Modulation of heat shock protein expression and cytokine levels in MCF-7 cells through photodynamic therapy.

In this study, we assess the impact of photodynamic therapy (PDT) using aluminum phthalocyanine tetrasulfonate (AlPcS4) on the viability and cellular stress responses of MCF-7 breast cancer cells. Specifically, we investigate changes in cell viability, cytokine production, and the expression of stress-related genes. Experimental groups included control cells, those treated with AlPcS4 only, light-emitting diode (LED) only, and combined PDT. To evaluate these effects on cell viability, cytokine production, and the expression of stress-related genes, techniques such as 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, enzyme-linked immunosorbent assays (ELISA), and real-time quantitative PCR (RT‒qPCR) were employed. Our findings reveal how PDT with AlPcS4 modulates mitochondrial activity and cytokine responses, shedding light on the cellular pathways essential for cell survival and stress adaptation. This work enhances our understanding of PDT's therapeutic potential and mechanisms in treating breast cancer.

Antifungal and antibiofilm effect of duloxetine hydrochloride against Cryptococcus neoformans and Cryptococcus gattii.

Cryptococcosis is an invasive mycosis caused mainly by Cryptococcus gattii and C. neoformans and is treated with amphotericin B (AMB), fluconazole and 5-fluorocytosine. However, antifungal resistance, limited and toxic antifungal arsenal stimulate the search for therapeutic strategies such as drug repurposing. Among the repurposed drugs studied, the selective serotonin reuptake inhibitors (SSRIs) have shown activity against Cryptococcus spp. However, little is known about the antifungal effect of duloxetine hydrochloride (DH), a selective serotonin and norepinephrine reuptake inhibitor (SSNRI), against C. neoformans and C. gattii. In this study, DH inhibited the growth of several C. neoformans and C. gattii strains at concentrations ranging from 15.62 to 62.50 µg/mL. In addition, DH exhibited fungicidal activity ranging from 15.62 to 250 µg/mL. In biofilm, DH treatment reduced Cryptococcus spp. biomass at a level comparable to AMB, with a significant reduction (85%) for C. neoformans biofilms. The metabolic activity of C. neoformans and C. gattii biofilms decreased significantly (99%) after treatment with DH. Scanning electron micrographs confirmed the anti-biofilm activity of DH, as isolated cells could be observed after treatment. In conclusion, DH showed promising antifungal activity against planktonic cells and biofilms of C. neoformans and C. gattii, opening perspectives for further studies with DH in vivo.

Sputtering of micro-carbon-silver film (µC-Ag) for endotracheal tubes to mitigate respiratory infections.

Polyurethane (PU) substrates are biocompatible materials widely used to manufacture endotracheal tubes. However, in common with other biomedical materials, they are liable to the formation of microbial films. The occurrence of pneumonia in intubated patients treated at intensive care units often takes the form of ventilator-associated pneumonia (VAP). The issue relates to the translocation of pathogenic microorganisms that colonize the oropharyngeal mucosa, dental plaque, stomach, and sinuses. New protective materials can provide a more effective therapeutic approach to mitigating bacterial films. This work concerns microcrystalline carbon film containing dispersed silver nanoparticles (µC-Ag) deposited on PU substrates using a physical vapor deposition sputtering process. For the first time, carbon paper was used to produce a carbon target with holes exposing a silver disk positioned under the carbon paper, forming a single target for use in the sputtering system. The silver nanoparticles were well distributed in the carbon film. The adherence characteristics of the µC-Ag film were evaluated using a tape test technique, and electron dispersive x-ray mapping was performed to analyze the residual particles after the tape test. The microbicidal effect of the thin film was also investigated using speciesS. aureus, a pathogenic microorganism responsible for most infections of the lower respiratory tract involving VAP and ventilator-associated tracheobronchitis (VAT). The results demonstrated that µC-Ag films on PU substrates are promising materials for mitigating pathogenic microorganisms on endotracheal tubes.

Alteration of Surface Glycoproteins After Photodynamic Therapy.

BACKGROUND: Cell membranes have been identified as an important intracellular cancer treatment target, since the glycoconjugates present on the cell surface are involved in numerous cell functions. Photodynamic therapy (PDT) is a therapeutic modality employed in the treatment of tumors that uses visible light to activate a photosensitizer. OBJECTIVE: This study analyzed the expression of surface carbohydrates after PDT with two different photosensitizers, 5-aminolevulinic acid (ALA) and Photosan-3. METHODS: Mice were injected subcutaneously with 2 × 105 B16 cells. After 7-10 days, the presence of a tumor with a diameter of 3.6 mm was observed. Photosan-3® and 5-aminolevulinic acid-ALA were used in the PDT treatment. Control animals (not submitted to either laser treatment or photosensitizer injection) and treated animals were euthanized 15 days post-treatment. The tumors were irradiated with a red diode laser, λ = 655 nm, energy density of 10 J.cm-2, and power density of 45 mW.cm-2. After 2 weeks of treatment with PDT, the mice were euthanized, the tumors were collected, and the cell surfaces were labeled with lectins concanavalin A (ConA) and wheat germ agglutinin (WGA). RESULTS: Fluorescence microscopy analysis of the cell surfaces with lectins ConA and WGA showed the presence of α-mannose and α-glucose. CONCLUSIONS: The combined effects of either Photosan-3 or ALA and red laser light on melanoma suggest an inhibitory glycosylation action from PDT on the surface of B16-F10 cells.

Evaluation of the photobiomodulation in L929 cell culture.

Low-level laser therapy has become an important tool for bio-modulation process. It can induce stimulatory or inhibitory effects according to cell behavior at specific irradiation. Our objective was to determine L929 cell line response to irradiation at λ 685 and 830 nm, concentrations of 5 and 10% fetal bovine serum and different energy densities of 0.1, 0.5, 1, 2, 3, 5, 7, 10, 20, and 30 J/cm(2). Thus, cells were plated at 1 × 10(5) cells/mL and irradiated with semiconductor laser As-Ga-Al. Twenty-four hours after irradiation, cells were subjected to MTT, neutral red, crystal violet tests, and cell staining was performed using the kit Alexa Fluor 488 Annexin V-FITC and propidium iodide. Our results showed that low-level laser therapy stimulates effect when the energy density is 5 to 3030 J/cm(2) and inhibits effects on energy density 0.1 to 3 J/cm(2). This inhibitory effect was evidenced by the absence of dead cells labeled, decreased cell density, and by the absorption of neutral red in intact cells. The study also demonstrated that fetal bovine serum, at different concentrations, did not affect response of the cells after irradiation.

Effect of photodynamic therapy supplemented with quercetin in HEp-2 cells.

Photodynamic therapy (PDT) is a technique that can be used as a complementary therapy in cancer treatment combined with other therapeutic modalities. Quercetin (QCT) is known to be effective in the treatment of cancer, by reducing the cell viability of different cancer cell lines. This study aimed to evaluate the influence of different concentrations of QCT in PDT on the viability, mitochondrial membrane potential and induction of apoptosis/necrosis in the human larynx carcinoma cells (HEp-2). The HEp-2 cells were treated with aluminum phthalocyanine tetrasulfonate (AlPcS4) and QCT and subsequently irradiated with a diode laser light (685 nm, 35 mW, 4.5 J/cm(2)). The results demonstrated that treatment of HEp-2 cells with high concentrations of QCT (at least 50 µM) reduced cell viability. This response was enhanced in cells subjected to PDT supplemented with high concentrations of QCT. In addition, was observed decrease in the mitochondrial membrane potential and characteristics of late apoptosis and/or initial necrosis process. QCT at concentrations from 50 µM improves PDT-induced cytotoxicity by significantly reducing cell viability by apoptosis and/or necrosis, and mitochondrial membrane potential of Hep-2 cells.

Photodynamic therapy in the cattle protozoan Tritrichomonas foetus cultivated on superhydrophilic carbon nanotube.

Superhydrophilic vertically aligned carbon nanotubes (VACNT-O2) were used for the first time as scaffolds for photodynamic therapy (PDT) to induce inhibition of cell division in eukaryotic cells. VACNT-O2 scaffolds were produced on Ti substrates using plasma enhanced chemical vapor deposition technique and functionalized by oxygen plasma. Scanning electron microscopy (SEM) analysis was performed to characterize the surface changes of the protozoan and interaction with VACNT-O2. Characterization of lipid and total protein expression was performed with protozoa that were or not treated with PDT. Quantification of protein was conducted using Qubit fluorometer and separated on a polyacrylamide gel. SEM analysis showed the release of lipid vesicles by protozoa after the PDT. These vesicles were characterized by the PKH26 fluorescent probe. The results demonstrated a greater amount of protein released after PDT than in the control. When analyzing the protein material in polyacrylamide gel, a significant protein expression of approximately 65 kDa was found. A model identified the programmed death of Tritrichomonas foetus after the PDT was also proposed.

Cellular changes after photodynamic therapy on HEp-2 cells using the new ZnPcBr(8) phthalocyanine.

OBJECTIVE: This study investigated the effects of photodynamic therapy (PDT) by using the new photosensitizer Octal-Bromide Zinc Phthalocyanine (ZnPcBr(8)) on the nucleus, mitochondria, and cytoskeleton of HEp-2 cells. BACKGROUND DATA: PDT has been widely used as a therapeutic method for tumor-selective treatment and for other diseases. The therapy requires a photosensitizer, molecular oxygen, and visible light. Different studies have demonstrated that cellular organelles are potential targets for PDT, and the results are dependent on the photosensitizer used in the treatment. In this study, we investigated changes in the nucleus, mitochondria, and cytoskeleton of HEp-2 cells after PDT with the new ZnPcBr(8) phthalocyanine. MATERIAL AND METHODS: HEp-2 cells were cultivated under standard conditions, and then incubated with ZnPcBr(8) (1 micromol/L) for 1 h, and subsequently irradiated with a diode laser light (676 nm, 30 mW, 4.5 J/cm(2)). The cells were further cultured for 1 and 24 h at 37 degrees C in a 5% CO(2) and analyzed with fluorescence microscopy by using specific probes for the investigated organelles. RESULTS: Before PDT, the photosensitizer showed a cytoplasmic diffuse distribution. After PDT, cells showed multinucleation, a punctuated mitochondrial distribution in the perinuclear region, and cellular retraction due to the cytoskeleton changes. All those cellular alterations disrupted homeostasis, contributing to cellular death, which is the major goal of PDT. CONCLUSION: Based on our results and the characteristics of the new ZnPcBr(8) phthalocyanine, mechanistic and biochemical studies must be performed, but it is tempting to consider the chemical as a promising agent for PDT.

Effect of GaAlAs laser irradiation on enzyme activity.

OBJECTIVE: The aim of this study was to determine the influence of laser irradiation on enzyme activity. BACKGROUND DATA: Enzymes are catalysts of extraordinary efficiency, able to accelerate reactions by manifold. Enzyme laser light activation is currently a fast-growing field and a large number of studies have been produced. MATERIALS AND METHODS: Liquid CNPG amylase and control serum (Qualitrol 1H) were used in the experiments. Laboratory analysis of alpha-amylase was performed on two sample groups: (i) E + S and (ii) E + S + L, in six repetitions per irradiation dose. Group 2 was irradiated with gallium-aluminum-arsenide (GaAlAs) 904 nm at doses of 0.01, 0.1, 0.5, and 1 J/cm(2). Enzyme activity was read using a spectrophotometer equipped with a thermostatic chamber capable of precise absorbance measurement at 405 nm. RESULTS: The results were analyzed with the Student's t-test, and the percentage of enzyme activity was determined. Photomodulation of alpha-amylase activity by GaAlAs laser was analyzed following irradiation with different doses. Irradiation doses from 0.01 to 1 J/cm(2) led to differences in enzyme activity: 0.01 J/cm(2) (0.10%), 0.1 J/cm(2) (13.44%), 0.5 J/cm(2) (12.57%), and 1 J/cm(2) (-6.10%). CONCLUSION: Irradiation doses of 0.1 J/cm(2) and 0.5 J/cm(2) led to statistically significant increases in enzyme activity in comparison to the control. The similar curves of the effects of temperature and pH on enzymatic activity observed in this study suggest that laser irradiation also possess an optimum dose to modulate the enzymatic activity. That is, enzymes have an optimum laser dose (or range) at which their activity is maximal, whereas at higher or lower doses activity decreases.

Cellular and molecular studies of the initial process of the photodynamic therapy in HEp-2 cells using LED light source and two different photosensitizers.

Photodynamic therapy is an attractive therapeutic procedure for a variety of neoplastic and non-neoplastic disorders that involve a tumor localizing photosensitizer that induces cytotoxicity when activated by a particular wavelength of light. Considering the acceptability of the phthalocyanine photosensitizers, we began to address initial cellular and molecular aspects involved in the therapy that leads to apoptosis or necrosis. After HEp-2 cells had been incubated for 1h with aluminium tetrasulfonate chloride (AlPcS(4)) or zinc phthalocyanines (ZnPc) at 10microM, they were irradiated with an LED prototype (640nm, 70mW, 4.5J/cm(2)) and subsequently analyzed at 0 and 3h after the irradiation through MTT, fluorescence microscopy, Annexin V-PI staining and gene transcription analysis. Based on the results we can address that the two photosensitizers led to 2 different death pathways; for AlPcS(4), the results suggest an initial process of intrinsic death pathway; and for ZnPc, a prostanoid-mediated activation of death machinery is suggested.

Ultrastructural aspects of female aging Wistar rat epithelium tongue: a HRSEM and TEM study.

BACKGROUND: There is general consensus that the effects of intrinsic aging on the oral mucosa are relatively small, though potentially important to understanding the pathologies present in the aged animals. OBJECTIVE: In this paper, the development of dorsal surface of rat tongue was examined using transmission electron microscopy (TEM) and high-resolution scanning electron microscopy (HRSEM) in order to understand the age-related structural and ultrastructural changes experimentally. METHODS: In this study, we used female rats 75 and 720 days old (adult and aging). Tissues of rat tongue were prepared and the specimens submitted to HRSEM and TEM techniques. RESULTS: The analysis of HRSEM and TEM demonstrated that the same characteristic keratinous epithelium was found in aging animals, however with some modifications. CONCLUSION: We agree that there are obvious changes in the oral mucosa with aging and these modifications can be observed starting from the ultrastructural aspects.

Cytotoxicity of octal-bromide zinc phthalocyanine after photodynamic therapy with different light sources.

OBJECTIVE: The objective of this study was to investigate the cytotoxicity of octal-bromide zinc phthalocyanine (ZnPcBr8) at different concentrations (0.25, 0.5, and 1 microM) after irradiating HEp-2 cell cultures with two different light sources: a diode semiconductor laser (660 nm, 30 mW) or an LED (640 nm, 70 mW). In order to obtain comparative results, the irradiation parameters of both light sources were adjusted so that the amount of energy density delivered would be the same (4.5 J/cm2). BACKGROUND DATA: Numerous photosensitizers and light sources used in the treatment of human disease have been studied. Based on these studies, a comparative evaluation of two light sources used in photodynamic therapy (PDT) with ZnPcBr8 was proposed. MATERIALS AND METHODS: HEp-2 cells were incubated with ZnPcBr8 at different concentrations (0.25, 0.5, or 1 microM) for 1 h, irradiated with the diode semiconductor laser (660 nm at 30 mW for 300 sec; 4.5 J/cm2) or the LED laser (640 nm at 70 mW for 128 sec; 4.5 J/cm2), and then incubated in MEM medium for 1 or 24 h. The cells were analyzed using the MTT and trypan blue dye exclusion tests. RESULTS: The results demonstrated that the concentration of 1 microM of ZnPcBr8 was the most effective after PDT administered by both light sources. According to the MTT results, HEp-2-cell viability decreased by 97.96% 1 h after, and by 99.87% 24 h after irradiation with the diode semiconductor laser, and decreased by 94.03% 1 h after, and by 99.21% 24 h after irradiation with the LED. The results obtained using the trypan blue dye exclusion test confirmed the photodynamic efficacy of ZnPcBr8 employed with both light sources. With regard to HEp-2-cell viability, the following results were observed: a decrease of 98.73% 1 h after, and of 99.49% 24 h after irradiation with the diode semiconductor laser; and a decrease of 98.76% 1 h after, and of 99.23% 24 h after irradiation with the LED. CONCLUSIONS: According to our results with the irradiation parameters studied here, both the LED and diode semiconductor laser can be used for PDT in vitro, since both light sources had excellent photodynamic efficacy.

Assessment of fibroblast cells submitted to ultrasonic irradiation.

Physiotherapists consider ultrasound an indispensable tool, which is commonly employed in clinical practice as a treatment aid for musculoskeletal dysfunctions. The aim of our study has been to analyze fibroblast cell structures following low-intensity pulsed ultrasonic irradiation. Fibroblast cell cultures irradiated with ultrasound were analyzed through electron microscopy to determine an ideal irradiation beam that preserved cell morphology and integrity. Analysis by fluorescence microscopy and transmission electron microscopy was used to follow morphological changes of the nucleus and cytoskeleton following different ultrasound irradiation intensities. According to the parameters used in the pulsed irradiation of fibroblast cultures, control over the intensity employed is fundamental to the optimal use of therapeutic ultrasound. Cell cultures submitted to low-intensity pulsed ultrasonic irradiation (0.2-0.6 W/cm2) at 10% (1:9 duty cycle) and 20% (2:8 duty cycle) maintained shape and cellular integrity, with little damage. In the group irradiated with an intensity of 0.8 W/cm2, a loss of adhesion was observed along with an alteration in the morphology of some cells at an intensity of 1.0 W/cm2, which resulted in the presence of cellular fragments and a decrease of adhering cells. In cells irradiated at 2.0 W/cm2, there was a complete loss of adhesion and aggregation of cellular fragments. The present study confirms that biophysical properties of pulsed ultrasound may accelerate proliferation processes in different biological tissues.

Mitochondria, endoplasmic reticulum and actin filament behavior after PDT with chloroaluminum phthalocyanine liposomal in HeLa cells.

Photodynamic therapy (PDT) for cancer is a therapeutic modality in the treatment of tumors in which visible light is used to activate a photosensitizer. Cell membranes have been identified as an important intracellular target for singlet oxygen produced during the photochemical pathway. This study analyzed the cytotoxicity in specific cellular targets of a photosensitizer used in PDT in vitro. The photosensitizing effects of chloroaluminum phthalocyanine liposomal were studied on the mitochondria, cytoskeleton and endoplasmic reticulum of HeLa cells. Cells were irradiated with a diode laser working at 670 nm, energy density of 4.5 J/cm2 and power density of 45 mW/cm2. Fluorescence microscopic analysis of the mitochondria showed changes in membrane potential. After PDT treatment, the cytoskeleton and endoplasmic reticulum presented basic alterations in distribution. The combined effect of AlPHCl liposomal and red light in the HeLa cell line induced photodamage to the mitochondria, endoplasmic reticulum and actin filaments in the cytoskeleton.

Photodynamic therapy with a new photosensitizing agent.

OBJECTIVE: The aim of this study was to investigate the cytotoxicity of octal-bromide zinc phthalocyanine (ZnPcBr(8)) before and after irradiation with a low-power laser (AsGaAl) and analyze the effects of photodynamic therapy (PDT) on the nucleus of L929 cells. BACKGROUND DATA: One of the most recent and promising applications of phthalocyanine in medicine is in the detection and cure of tumors. We studied the ZnPcBr(8) in agreement with the development of new photosensitizing agents for curing tumors. METHODS: L929 cells were cultivated at standard conditions, incubated with ZnPcBr(8) for 1 h at different concentrations, irradiated with a semiconductor laser, and incubated in MEM medium for 1, 12, or 24 h. Cells were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) technique and fluorescence microscopy. RESULTS: The results demonstrated that ZnPcBr(8) at 1 microM was the most effective concentration for PDT, with a decrease of 63% after 1 h, 99% after 12 h, and 100% after 24 h in relation to the control group. The fluorescence microscopy results showed that ZnPcBr(8) was localized in the perinuclear region when analyzed 1 h after incubation. Nucleus staining with DAPI made it possible to observe that nuclear fragmentation occurred 24 h after PDT, cytoplasm retraction at 1, 12, and 24 h after PDT, and vacuoles along the cytoplasm at 12 and 24 h after PDT. CONCLUSION: According to the results obtained in this study, L929 cell death caused by PDT with ZnPcBr(8) possesses characteristics of apoptosis mediated by the mitochondria, due to the decrease in cells viability, the subcellular localization, and the photodamage found.

Infrared laser photobiomodulation (lambda 830 nm) on bone tissue around dental implants: a Raman spectroscopy and scanning electronic microscopy study in rabbits.

OBJECTIVE: The aim of this study was to assess, through Raman spectroscopy, the incorporation of calcium hydroxyapatite (CHA; approximately 960 cm(1)), and scanning electron microscopy (SEM), the bone quality on the healing bone around dental implants after laser photobiomodulation (lambda830 nm). BACKGROUND DATA: Laser photobiomodulation has been successfully used to improve bone quality around dental implants, allowing early wearing of prostheses. METHODS: Fourteen rabbits received a titanium implant on the tibia; eight of them were irradiated with lambda830 nm laser (seven sessions at 48-h intervals, 21.5 J/cm(2) per point, 10 mW, phi approximately 0.0028 cm(2), 86 J per session), and six acted as control. The animals were sacrificed 15, 30, and 45 days after surgery. Specimens were routinely prepared for Raman spectroscopy and SEM. Eight readings were taken on the bone around the implant. RESULTS: The results showed significant differences on the concentration of CHA on irradiated and control specimens at both 30 and 45 days after surgery (p < 0.001). CONCLUSION: It is concluded that infrared laser photobiomodulation does improve bone healing, and this may be safely assessed by Raman spectroscopy or SEM.

Ultrastructural changes in Tritrichomonas foetus after treatments with AlPcS4 and photodynamic therapy.

The Tritrichomonas foetus is an amitochondrial parasitic protist which causes bovine trichomoniasis, a major sexually transmitted disease in cattle. No effective drugs for this disease have been approved to this date. Photodynamic therapy (PDT) is an experimental treatment that shows great potential for treating bacteria, fungi, yeasts, and viruses. However, the cytotoxic effect of PDT on protozoan has been poorly studied. In this study, PDT with aluminum phthalocyanine tetrasulfonated (AlPcS4) photosensitizer was efficient in killing T. foetus. The mode of cell death in T. foetus after PDT was investigated by transmission electron microscopy. Morphological changes, such as membrane projections, nucleus fragmentation with peripheral masses of heterochromatin, endoplasmic reticulum proliferation, intense cytoplasmic vacuolization, fragmented axostyle-pelta complex, and internalized flagella could be observed. This is the first report to demonstrate cell death in T. foetus after PDT, and thus will open up new lines of investigation to develop new treatments for bovine trichomoniasis.