Phenothiazine Photosensitizers Research Articles

Comparative effects of different phenothiazine photosensitizers on experimental periodontitis treatment.

The aim of the present study was to compare the effects of the phenothiazine photosensitizers methylene blue (MB), toluidine blue-O (TBO) and butyl toluidine blue (BuTB) in antimicrobial photodynamic therapy (aPDT), as adjuvant therapy to scaling and root planing (SRP) in the treatment of experimental periodontitis (EP) in rats. 120 Wistar rats underwent ligation around the lower left molar. After seven days, the ligature was removed. The animals were separated into the following groups (n = 15): EP, no treatment; SRP, SRP and irrigation with saline solution; MB, SRP and deposition of MB; TBO, SRP and deposition of TBO; BuTB, SRP and deposition of BuTB; MB-aPDT, SRP and aPDT with MB; TBO-aPDT, SRP and aPDT with TBO and; BuTB-aPDT, SRP and aPDT with BuTB. The aPDT session was performed after SRP, with deposition of the photosensitizer and irradiation with a diode laser (DL; InGaAlP, 660 nm, 40 mW, 60 s, 2.4 J). Histological and histometric analysis was performed. BuTB-aPDT group had a lesser extent of the inflammatory process compared to the EP, SRP, MB and TBO at all experimental periods (p < 0.05). At 15 days, the aPDT treated groups had a greater bone tissue structure than groups EP and SRP (p < 0.05) The BuTB showed lower Alveolar Bone Loss (ABL) compared to the TBO-aPDT group at 30 days (p < 0.05). aPDT using the photosensitizer BuTB proved to be the adjuvant therapy that most favored the reduction of inflammatory infiltrate in the furcation area and ABL.

Evaluation of physicochemical properties and bacterial photoinactivation of phenothiazine photosensitizers.

We report herein the physicochemical properties and antimicrobial activity of a new monobrominated derivative of Azure B and its parent compound. These dyes are used as photosensitizers for photodynamic therapy and photodynamic antimicrobial chemotherapy. Relevant pharmaceutical properties (pKa, chemical and photochemical stability, and in vitro antimicrobial activity) were determined. A UV-visible spectrophotometry method was developed and validated according to the International Conference on Harmonization (ICH) guidelines for use in stability indicating studies and determination of the acid dissociation constant of Azure B and its monobrominated derivative. The results showed that both dyes were chemically stable. In addition, bromination of the phenothiazine dye decreased its photochemical stability and pKa value without affecting the ionization rate at physiological pH. The analytical parameters for validation of the method were linearity (r2 > 0.9981), limit of detection (LOD) (0.2-0.9 μM), limit of quantification (LOQ) (0.6-2.7 μM), and intra-day precision (0.76-1.40%) expressed as relative standard deviation (RSD). Recoveries ranging from 99.5 to 100.9% were obtained for the two dyes. Thus, this method provides a simple, sensitive, accurate, and precise assay for the determination of all compounds. The effect of photosensitizer concentration and visible irradiation time on lethal photosensitization against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli was investigated. Both photosensitizers were active against the evaluated bacteria. However, the new monobrominated derivative was more effective than its predecessor and managed to eradicate these microorganisms by using different doses of the dye and light. In other words, a lower concentration of AzBBr and irradiation time were required to cause bacterial death equal to or greater than its precursor. The photodynamic efficacy of the two photosensitizers presented the following order: S. aureus > E. coli > P. aeruginosa. These studies indicated that the tested dyes satisfy the conditions of potential photosensitizers in terms of physicochemical and antimicrobial properties.

Phospholipid multilamellar vesicles entrapping phenothiazine photosensitizers. Preparation, characterization and evaluation of their photodynamic properties

The aim of the present paper was to optimize the properties of phenothiazine photosensitizers (PSs), Azure B and its monobrominated derivative. They were entrapped in multilamellar liposomes of egg phosphatidylcholine (EPC) and dipalmitoylphosphatidylcholine (dpPC). The higher partition coefficient of both PSs in the EPC/water system compared with that of both PSs in the dpPC/water system allowed the selection of EPC to be used for the development of third generation photosensitizers. The optimal phospholipid/phenothiazine ratio and the location of these dyes in the lipid bilayer systems were established. These dyes were found to be located within the polar head group region of the lipid bilayer, which would enhance the effectiveness of the irradiation procedure. In addition, the encapsulation of both photosensitizers in liposomes led to a decrease in the aggregation of these compounds, and consequently to an increase in their singlet oxygen quantum yield (ϕΔ). The encapsulation of the PSs in lipid vesicles significantly increased their photochemical reactivity, doubling the ϕΔ value of AzBBr and increasing that of AzB by 60%. The results obtained demonstrate that the vehiculization of these PSs in liposomes allowed the development of third generation photosensitizers with better photochemical properties to be used in potential therapeutic applications.

Enhancing integration of articular cartilage grafts via photochemical bonding.

The integration of osteochondral grafts to native articular cartilage is critical as the lack of graft integration may lead to continued tissue degradation, poor load transfer and inadequate nutrient transport. Photochemical bonding promotes graft integration by activating a photosensitizer at the interface via a light source and avoids negative effects associated with other bonding techniques. We hypothesized that the bond strength depends on photosensitizer type and concentration in addition to light exposure. Photochemical bonding was evaluated using methylene blue (MB), a cationic phenothiazine photosensitizer, and two phthalocyanine photosensitizers, Al(III) phthalocyanine chloride tetrasulfonic acid (CASPc) and aluminum phthalocyanine chloride (AlPc). Exposure was altered by varying irradiation time for a fixed irradiance or by varying irradiance with a fixed irradiation time. MB was ineffective at producing bonding at the range of concentrations tested while CASPc produced a peak twofold bond strength increase over controls. AlPc produced substantial bonding at all concentrations with a peak 3.9-fold bond strength increase over controls. Parametric tests revealed that bond strength depended primarily on the total energy delivered to the bonding site rather than the rate of light delivery or light irradiance. Bond strength persisted for 1 week of in-vitro culture, which warrants further exploration for clinical applications. These studies indicate that photochemical bonding is a viable strategy for enhancing articular cartilage graft integration. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2406-2415, 2018.

APDT for periodontitis treatment in ovariectomized rats under systemic nicotine.

This study evaluated the antimicrobial photodynamic therapy (aPDT) as an adjunctive therapy to scaling and root planning (SRP) for the treatment of experimental periodontitis (EP) in ovariectomized rats under systemic nicotine. Female ovariectomized rats (n = 180) were divided into two groups: vehicle administration (Veh) and nicotine administration (Nic). Mini-pumps containing either vehicle or nicotine were inserted in the rats 30 days before the induction of EP, which was induced by placing a ligature around the left mandibular first molar. The rats were randomly divided into three treatment subgroups: SRP, SRP plus low-level laser therapy (LLLT), and SRP plus aPDT. aPDT consisted of the application of a phenothiazine photosensitizer followed by LLLT. Ten animals from each group were euthanized at days 7, 15, and 30 after periodontal treatment. The furcation region was evaluated using histological, histometric analyses and immunolabelling for PCNA, TRAP, RANKL, and OPG. Nicotine administration resulted in greater bone loss (BL). aPDT resulted in lower BL compared to SRP. aPDT showed higher quantities of PCNA-positive cells compared to SRP, regardless of the nicotine status. aPDT resulted in less recruitment of osteoclasts and lower RANKL immunolabelling compared to LLLT and SRP. aPDT was effective in animals treated with nicotine.

Effect of surgical periodontal treatment associated to antimicrobial photodynamic therapy on chronic periodontitis: A randomized controlled clinical trial.

This randomized controlled clinical trial evaluated the effects of an adjunctive single application of antimicrobial photodynamic therapy (aPDT) in Surgical Periodontal Treatment (ST) in patients with severe chronic periodontitis (SCP). In a split-mouth design, 20 patients with SCP were treated with aPDT+ST (Test Group, TG) or ST only (Control Group, CG). aPDT was applied in a single episode, using a diode laser and a phenothiazine photosensitizer. All patients were monitored until 90days after surgical therapy. Levels of 40 subgingival species were measured by checkerboard DNA-DNA hybridization at baseline, 60 and 150days. Clinical and microbiological parameters were evaluated. In deep periodontal pockets depth (PPD ≥5mm), Test Group presented a significantly higher decrease in PPD than Control Group at 90days after surgical therapy (p<.05). Test Group also demonstrated significantly less periodontal pathogens of red complex (Treponema denticola) (p<.05). A single episode of aPDT used in adjunct to open flap debridement of the root surface in the surgical treatment of SCP: i) significantly improved clinical periodontal parameters; ii) eliminates periodontal pathogens of the red complex more effectively (NCT02734784).

Antimicrobial photodynamic therapy minimizes the deleterious effect of nicotine in female rats with induced periodontitis

The aim of this study was to compare the use of antimicrobial photodynamic therapy (aPDT) as an adjunct to scaling and root planing (SRP) in the treatment of experimentally induced periodontitis in female rats that were systemically treated with or without nicotine. Female rats (n = 180) were divided into two groups: vehicle administration (Veh) and nicotine administration (Nic). Mini-pumps containing either vehicle or nicotine were implanted in the rats 30days before the induction of experimental periodontitis (EP). EP was induced by placing a cotton ligature around the left mandibular first molar. After 7days, the ligature was removed, and the rats were randomly divided into three treatment subgroups: SRP (only SRP), DL (SRP plus diode laser), and aPDT (SRP plus aPDT). The aPDT consisted of phenothiazine photosensitizer deposition followed by diode laser irradiation. Ten rats from each subgroup were euthanized at 7, 15, and 30days after treatment. Alveolar bone loss (ABL) in the furcation region was evaluated using histological, histometric, and immunohistochemical analyses. The rats that were treated with nicotine showed more ABL compared to those treated with vehicle. In both the Veh and Nic groups, SRP plus aPDT treatment resulted in reduced ABL, smaller numbers of both TRAP- and RANKL-positive cells, and higher numbers of PCNA-positive cells compared to SRP treatment alone. aPDT was an effective adjunctive therapy for the treatment of periodontitis in female rats regardless of whether they received nicotine.

Antimicrobial photodynamic therapy associated to nonsurgical periodontal treatment in smokers: Microbiological results

BackgroundStudies suggest that smokers present more or greater numbers of potential periodontal pathogens than non-smokers. The aim of this study was to investigate the microbiologic effects of adjunctive aPDT on nonsurgical periodontal treatment in smokers with chronic periodontitis (CP). MethodsTwenty smokers with CP had two contralateral teeth randomly assigned in a split-mouth design to receive SRP (CG) or SRP+a single episode of aPDT (TG), with a diode laser and a phenothiazine photosensitizer. Levels of 40 subgingival species were measured using checkerboard DNA–DNA hybridization at baseline and 1, 4 and 12weeks after periodontal treatment. ResultsAll 40 bacterial species evaluated were detected in different levels at baseline, with no statistic significant differences between groups. After periodontal treatment, the levels of some bacterial species decreased, while some other species presented an increase. Despite this variation, the statistical analysis was not able to identify significant differences neither at intragroup nor at intergroup comparisons. ConclusionPeriodontal treatment with SRP or SRP+aPDT was not able to reduce levels of 40 subgingival species in smokers with CP. These outcomes indicate that smoking impairs periodontal healing after nonsurgical treatment even associated with aPDT.

Effect of the concentration of phenothiazine photosensitizers in antimicrobial photodynamic therapy on bone loss and the immune inflammatory response of induced periodontitis in rats

Antimicrobial therapy can suppress periodontal pathogens and increase the effectiveness of conventional mechanical treatment. The aim of this study was to assess bone loss and the immune inflammatory response of rats under the influence of two photosensitizing agents (MB and TBO) at two different concentrations in antimicrobial photodynamic therapy (aPDT), used as an adjuvant therapy in the treatment of periodontitis. Periodontitis was induced in the mandibular first molars of 162 rats. The animals were divided into nine groups: G1 - scaling and root planing (SRP); G2 - SRP plus 100μg/mL of methylene blue (MB); G3 - SRP plus 10mg/mL of MB; G4 - SRP plus 100μg/mL of toluidine blue (TBO); G5 - SRP plus 10mg/mL of TBO; G6 - SRP plus 100μg/mL of MB and laser; G7 - SRP plus 10mg/mL of MB and laser; G8 - SRP plus 100μg/mL of TBO and laser; and G9 - SRP plus 10mg/mL of TBO and laser. Six animals from each group were euthanized 7, 15, or 30d after treatment. Bone loss (BL) in the furcation region was evaluated using histomorphometric and immunohistochemical analyses to detect the receptor activator of nuclear factor-Κappa B ligand (RANKL), osteoprotegerin (OPG) and tartrate-resistant acid phosphatase (TRAP). There was significantly less BL in animals treated with aPDT using low concentrations of MB and TBO at 7, 15 and 30d. Immunohistochemical analysis revealed decreased RANKL and increased OPG in the aPDT groups and decreased TRAP-positive cells in G6 and G8. aPDT, using low concentrations of MB and TBO, was the most effective adjuvant therapy to SRP, acting indirectly as a downregulator of the molecular mechanisms that control bone resorption in periodontitis.

Adjunctive effect of antimicrobial photodynamic therapy to nonsurgical periodontal treatment in smokers: a randomized clinical trial

The aim of this study was to investigate the adjunctive effect of antimicrobial photodynamic therapy (aPDT) to scaling and root planing (SRP) in smokers with chronic periodontitis. Twenty subjects had two contralateral teeth randomly assigned to receive SRP (SRP group) or SRP + a single episode of aPDT (SRP + aPDT group), with a diode laser and a phenothiazine photosensitizer. Plaque index, bleeding on probing, probing depth (PD), clinical attachment level (CAL), and gingival recession were recorded, and gingival crevicular fluid was collected for assay of IL-1β and matrix metalloproteinase (MMP)-8 levels. There was a significant PD reduction (SRP 1.81 ± 0.52mm/SRP + aPDT 1.58 ± 1.28mm; p < 0.001) and a significant CAL gain (SRP 1.60 ± 0.92mm/SRP + aPDT 1.41 ± 1.58mm; p < 0.001) for both groups. Significant differences were not observed in between-group comparisons. IL-1β level in gingival crevicular fluid was higher in SRP group after 1week (SRP 24.65 ± 18.85pg/μL/SRP + aPDT 34.07 ± 24.81pg/μL; p = 0.048), and MMP-8 level was higher in SRP group after 12weeks (SRP 303.31 ± 331.62pg/μL/SRP + aPDT 534.23 ± 647.37pg/μL; p = 0.024). There were no statistically significant differences in intragroup comparisons. The adjunctive effect of aPDT did not warrant improvements on clinical parameters in smokers. However, it resulted in a suppression of IL-1β and MMP-8 when compared with SRP alone.

Nanoparticle Platform to Modulate Reaction Mechanism of Phenothiazine Photosensitizers

Herein, we report on the synthesis of photosensitizing nanoparticles in which the generation of different oxidizing species, i.e., singlet oxygen (1O2) or radicals, was modulated. Sol gel and surface chemistry were used to obtain nanoparticles with specific ratios of dimer to monomer species of phenothiazine photosensitizers (PSs). Due to competition between the reactions involving electron transfer within dimer species and energy transfer from monomer triplets to oxygen, the efficiency of 1O2 generation could be controlled. Nanoparticles with an excess of dimer have an 1O2 generation efficiency (S(delta)) of 0.01 while those without dimer have a S(delta) value of 0.4. Furthermore, we demonstrate that the PS properties of the nanoparticles are not subjected to interference from the external medium as is commonly the case for free PSs, i.e., PS ground and triplet states are not reduced by NADH and ascorbate, respectively, and singlet excited states are less suppressed by bromide. The modulated 1O2 generation and the PS protection from external interferences make this nanoparticle platform a promising tool to aid in performing mechanistic studies in biological systems. Also, it offers potential application in technological areas in which photo-induced processes take place.

Optimization of topical photodynamic therapy with 3,7-bis(di-n-butylamino)phenothiazin-5-ium bromide for cutaneous leishmaniasis.

Photodynamic therapy (PDT) has evolved as a promising therapeutic measure for the treatment of cutaneous leishmaniasis (CL). In particular, phenothiazine compounds have demonstrated efficacy for PDT of CL. The objective of our present study is to define the use of a new specific phenothiazine photosensitizer, 3,7-bis(di-n-butylamino)phenothiazin-5-ium bromide (PPA904) applied topically as a cream to treat CL. To establish the optimal conditions for this treatment, we compared two different ways to improve current regimens of PDT with PPA904 cream (500 microM of PPA904 in Unguentum M) by changing the duration of topical application, and by administration of several consecutive PDT procedures. An initial regimen recommended by the manufacturer (Photopharmica Co. Ltd., Leeds, UK) was maintained as a control: the cream was applied topically for 30 minutes at a final concentration of PPA904 at 500 microM, and the designated treatment area was irradiated with a broad band light source of 665+/-15 nm at a fluence of 50 J/cm(2) (50 mW/cm(2)). The best curative PPA904-PDT regimen was achieved under the conditions of a longer duration of topical application time (90 minutes) and several (three) consecutive treatments with 4-day intervals between treatments. The mechanisms responsible for such improvements (kinetics of drug penetration, depth of necrosis of the CL lesions after PDT, and daily changes in the parasitic load after PDT) are discussed in the present study. Topical PPA904-PDT, implemented as described above, is a promising treatment for CL, and clinical studies will be initiated to establish efficacy in humans.

Real-time fluorescence monitoring of phenothiazinium photosensitizers and their anti-mycobacterial photodynamic activity against Mycobacterium bovis BCG in in vitro and in vivo models of localized infection

An objective was to explore the photodynamic activity of two cationic photosensitizers (PS) (benzo[a]phenothiazinium chloride and benzo[a]phenoselenazinium chloride) against Mycobacterium bovis BCG both in vitro and in a murine model of BCG-granuloma. The hypothesis being tested in this study was that cationic molecules could best interact with the negatively charged membrane of BCG as a model for mycobacterial infection. Cells in culture were incubated with various concentrations of PS and subsequently illuminated using a 635 nm diode laser. Dark- and light-induced killing profiles were generated as a function of fluence and dye concentration. In vivo, local injection of the PS into subcutaneous Mycobacterium-induced granuloma sites in murine model was followed by red light illumination of the same area. A special microscope was fabricated for real-time in vivo fluorescent microscopy to monitor EtNBS delivery to subcutaneous murine granulomata. Both PS demonstrated good in vitro antimycobacterial photodynamic activity with varying degrees of toxicity under dark conditions. Real time in vivo monitoring of benzophenothiazine chloride in the mouse model indicated that this fluorescent photosensitizer was delivered rapidly to the subcutaneous granuloma site. In vivo, photosensitizer specific dark- and photo-toxicities depended on the structure, concentration of the photosensitizer and the light dose utilized. Cationic phenothiazine photosensitizers are promising candidates for use in anti-mycobacterial PDT for localized diseases such as cutaneous and pulmonary granulomata.

Antimicrobial Photodynamic Therapy in the Non‐Surgical Treatment of Aggressive Periodontitis: A Preliminary Randomized Controlled Clinical Study

The treatment of aggressive periodontitis has always presented a challenge for clinicians, but there are no established protocols and guidelines for the efficient control of the disease. Ten patients with a clinical diagnosis of aggressive periodontitis were treated in a split-mouth design study to either photodynamic therapy (PDT) using a laser source with a wavelength of 690 nm associated with a phenothiazine photosensitizer or scaling and root planing (SRP) with hand instruments. Clinical assessment of plaque index (PI), gingival index (GI), bleeding on probing (BOP), probing depth (PD), gingival recession (GR), and relative clinical attachment level (RCAL) were made at baseline and 3 months after treatment with an automated periodontal probe. Initially, the PI was 1.0 +/- 0.5 in both groups. At the 3-month evaluation, the plaque scores were reduced and remained low throughout the study. A significant reduction of GI and BOP occurred in both groups after 3 months (P <0.05). The mean PD decreased in the PDT group from 4.92 +/- 1.61 mm at baseline to 3.49 +/- 0.98 mm after 3 months (P <0.05) and in SRP group from 4.92 +/- 1.14 mm at baseline to 3.98 +/- 1.76 mm after 3 months (P <0.05). The mean RCAL decreased in the PDT group from 9.93 +/- 2.10 mm at baseline to 8.74 +/- 2.12 mm after 3 months (P <0.05), and in the SRP group, from 10.53 +/- 2.30 mm at baseline to 9.01 +/- 3.05 mm after 3 months. PDT and SRP showed similar clinical results in the non-surgical treatment of aggressive periodontitis.

An Overview of Synthetic Approaches to Porphyrin, Phthalocyanine, and Phenothiazine Photosensitizers for Photodynamic Therapy

The mechanistic basis of photodynamic therapy is reviewed briefly, and the factors that are important in improving photosensitizers are examined. The methods for preparing the three main classes of PDT photosensitizers, namely, porphyrins, phthalocyanines, and phenothiazines, are reviewed.

Monomeric and Polymeric Phenothiazine Photosensitizers for Photoinitiated Cationic Polymerization

The syntheses of several phenothiazine compounds bearing cationically polymerizable vinyl, vinyl ether and epoxide groups are described. These monomers were rapidly and efficiently polymerized or copolymerized by UV irradiation in the presence of onium salt photoinitiators. In addition, the monomers simultaneously function as efficient photosensitizers for the photolysis of the onium salts. In this article, the use of these polymerizable photosensitizers for the photopolymerizations of epoxide and vinyl ether monomers is reported. The inclusion of a small amount of the monomeric photosensitizer is effective in markedly accelerating the cationic photopolymerization of the bulk monomer. Polymers prepared by the polymerization of the monomeric phenothiazine photosensitizers also exhibit excellent photosensitization activity when used in combination with diaryliodonium, triarylsulfonium, and dialkylphenacylsulfonium salt photoinitiators.

Virus inactivation in blood components by photoactive phenothiazine dyes

The virucidal properties of photoactive phenothiazine dyes, such as methylene blue, have been known approximately 70 years. The mechanism of virus inactivation involves binding of the dye to nucleic acid, absorption of light, generation of reactive oxygen species, guanine oxidation in the viral genome. The first practical research involving phenothiazine photosensitizers in transfusion medicine was sparked by efforts to prevent transmission of hepatitis from pooled plasma administered during the Korean War. During the last decade, methylene blue was used by some European countries for virus photoinactivation in fresh frozen plasma, whereas other phenothiazine derivatives with greater affinities for nucleic acids and capabilities to inactivate intracellular virus have been investigated for photochemical decontamination of red cell suspensions. The toxicity of methylene blue is well characterized; the drug has been used for many years to treat methemoglobinemia using concentrations orders of magnitude greater than those used for virus photoinactivation, and several filters have been developed to remove the dye from plasma. Future widespread use of phenothiazines for virus photoinactivation in blood components may depend on whether the risk inherent in their use is less than the residual risk from transfusion-transmitted viruses.

Phenothiazine photosensitizers for onium salt photoinitiated cationic polymerization

AbstractPhenothiazine compounds bearing a wide range of different substituents are excellent photosensitizers for onium salt cationic photoinitiators. These photosensitizers are generally operative in the mid‐ and long‐range regions of the UV spectrum and are especially useful for enhancing the rate of photoinitiated cationic polymerization carried out utilizing both filtered and broadband UV emission sources. In this article, the syntheses of several different substituted phenothiazines are described and the ability of these compounds to photosensitize the photolysis of different onium salt photoinitiators is evaluated. Attempts were made to correlate the structure and spectral characteristics of the phenothiazines with their efficiency of photosensitization in the cationic photopolymerizations of several typical epoxide and vinyl ether monomers. © 2001 John Wiley &amp; Sons, Inc. J Polym Sci A: Polym Chem 39: 1187–1197, 2001

Phenothiazine Photosensitizers. III. Activity of Methylene Blue Derivatives against Pigmented Melanoma Cell Lines

The cytotoxicity and photocytotoxicity of methylene blue and several of its derivatives against two pigmented melanoma cell lines (SK-23 murine melanoma and SK-Mel 28 human melanoma) were investigated in culture. The derivatives were all more effective photosensitizers than methylene blue in both cell lines over a range of light doses (3.6-17.6 J cm−2). The increased activity correlated with increased cellular uptake and inherent photosensitizing efficacy. The photosensitizers also showed varying levels of interaction with the biopoly-mer melanin and although this appeared to affect uptake and activity, there was no direct correlation with toxicity.