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

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

Photodynamic therapy using methylene blue in lung adenocarcinoma xenograft and hamster cheek pouch induced squamous cell carcinoma

BackgroundPhotodynamic therapy (PDT) is used to treat early proximal bronchial cancer during a flexible bronchoscopy. The technique relies on the excitation of a photosensitizer by an appropriate wavelength, which is delivered into the bronchus in close contact with the tumor.ObjectiveTo assess methylene blue (MB) as a PDT agent for the treatment of respiratory tract cancer in animal models.MethodsMB-induced PDT was performed on 7 subcutaneous NCI-H460 lung adenocarcinoma xenografts in nude mice and 9 induced squamous cell cancer in the hamster cheek pouch model. In mice, PDT was carried out on right-sided tumors after intratumoral injection of methylene blue 1% (w/v) and illumination at 630 nm at 200 J/cm (Diomed PDT 630), with the left tumor used as control (illumination alone or MB alone). The tumoral volume was assessed before and 15 days after PDT.ResultsFourteen xenografts were treated in mice, including seven treated with MB-PDT, producing a 52% mean tumor volume regression (1568 mm³ vs. 544 mm³) compared to seven control cases in which tumor volume increased (p = 0.007; Mann-Whitney test). Nine cheek pouch induced carcinomas were treated in the hamster group, with a mean volume decrease of 85.8% (from 44.8% to 100%) (initial mean volume = 210 mm³ vs. post PDT mean volume = 97 mm³). Histology analysis showed 4/9 complete responses.ConclusionIntratumoral MB appears efficient as PDT agent for cancer treatment in animal models. Further studies are needed to assess the safety and efficacy of MB-associated PDT for the treatment of lung cancer in humans.     

Effect of photodynamic therapy on expression of HRAS,NRAS and caspase 3 genes at mRNA levels,apoptosis of head and neck squamous cell carcinoma cell line

ObjectivesThis study aimed to assess the effect of photodynamic therapy (PDT) on expression of CASP3, NRAS and HRAS genes at mRNA levels, and apoptosis of head and neck squamous cell carcinoma (HNSCC) cell line.Materials and methodsIn order to complete the present in vitro study, HNSCC cell line (NCBI C196 HN5) purchased from Pasteur Institute. Cells were divided into four groups; Group 1: photodynamic treatment (laser + methylene blue (MB) as photosensitizer), group 2: MB, group 3: laser (with 660 nm wavelength), and group 4: control (without any treatment). To determine the optimal concentration of MB, in a pilot study, toxicity of MB in different concentration was assessed using MTT assay. Cells in group 1, 2 and 3 was treated at optimal concentration of MB (1.6 μg/mL). Gene expression at mRNA levels was assessed after 24 h incubation, using real-time (qRT)-PCR. The expression of BAX and BCL2 genes at the mRNA levels was analyzed to evaluate apoptosis. 2^−ΔΔCt values of BCL2, BAX, CASP3, NRAS, and HRAS in groups was analyzed using ANOVA. Tukey’s HSD and Games Howell test was used to compare between two groups.ResultsOver-expression of BAX (p < 0.001), CASP3 (p < 0.001) and down-regulation of BCL2 (p = 0.004), HRAS (p = 0.023) and NRAS (p = 0.045) were noted in group 1 (PDT), compared with the control group. Treatment by laser alone induce down-regulation of CASP3 (p < 0.05), BAX (p < 0.05), BCL2 (p > 0.05), HRAS (p > 0.05) and NRAS (p > 0.05).ConclusionPDT caused down-regulation of NRAS, HRAS and BCL2 and over-expression of CASP3 and BAX genes at mRNA levels in HNSCC cell line. The present study raises the possibility that the role of MB on BCL2 down-regulation and BAX and CASP3 over-expression was higher than laser alone while it seems that laser alone was more effective than MB in HRAS and NRAS down-regulation.     

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.     

Antiviral photodynamic therapy: Inactivation and inhibition of SARS-CoV-2 in vitro using methylene blue and Radachlorin

IntroductionRecently, the COVID-19 pandemic has spread globally, necessitating the development of new methods for its prevention and treatment. The purpose of this study was to evaluate the antiviral activity of photodynamic therapy (PDT) against SARS-CoV-2 in vitro.MethodsVero E6 cells and SARS-CoV-2 isolated in Russia were used for PDT with methylene blue (MB) and Radachlorin. A continuous laser with wavelength λ = 662 nm in doses of 16 J/cm² and 40 J/cm² laser irradiation was used for PDT of a viral suspension and SARS-CoV-2-infected cells. The direct cytopathogenic effect of SARS-CoV-2 was evaluated via light microscopy to calculate the TCID₅₀ in the samples and perform statistical analysis.ResultsViral suspensions of SARS-CoV-2 that had a TCID₅₀ greater than 10³ were inactivated by PDT in the presence of MB and Radachlorin. Vero E6 cells were protected from 10⁴ TCID₅₀ of SARS-CoV-2 by PDT post infection. The range of protective concentrations was 1.0–10.0 μg/ml and 0.5–5.0 μg/ml for MB and Radachlorin, respectively. Additionally, it was found that MB and Radachlorin also possess significant antiviral activity even without PDT. The 50 % inhibitory concentration (IC₅₀) against 10² TCID₅₀ of SARS-CoV-2 was found to be 0.22 and 0.33 μg/mL with the addition of MB and Radachlorin, respectively, to cells concomitantly with virus, whereas in the case of applying the photosensitizers at 3.5 h post infection, the IC₅₀ was 0.6 and 2.0 μg/mL for MB and Radachlorin, respectively.ConclusionPDT shows high antiviral activity against SARS-CoV-2 when combined with MB and Radachlorin in vitro.     

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

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

Application of photodynamic therapy,laser therapy,and a cellulose membrane for calcaneal pressure ulcer treatment in a diabetic patient: A case report

Diabetes mellitus is a metabolic disorder in which a person has high blood glucose levels due to inadequate insulin production by the pancreas. Wounds in these individuals cannot heal properly over time due to circulatory changes that hinder and stagnate the healing process. We report the case of an 82-year-old female type 2 diabetes mellitus carrier, presenting to clinical-dermatological examination pressure ulcer (PU) in the right calcaneus region. The patient was treated with photodynamic therapy using curcumin and blue light-emitting diodes (LEDs), laser therapy, and the application of a cellulose membrane in order to promote ulcer decontamination by local action, accelerate wound healing, and maintain favorable conditions of asepsis and moisture, respectively. The ulcer healing occurred after 30 days of treatment and total epithelialization was observed. From the results obtained in this case report, we conclude that the combination of photodynamic therapy, laser therapy, and coating with a cellulose membrane is a promising treatment for the healing of PU in diabetic patients.     

Photo-toxic effects of 809-nm diode laser and indocyanine green on MDA-MB231 breast cancer cells

Photodynamic therapy (PDT), due to its positive outcomes in clinical applications, easiness of practice and few side effects, is a good candidate for an efficient treatment of cancer. Indocyanine green (ICG), a water-soluble, anionic tricarbocyanine and non-toxic molecule is a promising photosensitive agent for PDT applications on tumor cells. ICG exhibits strong maximum absorption at around 805 nm which will be an advantage for its use in PDT; light at that wavelength can be used to treat deeper tumors. In this study the inhibitory growth effects of ICG-PDT on MDA-MB231 human breast cancer cells were investigated in a time course experiment. Cells were irradiated with a continuous wave diode laser (λ = 809 nm, 60 mW, 24 J cm⁻²). Cell viability was measured by MTT assay 0, 3, 6, 9, 12, 24 and 48 h after light irradiation. The results showed that ICG-PDT application exerted its photo-oxidative effect on MDA-MB231 breast cancer cells immediately. Relative cell viability was determined throughout the 48 h time course, and a consistent decrease was observed after ICG-PDT applications. In conclusion, ICG when used in combination with near-infrared light showed a very fast (within 3 h) and persistent (up to 48 h) photo-toxic effect on MDA-MB231 human breast cancer cells.     

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

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

Comparative effect of photodynamic therapy on separated or mixed cultures of Streptococcus mutans and Streptococcus sanguinis

Antimicrobial photodynamic therapy (aPDT) has shown to exert a bactericidal effect against Streptococcus sanguinis and Streptococcus mutans. However, this efficacy has been reported for either type of bacteria separately. Bacterial suspensions of both strains, separately or together, were treated with concentrations of methylene blue (MB) and rose bengal (RB). Suspensions were irradiated with a light–emitting diode lamp (λ center at 625 nm for MB and λ center at 515 nm for RB) using a fluence of 18 J/cm². RB-aPDT at concentrations of 0.16–0.62 and 0.16–0.31 μg/mL, and MB-aPDT at concentrations of 0.62–1.25 and 0.31–1.25 μg/mL inhibited the growth of S. mutans and S. sanguinis respectively by 6 log₁₀. In suspensions of both strains together, the same 6 log₁₀ reduction in bacterial growth was achieved using the same concentrations of each photosensiziser.In conclusion, RB-aPDT and MB-aPDT appear to exert the same bactericidal effect against suspensions of S. sanguinis and S. mutans either for single strain treatment or for samples constituted by both bacteria mixed together. RB shows to be slightly more efficient than MB.     

Delivery of lipophilic porphyrin by liposome vehicles: Preparation and photodynamic therapy activity against cancer cell lines

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Palmatine hydrochloride mediated photodynamic inactivation of breast cancer MCF-7 cells: Effectiveness and mechanism of action

Breast cancer is one of the commonest malignant tumors threatening to women. The present study aims to investigate the effect of photodynamic action of palmatine hydrochloride (PaH), a naturally occurring photosensitizer isolated from traditional Chinese medicine (TCM), on apoptosis of breast cancer cells. Firstly, cellular uptake of PaH in MCF-7 cells was measured and the cytotoxicity of PaH itself on breast cancer MCF-7 cells was estimated using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Subcellular localization of PaH in MCF-7 cells was observed using confocal laser scanning microscopy (CLSM). For photodynamic treatment, MCF-7 cells were incubated with PaH and then irradiated by visible light (470 nm) from a LED light source. Photocytotoxicity was investigated 24 h after photodynamic treatment using MTT assay. Cell apoptosis was analyzed 18 h after photodynamic treatment using flow cytometry with Annexin V/PI staining. Nuclear was stained using Hoechst 33342 and observed under a fluorescence microscope. Intracellular production of reactive oxygen species (ROS) was studied by measuring the fluorescence of 2, 7-dichlorofluorescein (DCF) using a flow cytometry. Results showed that PaH treatment alone had no or minimum cytotoxicity to MCF-7 cells after incubation for 24 h in the dark. After incubation for 40 min, the cellular uptake of PaH reached to the maximum, and PaH mainly located in mitochondria and endoplasmic reticulum of MCF-7 cells. Photodynamic treatment of PaH demonstrated a significant photocytotoxicity on MCF-7 cells, induced remarkable cell apoptosis and significantly increased intracellular ROS level. Our findings demonstrated that PaH as a naturally occurring photosensitizer induced cell apoptosis and significantly killed MCF-7 cells.     

Influence of photodynamic therapy on peripheral immune cell populations and cytokine concentrations in head and neck cancer

BackgroundPhotodynamic therapy (PDT) represents a palliative treatment option for a selected group of patients with head and neck squamous cell carcinoma (HNSCC). PDT induces a local inflammatory reaction with the potential to initiate antitumor immune responses. However, the systemic impact on peripheral immune cells has not been described so far.MethodsHNSCC patients (n = 9) were treated with PDT in a palliative setting. All patients had previously undergone several oncologic treatment regimens. Blood samples were taken before, during and after PDT. Age-matched healthy donors served as control group (NC, n = 15). The frequency and absolute number of T- and B-lymphocytes, CD4⁺CD39⁺ regulatory T-cells (Treg) and NK-cells were measured by 10-color flow cytometry. Serum concentrations of T cell related cytokine panel, including HMGB1, IL-6, IL-10 and perforin were measured by bead array and ELISA.ResultsIn heavily pretreated HNSCC patients, the number and frequency of Treg and NK-cells were increased as compared to NC. PDT induced a further increase of the frequency of Treg and NK-cells in the peripheral blood. Additionally, the serum concentrations of HMGB1, IL-6 and IL-10 showed a significant elevation after treatment with simultaneously decreased perforin levels.ConclusionAlthough PDT is a local treatment regimen, a systemic inflammatory response with altered peripheral immune cell populations and cytokine concentrations is visible. The increased Treg and NK cell numbers after PDT support the hypothesis that PDT may successfully be combined with NK cell or T cell activating immune checkpoint modulators in HNSCC patients to improve HNSCC specific immunity.     

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

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

In vitro assessment of anti-tumorigenic mechanisms and efficacy of NanoALA,a nanoformulation of aminolevulic acid designed for photodynamic therapy of cancer

BackgroundThe development of nanocarriers is an important approach to increase the bioavailability of hydrophilic drugs in target cells. In this work, we evaluated the anti-tumorigenic mechanisms and efficacy of NanoALA, a novel nanoformulation of aminolevulic acid (ALA) based on poly(lactide-co-glycolide) (PLGA) nanocapsules designed for anticancer photodynamic therapy (PDT).MethodsFor this purpose, physicochemical characterization, prodrug incorporation kinetics, biocompatibility and photocytotoxicity tests, analysis of the cell death type and mitochondrial function, measurement of the intracellular reactive oxygen species production and DNA fragmentation were performed in murine mammary carcinoma (4T1) cells.ResultsNanoALA formulation, stable over a period of 90 days following synthesis, presented hydrodynamic diameter of 220 ± 8.7 nm, zeta potential of −30.6 mV and low value of polydispersity index (0.28). The biological assays indicated that the nanostructured product promotes greater ALA uptake by 4T1 cells and consequently more cytotoxicity in the PDT process. For the first time in the scientific literature, there is a therapeutic efficacy report of approximately 80%, after only 1 h of incubation with 100 μg mL⁻¹ prodrug (0.6 mM ALA equivalent). The mitochondria are probably the initial target of treatment, culminating in energy metabolism disorders and cell death by apoptosis.ConclusionsNanoALA emerges as a promising strategy for anticancer PDT. Besides being effective against a highly aggressive tumor cell line, the treatment may be economically advantageous because it allows a reduction in the dose and frequency of application compared to free ALA.