iron buildup detected in iron-overloaded mice. BCA can reduce iron deposition and the severity of KOA. Toluidine blue staining and the CCK-8 assay indicated that BCA could rescue chondrocytes killed by iron. Cell apoptosis rates were increased due to iron overload but improved by BCA. Further, the intracellular content of iron, ROS, and lipid-ROS was increased with ferric ammonium citrate (FAC) treatment but restored after treatment with different concentrations of BCA. JC-1 staining revealed that BCA could reduce mitochondrial damage induced by iron overload., Conclusion: Iron overload was shown to promote chondrocyte ferroptosis in vivo and in vitro. Moreover, iron overload suppressed the expression of collagen II and induced MMP expression by catalyzing ROS generation with mitochondrial dysfunction. Our results showed that BCA could directly reduce intracellular iron concentration by inhibiting TfR1 and promoting FPN but also target the Nrf2/system xc-/GPX4 signaling pathway to scavenge free radicals and prevent lipid peroxidation. The results of this research indicate that BCA regulates iron homeostasis during the progression of osteoarthritis, which can open a new field of treatment for KOA., Competing Interests: Conflict of interest statement All the authors have declared that no competing interest exists., (Copyright © 2022. Published by Elsevier Masson SAS.)

Background: Medication-related osteonecrosis of the jaws (MRONJ) is difficult to treat, therefore, prevention would be the ideal clinical approach. This study evaluated the effectiveness of antimicrobial photodynamic therapy (aPDT), mediated by butyl toluidine blue (BuTB) in the prevention of MRONJ-like lesions after tooth extraction in rats., Methods: Twenty-eight senescent female rats were distributed in groups: VEH and VEH-aPDT, treated with vehicle, ZOL and ZOL-aPDT, treated with 100 µg/Kg of zoledronate, both treatments every three days over seven weeks. After three weeks from the commencement of treatment, the mandibular first molar was extracted. For the VEH and ZOL groups, no local treatment was performed, while with the VEH-aPDT and ZOL-aPDT groups, photodynamic treatment was carried out at 0, 2, and 4 days after extraction. For aPDT, 500μl of BuTB solution was deposited on the dental extraction site (0.5 mg/ml; 60 s), followed by irradiation with low-level laser (InGaAIP; 660 nm; 35 mW; 74.2 J/cm²; 60 s). After 28 postoperative days, euthanasia was performed. The hemimandibles were processed to: (1) histological analysis of tissue repair; (2) histometric analysis of the percentage of newly formed bone tissue (PNFBT) and percentage of non-vital bone tissue (PNVBT); (3) immunohistochemical analysis for tartrate-resistant acid phosphatase (TRAP)., Results: The ZOL and ZOL-aPDT groups showed less TRAP-positive cells when compared with VEH and VEH-aPDT. The ZOL group demonstrated great compromise in the tissue repair process, consistent with MRONJ-like lesions. VEH, VEH-aPDT and ZOL-aPDT presented a favorable tissue repair process. PNFBT in the ZOL group was lower than in the VEH, VEH-aPDT and ZOL-aPDT groups, whereas PNVBT in the ZOL group was higher than in the VEH, VEH-aPDT and ZOL-aPDT groups., Conclusion: aPDT mediated by BuTB prevented the occurrence of MRONJ-like lesions after tooth extraction in rats., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Osteoarthritis (OA) is a degenerative disease caused by the progressive destruction of cartilage and subchondral bone [1]. Studies have shown that by inhibiting the degradation of cartilage cells and the loss of subchondral bone, OA can be prevented and treated. Neratinib, as a small molecule compound with anti-inflammatory and anti-tumor properties, is a very effective inhibitor of IL-1β-induced chondrocyte inflammation and anabolic metabolism. By investigating the effect of neratinib in ATDC5 chondrocytes, the study finds that neratinib reduces inflammation by inhibiting the MAPK and NF-κB signaling pathways, and at the same time reduces pyrolysis (indicated by the results of reverse transcription quantitative PCR and western blotting). For anabolic metabolism, after high-density cell culture, IL-1β-induced catalytic changes and degradation of the extracellular matrix were evaluated by toluidine blue staining. Since osteoclasts are key participants in the process of subchondral bone remodeling in OA, we also studied the effect of neratinib on the maturation of osteoclasts. The results showed that neratinib also acts as an anti-osteoclast agent in vitro. By inhibiting the NF-κB and MAPK pathways, it reduces the expression of osteoclast-related genes, thereby inhibiting RANKL-induced osteoclastogenesis. The results of in vivo animal experiments supported the conclusions from the experiments in vitro. Neratinib inhibited both the destruction of medial meniscus induced cartilage degradation and osteoclast formation, which proves that neratinib has a dual effect, protecting cartilage and inhibiting osteoclast formation. These results indicate that neratinib can be a brand-new latent strategy for the treatment of OA., 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 © 2022. Published by Elsevier Inc.)

Polyaryletherketone (PAEK) has good biocompatibility and mechanical properties and thus may have great potential in the fields of reparative medicine and bone intervention. In this study, the key representative PAEKs, polyetheretherketone (PEEK) and polyetherketoneketone (PEKK), were modified by UV grafting with sodium polystyrene sulfonate (polyNaSS) to improve their biocompatibility. Toluidine blue staining and Fourier transform infrared spectroscopic analyses showed that sulfonic acid groups were successfully introduced into PAEK, and the hydrophilicity and protein adsorption capacity of the materials were enhanced in a concentration-dependent manner. The effects of the grafted polyNaSS on osteoinduction and antibacterial properties of PAEK were analyzed in detail. We found that polyNaSS enhanced the viability, alkaline phosphatase activity, calcium mineral deposition, and levels of expression of osteoblast-related genes and proteins of adherent human umbilical cord Wharton's jelly-derived mesenchymal stem cells. In addition, when Escherichia coli , Staphylococcus aureus and Porphyromonas gingivalis were incubated with the materials, bacterial colony counting revealed that grafting of polyNaSS onto PAEK led to more potent inhibition of bacterial adhesion, and polyNaSS-grafted PEKK had stronger antibacterial performance than did polyNaSS-grafted PEEK fabricated under the same grafting conditions. These data show that polyNaSS-grafted PAEK, and particularly polyNaSS-grafted PEKK, may be useful as orthopedic and dental implant materials.

We generated a rat model of sciatic nerve crush injury and characterized the effects of curcumin on sciatic nerve recovery by using behavioral experiments, hematoxylin-eosin staining, toluidine blue staining, and immunohistochemical. Proteomic analysis using tandem mass tagging was performed to determine differentially expressed proteins (DEPs), and GO and KEGG pathway analyses of overlapping DEPs was conducted, following which, qPCR, western blotting, and immunofluorescence were further performed to validate the proteins of interest. Finally, a Schwann cell injury model was used to verify the effect of curcumin on potential targets. The rat model was successfully established and curcumin improved the sciatic nerve function index of rats with sciatic nerve injury (SNI) and increased the number and diameter of myelinated axons in the sciatic nerve. In the Sham group versus the Injured group and in the Injured group versus the Curcumin group, we identified a total of 4,175 proteins, of which 953 were DEPs, and 218 were known overlapping DEPs. Ten associated pathways, such as calcium signaling pathway, biosynthesis of antibiotics, and long-term potentiation, were identified. The 218 overlapping DEPs were primarily involved in negative regulation of apoptotic process, biological processes, cytoplasm cellular component, and protein binding molecular function based on GO annotation. Curcumin promoted increased expression of ApoD and inhibited the expression of Cyba in vivo and in vitro. These results indicated that curcumin promoted sciatic nerve repair through regulation of various proteins, targets, and pathways. Cyba and ApoD may be potential targets of curcumin in the treatment of SNI., (Copyright © 2022 IBRO. Published by Elsevier Ltd. All rights reserved.)

Alzheimer's disease (AD) is characterized by extracellular amyloid plaques composed of amyloid-β peptide (Aβ), intracellular neurofibrillary tangles containing hyperphosphorylated tau protein and neuronal loss. Most of the FDA-approved AD drugs currently on the market are cholinesterase inhibitors, which are only effective in relieving the symptoms of AD. However, recent studies in AD drug discovery focus on multi-targeted strategies, including anti-amyloid and anti-tau therapy. In the current study, we have investigated the effects of toluidine blue O (TBO), a cholinesterase inhibitor, on amyloid precursor protein (APP) processing, tau phosphorylation, and tau kinases/phosphatase in N2a mouse neuroblastoma cells stably expressing the Swedish mutation of human APP695 (N2a-APPSwe). The results demonstrated that TBO reduces Aβ40/42 levels by decreasing expression levels of β-secretase 1 (BACE1), presenilin 1 (PS1) and total APP without causing cytotoxic effects in N2a-APPSwe cells. TBO also decreased the levels of both total tau and phosphorylated tau at residues Ser202/Thr205, Thr181, Ser396 and Ser 396/Ser404. Moreover, when the possible mechanisms underlying its effects on tau pathology were explored, TBO was found to decrease tau phosphorylation at those sites by reducing the expression levels of Akt, GSK-3β, Cdk5, inactive p-PP2A and increasing the expression levels of p-Akt Ser473 and inactive p-GSK-3β Ser9. Our new data support the idea that TBO may be a promising multi-target drug candidate for the treatment of AD., 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 © 2022 Elsevier B.V. All rights reserved.)

Asthma is a high-incidence disease in the world. Oxysophocarpine (OSC), a quinolizidine alkaloid displays various pharmacological functions including anti-inflammation, neuroprotective, anti-virus and antioxidant. Here, we established mice and cell asthmatic model to explore the effects of OSC for asthma treatment. Mice were sensitized and challenged with ovalbumin (OVA) and treated with OSC before challenge. Enzyme-linked immuno sorbent assay (ELISA), hematoxylin and eosin (H&E), periodic acid-schiff (PAS), tolonium chloride staining and immunohistochemical assay were performed. OSC treatment inhibited inflammatory cell infiltration and mucus secretion in the airway, reduced IgE level in mouse serum and decreased IL-4, IL-5 production in bronchoalveolar lavage fluid (BALF). OSC also reduced the spleen index to regulate immune function. Meanwhile, NCI-H292 cells were induced by lipopolysaccharide (LPS) to simulate airway epithelial injury. OSC pretreatment decreased the IL-6 and IL-8 cytokine levels, mucin 5 AC expression, and mucin 5 AC mRNA level in the cell model. Further, OSC suppressed the phosphorylation of c-Jun N-terminal kinase (JNK), and activator protein 1 (AP-1, Fos and Jun). These findings revealed that OSC alleviated bronchial asthma associated with JNK/AP-1 signaling pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interests., (Copyright © 2022. Published by Elsevier B.V.)

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: Multi-resistant bacteria, partially a result of the abuse of antibiotics, have greatly diminished the effectiveness of antibiotics. The combination of antibiotics with other therapies like antimicrobial photodynamic therapy (aPDT) may provide a useful strategy for fighting resistant bacteria. Here, the synergistic bactericidal effects of toluidine blue (TB)-aPDT and gentamicin (GEN) were evaluated in vitro and in vivo., Methods: The Post-antibacterial effects were measured at 600 nm (OD600) by a microplate reader. The bacterial envelope and biofilm were observed by a field emission scanning electron microscope. The expression of oxidative stress and Agr system-related genes was analyzed by qRT-PCR after GEN combined with TB-aPDT (GEN&aPDT). Besides, the burn infection model was established to investigate the cloning efficiency of immobilized bacteria, wound healing and inflammatory factors in the lesions., Results: GEN&aPDT could inhibit the growth of Staphylococcus aureus (S. aureus) and multidrug-resistant S. aureus (MDR S. aureus) for up to 15 h, and destroyed the cell envelope and biofilm structure of S. aureus and MDR S. aureus. During the process, ROS played an important role, inducing oxidative stress and downregulating the expression of AgrA, AgrB and PSM in the Agr system, resulting in decreased bacterial virulence and infectivity. In addition, GEN&aPDT cotreatment could effectively promoted wound healing in burn-infected mice by reducing the numbers of bacterial colonization in the wound, decreasing the content of inflammatory factors, and increasing the expression of growth factors., Conclusion: The present study confirmed a bactericidal synergy between GEN and aPDT in vitro and in vivo, therein, the oxidative stress exhibited an important role in decreasing bacterial virulence and infectivity, which may bring new ideas for the treatment of bacterial resistance., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Introduction: Considering the importance of disinfecting dentin and enamel after cavity preparation and the possible effect of disinfection methods on induction of various reactions in the tooth structure the aim of the present study was to evaluate microleakage of composite resin restoration after disinfecting the prepared dentin and enamel surface with antimicrobial photodynamic therapy (aPDT) with toluidine blue (TBO) and indocyanine green (ICG)., Materials and Methods: Standard class V cavities were prepared on buccal surface of 71 human premolar teeth. The samples were randomly divided into 3 groups based on disinfection method: Group 1: conventional disinfection method with Phosphoric acid 37% as the control; Group 2: aPDT with TBO and diode laser with wavelength of 635 nm; Group 3: aPDT with ICG and diode laser with wavelength of 808 nm. All the cavities were restored with composite resin (3M™ Filtek™ Z250). After thermocycling and immersing in 0.5% basic fuchsin, the samples were prepared for microleakage evaluation under a stereomicroscope. Data was analyzed with Kruskal-Wallis and Wilcoxon signed-rank tests at P < 0.05., Results: There were no significant differences in the microleakage of occlusal and gingival margins between the TBO and control groups (P > 0.05). Also, the microleackage of occlusal margins between groups was not significantly different (P > 0.05) but microleackage of gingival margins of ICG group was lower than two other groups in a meaningful way (P < 0.05)., Conclusion: Photodynamic therapy with ICG as disinfecting agent in cavity preparations before composite resin restorations decreases the microleackage of gingival margins., (Copyright © 2022. Published by Elsevier B.V.)

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.)

Ethnopharmacological Relevance: The Tiao-bu-fei-shen (TBFS) formula, extensively used in Traditional Chinese Medicine (TCM), can enhance therapeutic efficacy and reduce the frequency of acute exacerbations of lung-kidney Qi deficiency in patients with chronic obstructive pulmonary disease (COPD). According to both TCM theory and long-term observation of practice, TBFS has become an effective treatment for COPD-associated tracheobronchomalacia (TBM)., Aim of the Study: To investigate the mechanism of the TBFS formula in treating COPD-associated TBM based on caveolin 1-p38 MAPK signaling and apoptosis., Materials and Methods: A rat COPD model was prepared by exposure to smoking combined with tracheal lipopolysaccharide injection. The trachea or bronchus chondrocytes from COPD rats were isolated, cultured, and treated with 10 ng/mL IL-1β for 24 h to develop a model of COPD-associated TBM. Normal rats were administered TBFS to prepare drug-containing serum, and CCK8 assays were used to screen the optimal drug-containing serum concentration and SB203580 dose. TBFS drug-containing serum and SB203580 were processed separately for the control, model, drug-containing serum, blocker, and drug-containing serum combined with blocker groups. Flow cytometry and CCK8 assays were used to detect apoptosis and proliferative activity. Toluidine blue staining and immunohistochemistry were used to analyze the chondrocyte proteoglycan and type II collagen content. Western blotting was used to detect the expression of caveolin 1, p-p38 MAPK, TNF-α, IL-1β, MMP-13, Bax, and Bcl-2 proteins. Quantitative PCR was used to detect the expression of caveolin 1, p38 MAPK, IL-1β, MMP-13, Bax, Bcl-2, and miR-140-5p., Results: The isolation and identification of bronchial chondrocytes from COPD rats revealed that 10 ng/mL IL-1β can produce a stable COPD-associated TBM model. Screened via the CCK8 method, fourth-generation bronchial chondrocytes were determined as the optimal cells, and 5 μM SB203580 and 5% low-dose drug-containing serum were the optimal intervention doses. The experimental chondrocytes of each group were treated separately for 48 h. Toluidine blue staining and immunohistochemical analysis revealed that TBFS drug-containing serum, SB203580, and TBFS drug-containing serum combined with SB203580 can effectively increase the proteoglycan and type II collagen content after chondrocyte degradation. Flow cytometry of cells treated with SB203580 and TBFS drug-containing serum combined with SB203580 revealed significantly reduced cell apoptosis and enhanced cell proliferation activity. Western blot and qPCR analyses revealed that the TBFS drug-containing serum, SB203580, and TBFS drug-containing serum combined with SB203580 effectively inhibit the expression of caveolin 1, p-p38 MAPK, MMP-13, IL-1β, TNF-α, and Bax proteins while promoting Bcl -2 protein expression. Treatment with TBFS drug-containing serum and SB203580 effectively inhibited the expression of MMP-13, p38 MAPK, caveolin 1, and Bax genes, and promoted the expression of Bcl-2 and miR-140-5p genes., Conclusions: A concentration of 10 ng/mL of IL-1β can generate a stable COPD-associated TBM cell model. TBFS can improve the proteoglycan and type II collagen content, increase cell activity, and reduce the amount of chondrocyte apoptosis. The role of TBFS may be related to mechanisms of inhibiting the expression of the key signaling molecules caveolin 1 and p-p38 MAPK in the caveolin 1-p38 MAPK signaling pathway, thereby reducing the expression of the downstream effector products MMP-13, IL-1β, and TNF-α, while inhibiting the expression of the apoptotic gene Bax and improving the expression of Bcl-2 and miR-140-5p genes., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Photodynamic therapy is a two-step procedure, involving the use of photosensitizing agents followed by selective illumination of the target lesion with visible light. Photodynamic therapy has been described recently as a promising strategy for treatment of leishmaniasis. This study aims to evaluate the in vitro phototoxic, morphological, and apoptotic effect of methylene blue, toluidine blue, chloro-aluminum phthalocyanine, and pheophorbide a-mediated photodynamic therapy on the viability of Leishmania tropica promastigotes., Methods: Parasites were treated with methylene blue, toluidine blue, chloro-aluminum phthalocyanine, and pheophorbide a or/and methylene blue, toluidine blue, chloro-aluminum phthalocyanine, and pheophorbide a-mediated photodynamic therapy, and cell proliferation, morphological changes, and apoptosis were evaluated by XTT, giemsa staining, DAPI staining, and DNA fragmentation, respectively., Results: Parasite viability was significantly different in between the groups treated with methylene blue, toluidine blue, and pheophorbide a, with or without irradiation. chloro-aluminum phthalocyanine treatment did not lead to any alterations in cell viability in Leishmania tropica promastigotes with or without irradiation. DAPI staining results indicated that apoptotic bodies and nucleus fragmentation started to be visible in methylene blue, chloro-aluminum phthalocyanine, and pheophorbide a-mediated photodynamic therapy groups. DNA ladder pattern which is used to define apoptosis was observed in irradiated methylene blue, chloro-aluminum phthalocyanine, and pheophorbide a groups., Conclusions: The results revealed that apoptosis-induced cell death was observed in Leishmania tropica promastigotes after the application of photosensitizers in combination with light irradiation., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Amyloid precursor-like protein-2 (APLP2) and its C-terminal fragments (CTFs) are expressed at high levels in pancreatic cancer cells and knockdown of APLP2 expression inhibits tumor growth. CTFs are released from APLP2 by beta-secretase (BACE). In this study, our goal was to determine whether methylene blue (MethB) and toluidine blue O (TBO) could be used to slow down the growth and viability of pancreatic cancer cells (Hs 766T). We found that TBO and MethB decreased the growth and viability of Hs 766T cells in a dose- and time-dependent manner compared to vehicle-treated control, as demonstrated by MTT and trypan blue exclusion assays. Although TBO led to decreased expression of APLP2, MethB did not show any significant effect on APLP2. However, both MethB and TBO reduced BACE activity and the levels of APLP2 CTFs in Hs 766T cells. In conclusion, MethB and TBO may be valuable candidates for the treatment of pancreatic cancer by targeting APLP2 processing., (© 2022 Wiley Periodicals LLC.)

Objectives: The aim of this study is to evaluate the influence of the antimicrobial photodynamic therapy with phycocyanin and toluidine blue on the bond strength of resin composite to the sound dentin., Materials and Methods: The samples include 120 tooth blocks taken from 60 human extracted sound third molar teeth. They were randomly divided into 12 groups and disinfected with two different photosensitizers (Phycocyanin and Toluidine blue) activated by 635 nm diode laser for aPDT procedure. Then two different protocols (total-etch and self-etch) of universal adhesive system (G-Premio BOND, GC Dental Products CORP. Japan) were applied. The samples then filled with resin composite (Gradia® Direct, GC Dental Products CORP. Japan). After 10000 cycles of thermocycling, all samples were subjected to the micro shear bond strength (μSBS) test using a universal testing machine (1 mm/min). The data were analyzed with One Way ANOVA., Results: The effect of aPDT using both photosensitizers (phycocyanin and toluidine blue O) on the bonding strength to dentin was not significant (P>0.05). However, the bonding strength of total etch groups was reduced significantly (P<0.05) after aPDT using both photosensitizers. Phycocyanin self-etch group showed the greatest bonding strength (19.48 ± 3.89 MPa) in comparison to other aPDT groups. No significant reduction after thermocycling in dentin bond strength was observed in all groups (P>0.05), with exception of control total etch groups, which showed significant difference (P<0.05)., Conclusion: aPDT using toluidine blue O and phycocyanin has no adverse effect on bonding to sound dentin when using universal adhesive in self-etch protocol., Clinical Relevance: Phycocyanin is more recommended to be used in aPDT in comparison to toluidine blue as a natural photosensitizer., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

(1) Background and the aim: The appropriate incubation time in the antimicrobial photodynamic therapy protocol seems to have a huge impact on the efficacy of this process. This is particularly important in relation to Candida strains, due to the size of these cells and the presence of the cell wall. The aims of this study were to determine the optimal incubation time needed for the absorption of toluidine blue by cells of C. albicans , C. glabrata , C. krusei and C. parapsilosis using direct observation by optical microscopy, and to evaluate the efficacy of TBO-mediated aPDT on planktonic cells of these strains. (2) Methods: The microscopic evaluation consisted of taking a series of images at a magnification of 600× and counting the % of stained cells. The in vitro effect of TBO-mediated aPDT combined with a diode laser (635 nm, 400mW, 12 J/cm 2 , CW) on the viability of yeast cells with different incubation times was evaluated. (3) Results: The presence of TBO within the cytoplasm was observed in all tested Candida strains and at all microscopic evaluation times. However, the highest percentages of cells were stained at 7 and 10 min. The highest % reduction of CFU/mL after TBO-mediated aPDT against Candida was obtained for the strain C. albicans ATCC 10,231 and it was 78.55%. (4) Conclusions: TBO-mediated aPDT against Candida was effective in reducing the number of CFU/mL at all assessed incubation times. However, the most efficient period for almost all strains was 7-10 min., Competing Interests: The authors declare no conflict of interest.

Neospora caninum causes heavy losses related to abortions in bovine cattle. This parasite developed a complex defense redox system, composed of enzymes as glutathione reductase (GR). Methylene blue (MB) impairs the activity of recombinant form of Plasmodium GR and inhibits the parasite proliferation in vivo and in vitro. Likewise, MB and its derivatives inhibits Neospora caninum proliferation, however, whether the MB mechanism of action is correlated to GR function remains unclear. Therefore, here, N. caninum GR (NcGR) was characterized and its potential inhibitors were determined. NcGR was found in the tachyzoite cytosol and has a similar structure and sequence compared to its homologs. We verified the in vitro activity of rNcGR (875 nM) following NADPH absorbance at 340 nM (100 mM KH 2 PO 4 , pH 7.5, 1 mM EDTA, ionic strength: 600 mM, 25 °C). rNcGR exhibited a Michaelian behavior (K m(GSSG) :0.10 ± 0.02 mM; k cat(GSSG) :0.076 ± 0.003 s -1 ; K m(NADPH) :0.006 ± 0.001 mM; k cat(NADPH) : 0.080 ± 0.003 s -1 ). The IC 50 of MB,1,9-dimethyl methylene blue, new methylene blue, and toluidine blue O on rNcGR activity were 2.1 ± 0.2 μM, 11 ± 2 μM, 0.7 ± 0.1 μM, and 0.9 ± 0.2 μM, respectively. Our results suggest the importance of NcGR in N. caninum biology and antioxidant mechanisms. Moreover, data presented here strongly suggest that NcGR is an important target of phenothiazinium dyes in N. caninum proliferation inhibition., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Bacterial spores are an important consideration in healthcare decontamination, with cross-contamination highlighted as a major route of transmission due to their persistent nature. Their containment is extremely difficult due to the toxicity and cost of first-line sporicides., Methods: Susceptibility of Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli to phenothiazinium photosensitizers and cationic surfactants under white- or red-light irradiation was assessed by determination of minimum inhibitory concentrations, minimum bactericidal concentrations and time-kill assays. B. subtilis spore eradication was assessed via time-kill assays, with and without nutrient and non-nutrient germinant supplementation of photosensitizer, surfactant and photosensitizer-surfactant solutions in the presence and absence of light., Results: Under red-light irradiation, >5-log 10 colony-forming units/mL reduction of vegetative bacteria was achieved within 10 min with toluidine blue O (TBO) and methylene blue (MB). Cationic surfactant addition did not significantly enhance spore eradication by photosensitizers (P>0.05). However, addition of a nutrient germinant mixture to TBO achieved a 6-log 10 reduction after 20 min of irradiation, while providing 1-2 log 10 improvement in spore eradication for MB and pyronin Y., Conclusions: Light-activated photosensitizer solutions in the presence of surfactants and germination-promoting agents provide a highly effective method to eradicate dormant and vegetative bacteria. These solutions could provide a useful alternative to traditional chemical agents used for high-level decontamination and infection control within health care., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Photodynamic therapy (PDT) has become an advantageous therapeutic approach for the treatment of select cancers and microbial infections. PDT generates toxic reactive oxygen species as an end product of the interaction between the photosensitizer and light with an appropriate wavelength. Toluidine blue ortho is a photosensitizer that is commonly used in the photodynamic treatment of bacterial infection and a promising photosensitizer for cancer treatment. This study aims to evaluate the potential photo-cytotoxicity of toluidine blue ortho-mediated photodynamic therapy on PC-3 prostate cancer cells., Methods: In this study toluidine blue ortho-mediated photodynamic therapy was assessed on PC-3 cancer cells with various photosensitizer concentrations and light energy densities of the 655-nm diode laser. MTT analysis was used for the determination of the cytotoxicity on the cells and viability/cytotoxicity assay was used for live/dead cell staining after the applications. The mechanism of this application was further analyzed with the determination of intracellular reactive oxygen species and nitric oxide release., Results: The light applications and the photosensitizer alone did not inhibit the cell viability of PC-3 cells. 20 J/cm 2 laser energy density together with 100 μM photosensitizer concentration resulted in maximum cancer cell death with a rate of approximately 89 %. The level of intracellular reactive oxygen species increased with the increasing parameters of the applications that resulted in more cell death., Conclusion: This study showed the successful anticancer activity of toluidine blue ortho upon irradiation with 655 nm of laser light against PC-3 cancer cells and it was mediated with the production of reactive oxygen species., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cationic biocides are widely utilized for surface disinfection. Photosensitizers such as toluidine blue O (TBO) produce reactive oxygen species following light excitation and are being investigated as novel biocides for similar applications. Aspergillus brasiliensis conidia contain melanin which protects against environmental stressors. The negative charge and antioxidant properties of melanin may confer resistance to photosensitizers and other biocides. In this study, the yeasticidal and fungicidal activity benzalkonium chloride (BZC), sodium dichloroisocyanurate (NaDCC) and TBO with red light were examined using quantitative suspension tests. All three biocides were highly effective against Candida albicans and > 5·0 log 10 reductions in viability were attainable within 5 minutes. Wild-type A. brasiliensis conidia were highly tolerant to treatment and 0·4 log 10 reductions in viability were observed within the same time frame when treated with TBO or BZC. NaDCC was markedly more effective. Inhibition of melanin biosynthesis by culturing with 100 μg ml -1 kojic acid resulted in a hypopigmented phenotype with significantly increased sensitivity to all three biocides. These observations indicate that melanin is a significant contributor towards A. brasiliensis tolerance of biocides and photosensitizers and demonstrate that cationic biocides are poorly suited to applications where the control of A. brasiliensis is required., (© 2020 The Authors. Letters in Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

In vitro experiments confirmed that antibacterial photodynamic treatment (aPDT) inactivates periodontal pathogens. However, more effective sterilization is needed in the complex oral environment. This study tested whether dihydroartemisinin (DHA) enhanced the photokilling effect of aPDT on Porphyromonas gingivalis (P. gingivalis) in planktonic and biofilm states. aPDT combining toluidine blue O (TBO) with 630 nm red light was performed on bacterial suspensions and biofilms in vitro with different final concentrations of DHA (10, 20 and 40 μg mL -1 ). The sensitization mechanism was preliminarily investigated by uptake experiments. The above experiments were repeated with different incubation times (30, 60, 120 s). Porphyromonas gingivalis biofilms exhibited significantly higher resistance to aPDT than P. gingivalis in suspension under the same experimental parameters. DHA alone had no cytotoxic effect on P. gingivalis with or without light irradiation. In either bacterial suspensions or biofilms, DHA concentration-dependently enhanced the photokilling effect of aPDT and increased TBO uptake by P. gingivalis. Prolonged incubation time enhanced the photokilling efficiency of aPDT until cellular TBO uptake reached saturation. DHA can enhance aPDT activity against P. gingivalis in planktonic and biofilm states. DHA also accelerated TBO uptake, reducing incubation time., (© 2020 American Society for Photobiology.)

Background: There are investigations on multiple photosensitizers for modulation of caries-related biofilms using PDT. However, much controversy remains about recommended parameters mostly on the selection of an efficient photosensitizer., Objective: The study performed a systematic review to identify the answer to the following question: What photosensitizers present high bactericidal efficacy against cariogenic biofilms?, Methods: Systematic review with meta-analyses were carried out for English language articles from October to December 2019 (PRISMA standards) using MEDLINE, Scopus, Biomed Central, EMBASE, LILACS, and Web of Science. Information on study design, biofilm model, photosensitizer, light source, energy delivery, the incubation time for photosensitizer, and bacterial reduction outcomes were recorded. We performed two meta-analyses to compare bacterial reduction, data was expressed by (1) base 10 Logarithm values and (2) Log reduction RESULTS: After the eligibility criteria were applied (PEDro scale), the selected studies showed that toluidine Blue Ortho (TBO) and methylene blue (MBO) (5-min incubation time and 5-min irradiation) demonstrated better bacterial reduction outcomes. For the data expressed by Log TBO, MBO, curcumin, and Photogem® presented a significant bacterial decrease in comparison to the control (p = 0.042). For the data represented by Log reduction, the bacterial reduction toward S.mutans was not significant for any photosensitizer (p = 0.679)., Conclusion: The lack of methodological standardization among the studies still hinders the establishment of photosensitizer and bactericidal efficiency. TBO, MBO, curcumin, and photogem generate greater PDT-based bacterial reduction on caries-related bacteria.. Further clinical studies are necessary in order to obtain conclusive results., (Copyright © 2020. Published by Elsevier B.V.)

Toluidine blue O (TBO) is a water-soluble photosensitizer that has been used in photodynamic antimicrobial and anticancer treatments, but suffers from limited solubility in hydrophobic media. In an effort to incrementally increase TBO's hydrophobicity, we describe the synthesis of hexanoic (TBOC6) and myristic (TBOC14) fatty acid derivatives of TBO formed in low to moderate percent yields by condensation with the free amine site. Covalently linking 6 and 14 carbon chains led to modifications of not only TBO's solubility, but also its photophysical and photochemical properties. TBOC6 and TBOC14 derivatives were more soluble in organic solvents and showed hypsochromic shifts in their absorption and emission bands. The solubility in phosphate buffer solution was low for both TBOC6 and TBOC14, but unexpectedly slightly greater in the latter. Both TBOC6 and TBOC14 showed decreased triplet excited-state lifetimes and singlet oxygen quantum yields in acetonitrile, which was attributed to heightened aggregation of these conjugates particularly at high concentrations due to the hydrophobic "tails." While in diluted aqueous buffer solution, indirect measurements showed similar efficiency in singlet oxygen generation for TBOC14 compared to TBO. This work demonstrates a facile synthesis of fatty acid TBO derivatives leading to amphiphilic compounds with a delocalized cationic "head" group and hydrophobic "tails" for potential to accumulate into biological membranes or membrane/aqueous interfaces in PDT applications., (© 2020 American Society for Photobiology.)

Aim: This study aimed to compare the antibacterial effect of antimicrobial photodynamic therapy (aPDT) by use of light emitting diode (LED) and diode laser light sources with toluidine blue (TBO) photosensitizer on Enterococcus faecalis (E. faecalis) biofilm in root canals of extracted single-canal human teeth., Methods: Eighty-five sound human single-canal teeth were chosen for this study and standardized to have 15 mm of root length. The root canals were prepared with ProTaper rotary files and inoculated and incubated with E. faecalis for one week. Samples were divided into five experimental (n = 15) groups of PDT with TBO/LED, TBO/diode laser, LED, TBO and diode laser and one negative (NaOCl) and one positive (no treatment) control group. Dentin chip samples were collected from inside the canals using size 40 hedstrom file. The number of colony forming units (CFUs) in each group was calculated., Results: Irrespective of the light source used for activation of photosensitizer (diode or LED), PDT was significantly more effective than other experimental groups (P < 0.001). No significant difference was noted between aPDT with diode laser or LED (P > 0.05). No significant difference was noted in colony count among other groups (LED, TBO, diode; P > 0.05)., Conclusion: The results showed that aPDT significantly decreased residual bacteria in the canal. Thus, it may be used as an adjunct for root canal disinfection. Both diode and LED are suitable light sources for this purpose and can be used alternatively., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Traditional chemomechanical treatment procedures are an indispensable part of endodontic treatment, however, additional treatment approaches such as antimicrobial photodynamic therapy (aPDT) may also be recommended for the elimination of residual microorganisms. In this study, the disinfection efficiency of aPDT performed using methylene blue (MB), toluidine blue (TB), and tetra 2-mercaptopyridine substituted zinc phthalocyanine (TM-ZnPc) was compared in the roots contaminated with Enterococcus faecalis (E. faecalis)., Materials and Methods: Forty-nine teeth with a single root and canal were included in this study. The roots were sterilized, and inoculated with E. faecalis. The roots were kept in an incubator for 30 days to form the biofilm. Forty-five teeth were prepared up to the F3 file of the ProTaperNext system under 2.5 % NaOCL irrigation. The samples were divided into three groups according to the type of used photosensitizer (PS) (n = 15); MB (313 μM), TB (327 μM), and TM-ZnPc (6μM). All PSs were irradiation with a light-emitting diode (LED) lamp (630 nm, 2-4 mW/cm²) for the 60 s. Two microbiological samples of the intracanal content were taken (one before and one immediately after additional aPDT in all groups) using sterile paper points. The colony-forming units per milliliter (CFU/mL) were calculated after 24 h of incubation at 37 °C., Results: After all aPDT protocols, intracanal bacterial load decreased significantly compared to the amount after chemomechanical preparation (P < 0.05). No significant difference in the reduction in intracanal bacterial load was found between the PSs (P < 0.05)., Conclusions: In the current study, the aPDT protocol performed with TM-ZnPc provided similar antimicrobial efficacy, although it was used at a lower concentration compared to MB and TB. Therefore, the use of TM-ZnPc in intra-canal disinfection in endodontics seems promising., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aim: This study evaluated three concentrations of butyl toluidine blue (BuTB) for antimicrobial photodynamic therapy (aPDT) in experimental periodontitis (EP) in rats., Material and Methods: EP was ligature-induced at the first mandibular molar in 105 rats. Ligature was removed after 7 days and animals were distributed into the following treatments: SRP, scaling and root planing (SRP) plus saline solution; BuTB-0.1, SRP plus BuTB at 0.1 mg/mL; aPDT-0.1, SRP plus BuTB at 0.1 mg/mL and InGaAlP diode laser (DL) irradiation; BuTB-0.5, SRP plus BuTB at 0.5 mg/mL; aPDT-0.5, SRP plus BuTB at 0.5 mg/mL and DL irradiation; BuTB-2.0, SRP plus BuTB at 2 mg/mL; aPDT-2.0, SRP plus BuTB at 2 mg/mL and DL irradiation. Five animals from each group were submitted to euthanasia at 7, 15 and 30 days post-treatment. The furcation area was submitted to histological, histometric and immunohistochemical (TGF-ß1, OCN and TRAP) analyses., Results: aPDT-0.5 group presented a better tissue remodeling in all periods, resolution of the inflammatory response and bone neoformation areas at 30 days. aPDT-0.5 also resulted in higher immunolabeling patterns of TGF-ß1 at all periods (p < 0.05) and of OCN at 30 days (p < 0.05)., Conclusion: aPDT-0.5 showed the best benefits for inflammatory response and periodontal repair process., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Chagas disease is endemic in Latin America and increasingly found in non-endemic countries. Its treatment is limited due to the variable efficacy and several side effects of benznidazole. Photodynamic antimicrobial chemotherapy (PACT) may be an attractive approach for treating Chagas disease. Here, the trypanocidal activity of PACT was investigated in vitro using phenothiazine derivatives. The cytotoxicity of both, methylene blue (MB) and toluidine blue (TBO), was determined on macrophages cultures using AlamarBlue method. The trypanocidal activity of the two photosensitizers was initially evaluated by determining their IC 50 values against trypomastigote forms. After this, the trypanocidal effect was evaluated in cultures of infected macrophages using an automatized image analysis protocol. All experiments were performed in the dark and in the clear phase (after a photodynamic exposure). The compounds showed no cytotoxicity in both phases at the tested concentrations. The IC 50 values for the sole use of MB and TBO were 2.6 and 1.2 μM, respectively. The photoactivation of the compounds using a fixed energy density (J/cm 2 ) caused a reduction of the IC 50 values to 1.0 and 0.9 μM, respectively. It was found that, on infected macrophage, the use of TBO significantly reduced the number of infected cells and parasitic load, and this effect was increased in the presence of light. The results of the present study are indicative that PACT may be considered as both selective and effective therapeutic intervention for treating Chagas disease.

Antibiotics and photodynamic therapy (PDT) are widely employed in curing acne. However, antibiotics as an effective treatment would lead to bacterial resistance and severe side effects. In this study, we aimed to develop a novel TBO hydrogel, which could prolong the retention time of photosensitizer (TBO) at the lesion site and improve therapeutic effect. In vitro antibacterial experiments (against Staphylococcus aureus and Escherichia coli), the response surface methodology was used to optimize the formulation of TBO hydrogel. The results indicated that the optimal formulation was 0.5% (v/v) carbomer, 0.01 mg/mL TBO, 0.5% (v/v) ethanol concentration, 0.5% (v/v) Tween 80, the mass ratio of NaOH to carbomer of 0.4 (w/w). The TBO hydrogel formulation showed the strong antibacterial activity for Propionibacterium acnes. The stability, pH, and antibacterial activity of TBO hydrogel did not significantly change under 4 °C, 25 °C, and 40 °C during 6-week storage. Furthermore, TBO combined with carbomer hydrogel showed the 51.28% (4 h) and 69.80% (24 h) release. In summary, the hydrogel TBO might be a vital therapeutic strategy to promote the PDT applied in the topical therapy of acne. Graphical abstract A TBO hydrogel for photodynamic therapy in the treatment of acne.

Background: The emergence of drug-resistant bacterial strains has raised the need to develop alternative treatment modalities to combat infectious diseases. Antimicrobial photodynamic therapy (aPDT) is an alternative to conventional treatment modalities. aPDT integrates a photosensitizer, which, after exposure to light of an appropriate wavelength, leads to the generation of cytotoxic reactive oxygen species (ROS)., Methods: The aim of the present study was to synthesize a toluidine blue/multiwalled carbon nanotube conjugate (TBCNT) for enhanced photoinactivation of Pseudomonas aeruginosa and Staphylococcus aureus. Synthesized TBCNT conjugate was characterized and its antibacterial and antibiofilm activity was determined., Results: During TBCNT synthesis, dye loading, and entrapment efficiency of the CNT were 12.04 ± 0.55% and 48.99 ± 2.33%, respectively. The photo-destruction of planktonic cells of the test bacteria was performed by exposure to a 125 mW red laser with a wavelength of 670 nm (radiant exposure of 58.49 J/cm 2 ) for 3 min. Photoinactivation using TBCNT resulted in a 4.91- and 5.47-log10 reduction in P. aeruginosa and S. aureus, respectively. The mechanism of this aPDT was studied by measuring intracellular ROS generation, protein leakage, and lipid peroxidation in the test bacteria after light irradiation. The antibiofilm activity of TBCNT after light exposure was 69.94% and 75.54% for P. aeruginosa and S. aureus, respectively. Photoinactivation of test bacteria treated with TBCNT reduced cell viability and exopolysaccharide production. Confocal laser-scanning microscopy revealed a significant biofilm inhibition efficacy of the TBCNT conjugate., Conclusion: Therefore, TBCNT conjugates may be used for the eradication of P. aeruginosa and S. aureus biofilms., (Copyright © 2019 Elsevier B.V. All rights reserved.)