Oxygen (and lack thereof) in photodynamic therapy

Photodynamic therapy is based on the photosensitized oxygenation of target cellular molecules, which consumes oxygen. Oxygen is therefore and essential component of the therapy and over the years, several efforts have been made either to ensure a sufficient oxygen supply during the illumination phase of the therapy, or, more recently, by developing photosensiting agents and strategies capable of performing the photodynamic effect under hypoxic conditions.Using examples from our laboratory and from the literature, this presentation will try to pinpoint the problems detracting from the efficiency of photodynamic treatments under hypoxic conditions and review some of the solutions proposed.Acknowledgments: This research was funded by the Agencia Estatal de Investigación and FEDER “Una manera de hacer Europa” (PID2020-115801RB-C22/MICIN/AEI/10.13039/501100011033).     

In vitro evaluation of ruthenium complexes for photodynamic therapy

BackgroundPhotodynamic therapy (PDT) is a promising anti-tumor treatment strategy. Photosensitizer is one of the most important components of PDT. In this work, the anticancer activities of PDT mediated by six new ruthenium porphyrin complexes were screened. The mechanisms of the most efficacious candidate were investigated.MethodsPhotocytotoxicity of the six porphyrins was tested. The most promising complex, Rup-03, was further investigated using Geimsa staining, which indirectly detects reactive oxygen species (ROS) and subcellular localization. Mitochondrial membrane potential (MMP), cell apoptosis, DNA fragmentation, c-Myc gene expression, and telomerase activities were also assayed.ResultsRup-03 and Rup-04 had the lowest IC₅₀ values. Rup-03 had an IC₅₀ value of 29.5 ± 2.3 μM in HepG2 cells and 59.0 ± 6.1 μM in RAW264.7 cells, while Rup-04 had an IC₅₀ value of 40.0 ± 3.8 μM in SGC-7901 cells. The complexes also induced cellular morphological changes and impaired cellular ability to scavenge ROS, and accumulated preferentially in mitochondria and endoplasmic reticulum. Rup-03 reduced MMP levels, induced apoptosis, and repressed both c-Myc mRNA expression and telomerase activity in HepG2 cells.ConclusionsAmong six candidates, Rup-03-mediated PDT is most effective against HepG2 and RAW264.7, with a similar efficacy as that of Rup-04-mediated PDT against SGC-7901 cells. Repression of ROS scavenging activities and c-Myc expression, which mediated DNA damage-induced cell apoptosis and repression of telomerase activity, respectively, were found to be involved in the anticancer mechanisms of Rup-03.     

卟吩姆钠漂白特性用于大鼠体外MLL细胞光动力学疗法治疗的单线态氧剂量估算

背景与目的 光动力学治疗作用是靠单线态氧的生成介导。作者将基于动力学的单线态氧剂量模型用于光敏剂卟吩姆钠（portimer sodium，Photofrin）。Photofrin的漂白机制不仅有单线态氧介导的漂白，还有不依赖氧的漂白机制，作者试图将后一特点纳入Photofrin的单线态氧剂量模型中。     

Oxygen consumption and ventilation during constant-load exercise in runners and cyclists

The effect of using specialized or no specialized muscle groups on ventilatory threshold (VT) and on maintenance of steady-state oxygen consumption during long term exercise on treadmill and on cycle ergometer was studied in ten endurance runners and nine cyclists. Initially, maximum oxygen consumption (VO2max) and VT were determined. Oxygen consumption (VO2) ventilation (VE) respiratory rate (f), and blood lactate (in the beginning and at the end of exercise) were measured during exercise at constant relative loads of 60, 70, and 80% of VO2max. In the runners, both VO2max and VT (expressed as % VO2max and VO2 l/min) were greater on treadmill than on cycle ergometer and in the cyclists, VO2max was higher on cycle ergometer than on treadmill, but no differences in VT were detected between the two tests. The VO2, VE, and f drifts correlated with blood lactate level as well as with the calculated VT. The results suggest that the effect of using specialized or no specialized muscle groups on the maintenance of VO2 steadiness is achieved through training-induced changes on the level of blood lactate and, in VT.     

In vitro effects of photodynamic therapy induced by chloroaluminum phthalocyanine nanoemulsion

BackgroundThe photodynamic therapy (PDT) has been used to treat cancer mainly by inducing oxidative stress. Our aim was to evaluate the effect of PDT and its combination with methoxyamine (MX), a blocker of base excision repair (BER), in cells expressing high levels of the APE1 protein, which is involved in cell oxidative damage response.MethodsThe HeLa and A549 cells were treated for 3 h with chloroaluminum phthalocyanine incorporated into a well-designed nanoemulsion (ClAlPc/NE); and then irradiated by visible light (@670 nm) with doses of 0.1, 0.5 and 1.0 J/cm². A simultaneous combination of MX + ClAlPc/NE was performed and then irradiated with the selected dose of 0.5 J/cm². The treatments were evaluated in terms of viability, clonogenicity, DNA fragmentation, and cell death mechanism by apoptosis and/or necrosis.ResultsThe APE1 protein expression observed was higher in HeLa than in A549. Both cell lines exhibited substantial differences in cell cytotoxicity. The PDT decreased the clonogenicity of HeLa by inducing apoptosis (sub-G1 and annexin detection). Additionaly, the MX potentiates the PDT-effects in HeLa. Otherwise, low cytotoxicity was observed in A549 cells.ConclusionThe PDT induced apoptosis in high APE1 expressive HeLa cells, and the blockage of BER by MX increased its effects.     

Bioluminescence-activated photodynamic therapy for luciferase transfected,grade 4 astrocytoma cells in vitro

Background. Grade 4 astrocytoma is incurable due to the diffusely infiltrative nature of the disease. Photodynamic therapy (PDT) is a promising therapeutic option, but external light delivery is not feasible when cancer cells infiltrate unknown areas of normal brain. Hence the search for endogenous sources such as bioluminescence that can generate light at cancer cells. This requires a substrate (a luciferin) and an enabling enzyme (a luciferase), neither seen in mammalian cells.Methods. Preliminary studies confirmed that U87 cells (derived from a human grade 4 astrocytoma) could be killed by conventional PDT using the photosensitizers hypericin or mTHPC. U87 cells were then transfected with firefly and other luciferases and light generating cell lines (U87-luc, U87-hRluc, U87-CBG68luc) identified using the appropriate substrate. Reagent doses and conditions were optimized and U87-luc cells incubated with hypericin or mTHPC with d-luciferin added to initiate bioluminescence activated PDT (bPDT). Cell survival was assessed by MTT assay, haemocytometry and growth assay. Control groups included U87-luc cells with no added active reagents, substrate only, photosensitizer only and non-transfected U87 cells. Results were expressed as a percentage of surviving cells compared with untreated U87-luc controls.Results. There was no bPDT effect on non-transfected cells. The mean survival of treated transfected cells was 36%, (P<0.001) using hypericin and 35% (P<0.001) using mTHPC, compared with untreated U87-luc cells. bPDT effects were suppressed by the anti-oxidant, lycopene.Conclusions. bPDT can kill Grade 4 astrocytoma cells transfected with luciferase in vitro. This justifies progression to in vivo studies.     

In vivo and in vitro models for photodynamic therapy by using novel photosensitizers

Photodynamic therapy (PDT) is an effective treatment for both malignant and non-malignant diseases, and new photosensitizers / chromophores are studied by confocal imaging and biological techniques determining cell survival with/without light. During PDT the activated PS transfers energy to nearby oxygen molecules, generating singlet oxygen (¹O₂) resulting in oxidative stress (ROS), which further elicit cell death by necrosis and apoptosis. Protoporphyrin IX (PpIX), is an efficient and widely used PS for bladder superficial bladder cancer treatment; either endogenously produced in the cancer cells by e. g. aminolaevulinic acid (ALA) or exogenously added as e. g. hexyl-ALA. The effects in vitro and in vivo are present by using an orthotopic rat cancer model; also included pphotochemical internalization (PCI). This is a new strategy for local enhancement of various types of drug molecules by employing a photosensitising compound and illumination of a diseased area in the body. The possibility of using PCI to enhance effects of the cytotoxic drug bleomycin is investigated, together with photophysical determinations and outlines of a treatment for intravesical therapy of bladder cancer. In vitro experiments indicate that employment of PCI technology using the novel photosensitizer TPCS_2a® enhance cytotoxic effects of bleomycin in bladder cancer cells. Furthermore, experiments in an orthotopic in vivo bladder cancer model show effective reduction in both necrotic area and bladder weight after TPCS_2a based photodynamic therapy (PDT). The tumor selectivity and PDT effects may be sufficient to destroy tumors without damaging detrusor muscle layers. Our results present a possible new treatment strategy for non-muscle invasive bladder cancer, with intravesical instillation of photosensitizer and bleomycin followed by illumination through an optic fiber by using a catheter.     

Nanodrug applications in photodynamic therapy

Photodynamic therapy (PDT) has developed over last century and is now becoming a more widely used medical tool having gained regulatory approval for the treatment of various diseases such as cancer and macular degeneration. It is a two-step technique in which the delivery of a photosensitizing drug is followed by the irradiation of light. Activated photosensitizers transfer energy to molecular oxygen which results in the generation of reactive oxygen species which in turn cause cells apoptosis or necrosis. Although this modality has significantly improved the quality of life and survival time for many cancer patients it still offers significant potential for further improvement. In addition to the development of new PDT drugs, the use of nanosized carriers for photosensitizers is a promising approach which might improve the efficiency of photodynamic activity and which can overcome many side effects associated with classic photodynamic therapy. This review aims at highlighting the different types of nanomedical approaches currently used in PDT and outlines future trends and limitations of nanodelivery of photosensitizers.     

Mechanism of mitochondrial membrane permeabilization during apoptosis under Photofrin-mediated photodynamic therapy

Photofrin-mediated photodynamic therapy (PF-PDT) can induce cell apoptosis via the mitochondria/caspase-3 pathway. Here, we further investigate the mechanism involved in the mitochondrial apoptotic process induced by PF-PDT. A high-level intracellular reactive oxygen species (ROS) generation in mitochondria, mitochondrial swelling, and dissipation of mitochondrial transmembrane potential were observed immediately after irradiation, indicating that mitochondria were the major ROS generation sites and also the first oxidative damage sites after PF-PDT treatment. For mitochondrial permeability detection, the decrease of calcein fluorescence emission intensity and release of cytochrome c were observed immediately after PF-PDT treatment, indicating the occurrence of mitochondrial inner membrane permeabilization (MIMP) and the mitochondrial outer membrane permeabilization (MOMP). However, cytochrome c release was not prevented by cyclosporine (CsA), a specific inhibitor of mitochondrial permeability transition (MPT). Taken together, these results demonstrated that PF-PDT caused simultaneous onset of MIMP and MOMP immediately after the treatment, and MOMP was independent of the MPT. Besides, inducible mitochondrial ROS generation played key roles in PF-PDT-induced cell apoptosis. This study will be benefit for understanding the mechanism involved in the initial mitochondrial oxidative damage by PF-PDT.     

Rutin-gallium complex mediated antimicrobial photodynamic therapy: An in vitro studies against Streptococcus mutans biofilms

BackgroundDiscoloration of teeth following antimicrobial photodynamic therapy (aPDT) is a serious concern. Common photosensitizers are colored, and access to a photosensitizer that does not leave color on the teeth or is the same color as the enamel and dentin is highly demanded. The physicochemical characterization, anti-virulence, and antimicrobial effects of a novel rutin-gallium (III) (Rt-Ga) complex as novel photosensitizer are presented herein.Materials and MethodsPhotophysical properties and cytotoxicity of the Rt-Ga complex were evaluated in comparison with the parental rutin. Intracellular reactive oxygen species (ROS) generation following Rt-Ga complex-mediated aPDT was measured using the fluorescent 2′,7′-dichlorofluorescein diacetate (H2DCF-DA) method. The anti-biofilm effects of Rt-Ga complex-mediated aPDT on Streptococcus mutans were assessed using a colorimetric assay. The virulence‑associated gtfB gene expression was assessed following Rt-Ga complex-mediated aPDT by quantitative real‑time PCR.ResultsThe photophysical properties of the Rt-Ga complex revealed a significant blue-shift in absorption (60 nm shift) and increased extinction coefficient (4100 M −1 cm −1; at λmax = 450 nm). Average (± SEM) DCF fluorescence intensities in an arbitrary unit (A.U.) were 7.1 ± 0.9, 4.1 ± 0.5, and 1.7 ± 0.3 for 10.0 μM of Rt-Ga complex-mediated aPDT, 7.5 μM of Rt-Ga complex-mediated aPDT, and 10.0 μM of Rt-Ga complex alone, respectively. The corresponding DCF fluorescence intensities were 710% (P = 0.001), 410% (P = 0.001), and 170% (P = 0.02) of the untreated S. mutans as the control group (1.0 ± 0.1 A.U.), respectively. The novel Rt-Ga complex-mediated aPDT exhibited no toxicity against primary human gingival fibroblast cells, a dose dependent decrease in S. mutans biofilm cell survival and virulence were observed (91.4% and 11.7-fold, respectively).ConclusionThe Rt-Ga complex-mediated anti-virulence and antimicrobial photodynamic effects were superior to the one caused by rutin alone making the Rt-Ga complex a more promising photosensitizer than the parent material.     

Novel benzoylthiourea derivatives had differential anti-inflammatory photodynamic therapy potentials on in vitro stimulated mammalian macrophages

Novel benzoylthioureas, N-((5-chloropyridin-2yl)carbamothioyl)benzamide, (HL¹), N-((2-chloropyridin-3yl)carbamothioyl)benzamide, (HL²), N-((5-bromopyridin-2yl)carbamothioyl)benzamide, (HL³) and N-(Naphthalene-1-yl(phenyl)carbamothioyl)benzamide, (HL⁴), were synthesized. Their characterizations were made by FT-IR,¹H NMR and ¹³C NMR spectrophotometric analysis. Single crystal X-ray diffraction measurements were conducted to determine the crystal structure of HL¹ and HL⁴.  The HL¹ crystallization conditions are: in the monoclinic crystal system with P2₁/c space group, Z = 2, a = 8.118(2) Å, b = 12.056(3) Å, c = 13.753(4) Å. HL⁴crystallization conditions are: in the orthorhombic crystal system with Pbca space group, Z = 8, a = 19.597(9) Å, b = 8.270(4) Å, c = 24.299(11) Å. Investigation of photodynamic and antiinflamatory effects of these compounds revealed that they are potent adducts. Using these derivatives, mammalian macrophages were stimulated with LPS to test their anti-inflammatory activity. Based on pro-inflammatory cytokine production levels, the photodynamic anti-inflammatory activity of these adducts were found to differ. Our results showedthat benzoylthioureas can be used as potential photodynamic therapy agents to suppress the excessive inflammatory reactions encountered in autoimmune and inflammatory disorders.     

Oxygen consumption in nonexercising muscle after exercise

Little is known about the effect of exercise intensity on post-exercise oxygen consumption in nonexercising muscle. This study examined the effect of exercise intensity on muscle oxygen consumption (VO2mus) in nonexercising forearm flexor muscles (nonexVO2mus) after cycling exercise. Eight healthy male subjects performed 20 min of cycling exercise at 30%, 50%, and 70% of maximal oxygen consumption (%VO2max) on separate days. The nonexVO2mus values at rest, at the end of exercise, and during recovery after exercise were measured by near-infrared spectroscopy. VO2mus was determined using the rate of decrease in oxygenated hemoglobin during arterial occlusion. The nonexVO2mus at the end of exercise significantly increased by 1.3 +/- 0.1, 2.0 +/- 0.3, and 2.2 +/- 0.3-fold over resting values at 30%, 50%, and 70% VO2max, respectively. NonexVO2mus returned to the resting value after 3 - 5 min of recovery and then showed no significant change for 120 min after exercise at all exercise intensities. NonexVO2mus at the end of exercise at 70% VO2max was significantly higher than that after exercise at 30% VO2max. These results show that 20 min of cycling exercise induced an increase in nonexVO2mus and that higher intensity exercise produces a larger increase in nonexVO2mus after exercise.     

Effect of photodynamic therapy with two photosensitizers on Streptococcus mutants: In vitro study

Background and objectivesStreptococcus mutans (S. mutans) colonizes the oral cavity and causes dental caries and periodontal diseases. Considering the importance of the treatments that decrease pathogenic microorganisms, the aim of the present research was the assessment of the antimicrobial effect of Photodynamic Therapy (PDT) with Methylene Blue (MB) and Indocyanine Green (IG) photosensitizers on S. mutans.Materials and methodsIn this In vitro experimental study, Sixty four caries-free first premolars were contaminated with 0.5 McFarland S.mutans suspension and were randomly assigned to 4 groups. The teeth in the first group were impregnated with 2% MB while the teeth in the second group were impregnated with 0.2% IG. The teeth in the first group were irradiated with continuous-wave 660 nm dod laser with 40 mw output power, energy density of 2.4 J/cm² and 100% duty cycle for 60 s, while the teeth in the second group were irradiated with continuous -wave 810 nm diode laser with 100 mw out power, density energy of 6 J/cm² and 100% duty cycle for 60 s in contact mode. In the third group, the teeth were suspended in 0.2% Chlorhexidine for 30 s. The fourth group was considered as the control. The teeth were sampled before and after the interventions and the samples were incubated in Blood Agar for 24 h. Afterwards, the number of S. mutans colonies were counted. Data were statistically analyzed by Kruskal-Wallis, Dunn's and Friedman tests.ResultsIn the groups treated with a combination of MB and IG and laser irradiation and also in the Chlorhexidine group, the final number of S. mutans colonies equaled zero. In “MB and IG groups without laser irradiation”, although the amount of microorganisms decreased, but the number of colonies did not reach zero. Pair comparisons by Dunn's test showed that there was a significant difference between “MB and IG groups without laser irradiation” and the other experimental groups p = 0.03).ConclusionPDT with MB and IG photosensitizers and also Chlorhexidine mouthwash, have the ability to completely eradicate S. mutans bacterial colonies.