Effect Of Photodynamic Treatment Research Articles

GJIC Enhances the phototoxicity of photofrin-mediated photodynamic treatment by the mechanisms related with ROS and Calcium pathways.

Despite initially positive responses, recurrences after Photodynamic treatment (PDT) can occur and there is need for improvement in the effectiveness of PDT. Our study uniquely showed that there was a significantly gap junctional intercellular communication (GJIC)-dependent PDT cytotoxicity. The presence of GJIC composed of Connexin 32 increased the PDT phototoxicity in transfected HeLa cells and in the xenograft tumors, and the enhanced phototoxicity of Photofrin-mediated PDT by GJIC was related with ROS and calcium pathways. Our study indicates the possibility that up-regulation or maintenance of gap junction functionality may be used to increase the efficacy of PDT. The phototoxicity effect of Photofrin was substantially greater in Dox-treated cells, which expressed the Cx32 and formed the GJ, than Dox-untreated.

Photodynamic and Nail Penetration Enhancing Effects of Novel Multifunctional Photosensitizers Designed for The Treatment of Onychomycosis

Novel multifunctional photosensitizers (MFPSs), 5,10,15-tris(4-N-methylpyridinium)-20-(4-phenylthio)-[21H,23H]-porphine trichloride (PORTH) and 5,10,15-tris(4-N-methylpyridinium)-20-(4-(butyramido-methylcysteinyl)-hydroxyphenyl)-[21H,23H]-porphine trichloride (PORTHE), derived from 5,10,15-Tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) and designed for treatment of onychomycosis were characterized and their functionality evaluated. MFPSs should function as nail penetration enhancer and as photosensitizer for photodynamic treatment (PDT) of onychomycosis. Spectrophotometry was used to characterize MFPSs with and without 532nm continuous-wave 5mWcm(-2) laser light (±argon/mannitol/NaN3 ). Nail penetration enhancement was screened (pH 5, pH 8) using water uptake in nails and fluorescence microscopy. PDT efficacy was tested (pH 5, ±argon/mannitol/NaN3 ) in vitro with Trichophyton mentagrophytus microconida (532nm, 5mWcm(-2) ). A light-dependent absorbance decrease and fluorescence increase were found, PORTH being less photostable. Argon and mannitol increased PORTH and PORTHE photostability; NaN3 had no effect. PDT (0.6Jcm(-2) , 2μm) showed 4.6 log kill for PORTH, 4.4 for Sylsens B and 3.2 for PORTHE (4.1 for 10μm). Argon increased PORTHE, but decreased PORTH PDT efficacy; NaN3 increased PDT effect of both MFPSs whereas mannitol increased PDT effect of PORTHE only. Similar penetration enhancement effects were observed for PORTH (pH 5 and 8) and PORTHE (pH 8). PORTHE is more photostable, effective under low oxygen conditions and thus realistic candidate for onychomycosis PDT.

The effect of photodynamic treatment on the morphological and mechanical properties of the HeLa cell line

High resolution imaging of biological structures and changes induced by various agents such as drugs and toxins is commonly performed by fluorescence and electron microscopy (EM). Although high-resolution imaging is possible with EM, the requirements for fixation and staining of samples for image contrast severely limits the study of living organisms. Atomic force microscopy (AFM), on the other hand, is capable of simultaneous nanometer spatial resolution and piconewton force detection, allowing detailed study of cell surface morphology and monitoring cytomechanical information. We present a method that images and studies mechanically characterized cells using AFM. We used a HeLa cell line (cervix carcinoma cell), which is sensitive to photodynamic treatment (PDT); growth media as a scanning surrounding; atomic force microscopy NT-MDT Aura for cytomechanical measurement; and scanning electron microscope Hitachi Su 6600 for control images of the cells. The modulus of elasticity for intact and photodynamically damaged cells can indicate mechanical changes to the main properties of cells. Cell elasticity changes can provide information on the degree or value of cell damage, for example after PDT. Measurements were carried out on approximately sixty cells, including three independent experiments on a control group and on sixty cells in a photodamaged group. Cells before PDT show higher elasticity: the median of Young´s modulus on the nucleus was 35.283 kPa and outside of the nucleus 107.442 kPa. After PDT, the median of Young's modulus on the nucleus was 61.144 kPa and outside of the nucleus was 193.605 kPa.

Effects of photodynamic treatment on mesenchymal stromal cells

Effects of photodynamic treatment on mesenchymal stromal cells

Choroidal thickness following extrafoveal photodynamic treatment with verteporfin in patients with central serous chorioretinopathy

To evaluate the effect of verteporfin photodynamic treatment (PDT) on choroidal thickness in patients with central serous chorioretinopathy (CSC). Choroidal thickness was measured with enhanced depth imaging- optical coherence tomography (EDI-OCT) before and after verteporfin PDT (full-dose verteporfin, half-light dose) in 16 eyes in 16 patients with serous detachment of the fovea secondary to extrafoveal angiographic fluorescein leakage. Treatment was confined to the area of leakage, whereas choroidal thickness before and after treatment was assessed over a larger area of the fundus using OCT. Complete resolution of the serous detachment was seen in all 16 eyes within 1 month of extrafoveal PDT, while choroidal thickness in the area where PDT was applied decreased from 407 μm [mean; 95% confidence interval (CI(95) ) 356-458 μm] to 349 μm (mean; CI(95) 300-399 μm; p < 0.0001), and subfoveal choroidal thickness was reduced from 421 μm (mean; CI(95) 352-489 μm) to 346 μm (mean; CI(95) 278-414 μm; p = 0.0001). Initially, subfoveal choroidal thickness was significantly increased in the treated eye compared with the healthy fellow eye (mean 324 μm; CI(95) 273-376 μm; p = 0.0003), but after treatment, the difference was not significant. Photodynamic therapy of active CSC was followed by choroidal thickness reduction, not only locally but also at considerable distance from the treated area. Thus, the process that causes choroidal thickening in CSC appears to spread laterally within the choroid.

Tumor regression induced by photodynamic treatment with chlorin p6 in hamster cheek pouch model of oral carcinogenesis: Dependence of mode of tumor cell death on the applied drug dose

We investigated tumor regression and the mode of tumor cell death induced by photodynamic treatment (PDT) with chlorin p(6) (Cp(6)) in hamster cheek pouch model of oral squamous cell carcinoma. Cp(6) was administered systemically through intraperitoneal injection and after 4h the tumors were subjected to photodynamic treatment using red light (660±25nm, fluence ∼100J/cm(2)). Tumor response to PDT was monitored by measuring the tumor volume before PDT and 1week after. Results show that smaller tumors (⩽80mm(3)) regressed completely after PDT with Cp(6) dose of 2.0mg/kg body weight and for the bigger tumors (∼180mm(3)) higher dose of Cp(6) (4.0mg/kg) was more effective. Tumors treated with lower Cp(6) dose showed infiltration of immune cells, absence of TUNEL labeling, smeared pattern of DNA fragmentation and no significant increase in caspase-3 activity suggestive of necrotic cell death and inflammation. In tumors treated with higher Cp(6) dose, features characteristic of apoptotic cell death such as extensive TUNEL positive labeling, increase in caspase-3 activity and laddered pattern of DNA fragmentation were observed and there was no infiltration of immune cells. PDT with Cp(6) was also found to lead to expression of matrix metalloprotease-9 (MMP-9) which was greater at lower drug dose PDT as compared to higher drug dose PDT. These results suggest that drug dose plays an important role in determining the mechanism of tumor cell death and effectiveness of PDT.

Conjugation of chlorin p 6 to histamine enhances its cellular uptake and phototoxicity in oral cancer cells

Our previous studies in hamster cheek pouch model have shown that chlorin p (6) (Cp (6)), a chlorophyll derivative is a suitable photosensitizer for photodynamic treatment (PDT) of small tumors (<5mm). However, for bigger tumors, the accumulation of Cp (6) was inadequate, which compromised the effectiveness of PDT. The purpose of present study was to investigate the possibility of improving the cellular uptake of Cp (6) by conjugating it to histamine, a biogenic amine that is known to modulate tumor growth and development via cell surface receptors. The conjugate of Cp (6) and histamine (Cp (6)-his) was prepared by carbodiimide coupling reaction. Cellular uptake, intracellular localization and cytotoxicity of both Cp (6) and its conjugate were investigated in two human oral cancer cell lines (4451 and NT8e). The percentage of necrotic and apoptotic cells after PDT were also estimated using Hoechst 33342-propidium iodide staining. In both the cell line, the cellular uptake of Cp (6)-his was found to be ~10 times higher when compared to Cp (6). Histamine led to a slight increase in intracellular uptake of Cp (6)-his, whereas ranitidine, a histamine H2 receptor antagonist, and incubation at lower temperature (~15°C) led to its inhibition, suggesting that uptake of Cp (6)-his is receptor mediated. Results on western blot confirmed the presence of H2 receptor in both the cell line. Observations on intracellular localization revealed that unlike Cp (6), which localized on multiple sites, Cp (6)-his showed localization on the cell membrane and around the perinuclear region. Moreover, the phototoxicity induced by Cp (6)-his was ~4 times higher when compared to Cp (6) in both the cell lines. There was, however, no significant difference in the mode of cell death. Results suggest that conjugating Cp (6) with histamine can help improve the effectiveness of PDT in oral cancer cells by enhancing its intracellular delivery.

Effect of photodynamic treatment on the secretion of tumor necrosis factor α by human vascular endothelial cells.

Objective To measure the changes in levels of tumor necrosis factor α(TNF-α) secreted by human endothelial cells EC-304 after hematoporphyrin monomethyl ether-photodynamic treatment (HMME-PDT),and to explore the relationship between cytokines and inflammation initiation after management of nevus flammeus with photodynamic therapy.Methods EC-304 cells were cultured in 6-well plates,and classified into 4 groups:HMME-PDT group pretreated with HMME followed by irradiation with laser,HMME control group treated with HMME only,laser control group irradiated with laser only,and blank control group without any treatment.Culture supematants of EC-304 cells were collected from HMME-PDT group at 12,24 and 48 hours after the irradiation,and from the other three groups at the same time points.The supernatant TNF-α level was measured with enzyme linked immunosorbent assay (ELISA).Results The difference was statistically significant in the supernatant TNF-α level between different time points in each group (F=62.276.P 0.05).Conclusion HMME-PDT promotes the secretion of TNF-α by EC-304 cells. Key words: Photochemotherapy; Tumor necrosis factor-alpha; Endothelial cells

Impact of Photodynamic Treatment with Meso‐substituted Porphyrin on the Immunomodulatory Capacity of White Blood Cell‐containing Red Blood Cell Products

After transfusion, the presence of contaminating white blood cells (WBC) in blood components may result in either deleterious or positive immunological responses. We have previously reported that photodynamic treatment (PDT) with meso-substituted mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin (Tri-P(4)) and red light can inactivate pathogens in red blood cell (RBC) products. The present study explored the effect of PDT on contaminating WBC in RBC products with varying hematocrit (Hct). After PDT, we evaluated adaptive and innate immunomodulation through allogeneic and mitogenic stimulation. PDT resulted in decreased T-cell proliferation which was more pronounced with lower Hct. Dark effect of porphyrin Tri-P(4) was remarkable on antigen-presenting cells affecting expression of co-stimulatory molecules CD80/CD86. Finally, cytokine profile after PDT revealed a mixed Th1/Th2 type response while surface antigen expression supported the development of alternatively activated macrophages (AAM phi or Type 2 macrophages) instead of dendritic cells. In conclusion, PDT with Tri-P(4) altered proliferation, allo-stimulation, cell surface antigen expression and cytokine profiles of the cells. These results suggest that PDT may be potentially useful in preventing transfusion-associated graft-versus-host disease and alloimmunization. It seems worthwhile to further explore PDT-induced immunomodulation to optimize conditions which may result in allo-tolerance by AAM phi.

Sequential effects of photodynamic treatment of basal cell carcinoma

Sequential effects of photodynamic treatment of basal cell carcinoma

Atomic force microscopic study on morphological alterations induced by photodynamic action of Toluidine Blue O in Staphylococcusaureus and Escherichiacoli

Topographical alterations induced by Toluidine Blue O (TBO) mediated photodynamic treatment in Staphylococcus aureus and Escherichia coli was studied using atomic force microscopy (AFM). The AFM images showed distinct differences in the effect of photodynamic treatment on the morphology of S. aureus and E. coli. In S. aureus, photodynamic treatment with TBO resulted in light dose dependent increase in surface bleb formation suggesting breakage in the contact between the cell wall and the membrane with no significant change in the cell dimensions. Photosensitization of E. coli, resulted in surface indentations, significant reduction in the mean cell height, and flattening of bacteria as compared to the bacteria treated with the photosensitizers in the dark. These results indicate damage to the bacterial membrane and reduction of cell volume due to the loss of cytoplasmic materials. Leakage of intracellular contents measured using absorption spectrophotometry was higher and occurred faster in E. coli as compared to S. aureus and correlated with the morphological alterations. The results suggest that with AFM imaging it is possible to distinguish the membranolytic action of TBO in Gram-positive and Gram-negative bacteria.

Expression of IL-10, TGF-β1and TNF-α in Cultured Keratinocytes (HaCaT Cells) after IPL Treatment or ALA-IPL Photodynamic Treatment

Depending on the light dose and concentration of photosensitizer for photodynamic treatment (PDT), a multitude of dose-related events are demonstrable in PDT-treated cells. Sublethal doses may result in the alteration of cytokine release and consequently modify immune actions, rather than cause cell death. The purpose of this study was to investigate cytokine expression in cultured HaCaT cells after intense pulse light (IPL) treatment or PDT utilizing 5-aminolevulinic acid (ALA) and IPL at sublethal doses. Cultured HaCaT cells were treated with either IPL only (4, 8 and 12 J/cm(2)) or ALA-IPL PDT (100micromol/L of ALA; 0, 4, 8, and 12 J/cm(2) of IPL). The expression of IL-10, TGF-beta(1) and TNF-alpha was investigated by reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay. IL-10 protein increased up to 5.95-fold after IPL treatment and up to 2.85-fold after PDT. TGF-beta(1) mRNA and protein showed slight increases after both IPL treatment and PDT, of which the latter induced slightly larger increases. TNF-alpha mRNA and protein showed no induction or reduction after PDT. Increased expressions of IL-10 and TGF-beta(1) was observed after PDT. The induction of IL-10 may contribute to the anti-inflammatory effect, which explains the therapeutic benefit of PDT for inflammatory dermatoses, and that of TGF-beta(1) may be related to the therapeutic effect for psoriasis. The finding that IL-10 induction was more marked after IPL treatment than after PDT suggests that other mechanisms than IL-10 induction in keratinocytes after PDT may participate in the anti-inflammatory effect of PDT.

The effect of photodynamic treatment on the activity of regulatory glycolytic enzymes in A431 cells

The effect of photodynamic treatment on the activity of regulatory glycolytic enzymes in A431 cells

A novel ex vivo skin model to study the susceptibility of the dermatophyte Trichophyton rubrum to photodynamic treatment in different growth phases

Dermatophytes are fungi that can cause infections of skin, hair and nails because of their ability to feed on keratin. Superficial mycoses are among the most prevalent infectious diseases worldwide. Two important restrictions of current therapeutic options are the recurrence of the infection and prolonged treatment. This is especially true for infections caused by Trichophyton rubrum, a widely distributed dermatophyte. The application of photosensitizers for treatment of fungal infections is, within the field of photodynamic treatment (PDT), relatively new. Recently, we demonstrated that the porphyrins 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) and deuteroporphyrin monomethylester (DP mme) were excellent photosensitizers towards T. rubrum when using red light. To evaluate the photodynamic effectiveness of the porphyrins in a situation that mimics the clinical situation, we developed an ex vivo model using human stratum corneum. This model offers the possibility of applying PDT at different time points during the germination and subsequent development of T. rubrum microconidia. The model was used for two different incubation media, Dulbecco's modified Eagle medium (DMEM) and distilled water. We demonstrated that the PDT susceptibility of T. rubrum depended on the time of PDT application after spore inoculation. A decrease in susceptibility was observed with increasing time of PDT application for both photosensitizers in DMEM. Changing the incubation medium to distilled water resulted in an increased fungicidal effect for Sylsens B and in a decreased effect for DP mme. We conclude that T. rubrum is susceptible to PDT in a situation that mimics the clinical situation. The fungicidal effect of PDT on fungal spores is of particular importance.

Photodynamic Treatment of Oral Lesions

Photodynamic treatment (PDT) was first started in the oral cavity in the mid 1980s. Hematoporphyrins were rapidly replaced by Photofrin and meta-tetrahydroxyphenylchlorin (mTHPC) as photosensitisers of choice, and over the years these two have been approved by several health authorities for PDT. 5-aminolevulinic acid (ALA) and some dyes (e.g., toluidine and methyene blue) have also been tested. Several different nonthermal lasers have been used and lately light-emitting diodes (LEDs) have been tried. Most of the clinical treatments have been carried out on oral squamous cell carcinoma (OSSC), either primary or metastatic lesions, with good results. The treatment leaves little scarring and can be used before, in conjunction with, and adjunctive to other treatment modalities. The greatest disadvantage is that the patients are photosensitive for several weeks following systemic administration of the photosensitiser. PDT is now an accepted palliative treatment. Systemic administration of ALA has been more successful than local application in the treatment of precancerous lesions such as oral leukoplakia. PDT following topical application of photosensitiser (metylene blue and methyl-ALA) has shown improvement in cutaneous diseases of the oral mucous membrane such as oral lichen planus. The bactericidal effect of PDT has also been tested on oral plaque, but little clinical work has been performed so far. Instead of mechanical cleaning or antibiotic therapy, PDT may also play a role in dental diseases.

Production of reactive oxygen species in mitochondria of HeLa cells under oxidative stress

Mitochondria can be a source of reactive oxygen species (ROS) and a target of oxidative damage during oxidative stress. In this connection, the effect of photodynamic treatment (PDT) with Mitotracker Red (MR) as a mitochondria-targeted photosensitizer has been studied in HeLa cells. It is shown that MR produces both singlet oxygen and superoxide anion upon photoactivation and causes photoinactivation of gramicidin channels in a model system (planar lipid bilayer). Mitochondria-targeted antioxidant (MitoQ) inhibits this effect. In living cells, MR-mediated PDT initiates a delayed (“dark”) accumulation of ROS, which is accelerated by inhibitors of the respiratory chain (piericidin, rotenone and myxothiazol) and inhibited by MitoQ and diphenyleneiodonium (an inhibitor of flavin enzymes), indicating that flavin of Complex I is involved in the ROS production. PDT causes necrosis that is prevented by MitoQ. Treatment of the cell with hydrogen peroxide causes accumulation of ROS, and the effects of inhibitors and MitoQ are similar to that described for the PDT model. Apoptosis caused by H 2O 2 is augmented by the inhibitors of respiration and suppressed by MitoQ. It is concluded that the initial segments of the respiratory chain can be an important source of ROS, which are targeted to mitochondria, determining the fate of the cell subjected to oxidative stress.

Electron microscopic study of monkey retina after photodynamic treatment.

The purpose of this histological study was to determine the effects of photodynamic treatment, using a hematoporphyrin derivative and argon laser, on normal retinas of monkeys. Ten cynomolgus monkeys were treated with a hematoporphyrin derivative, given intravenously at a dose of 2.5 mg/kg. Forty minutes or 1 or 3 days after the injection, argon laser photoradiation was given over a 2.0-mm-diameter with a 10-min exposure and at an intensity of 40, 100, or 200 mW. The eyes were enucleated 1, 3, 4, 15, 18, 21, 35, or 38 days after the photoradiation and tissue samples were observed under a transmission electron microscope. The most fragile regions in the retina were the retinal nerve fibers, the outer segments of the visual cells, and the retinal pigment epithelium. Vascular endothelial cells were also fragile. The retinal capillary was easily obstructed, and the choriocapillaris was also occluded in an animal with severe retinal damage. The Mueller cells had the highest tolerance to the photodynamic treatment. Thus, exposing the normal part of the retina to light during photodynamic therapy should be avoided.

Photodynamic Diagnosis and Treatment for Atherosclerosis by an Endoscopic Approach

The photosensitizer, mono-L-aspartyl chlorin e6 (NPe6), specifically accumulates in the atheromatous plaque. We detected the fluorescence spectra of NPe6 emitted from atheromatous plaques on the descending thoracic aorta by an angioscopic approach using the animal model of atherosclerosis. We also showed that a fluorescence spectrum peak at 675 nm was obtained laparoscopically only in parts of the abdominal aorta with an atheromatous plaque. By a fluorescence endoscope, atheromatous plaques on the carotid artery were recognized as reddish spots from outside the artery. In addition, we visualized specifically at the beating heart surface small coronary atherosclerosis using an epifluorescence stereoscope system. We examined the effects of photodynamic treatment with NPe6 on the atheromatous plaque. The change in the elastic framework in the atheromatous plaque after photodynamic treatment was evaluated using scanning electron microscopy. The destruction of the architecture of the elastic fiber network in the atheromatous plaque was revealed. We also studied the change in the lipid components of the atheromatous plaque using Fourier transform infrared (FTIR) microspectroscopy. FTIR microspectroscopic analysis showed a dissociation of ester bonds of cholesterol esters in the atheromatous plaque after photodynamic treatment. The framework of the atheromatous plaque and the lipids accumulated in the plaque could be destroyed following such treatment.

Herpes simplex virus proteins are damaged following photodynamic inactivation with phthalocyanines

The photodynamic inactivation of herpes simplex virus type 1 (HSV-1) by two phthalocyanines (Pcs), the cationic dye HOSiPcOSi(CH 3) 2(CH 2),N + (CH 3) 3I − (Pc5) and the amphiphilic dye aluminum dibenzodisulfophthalocyanine hydroxide ( AlN 2SB 2POH), has been compared with that by the anionic dye, Merocyanine 540 (Mc540). Both Pc derivatives demonstrate a remarkable virucidal activity upon light activation even 3 h after the onset of HSV-1 adsorption, while Mc540 is effective for only 30 min after adsorption. Since fusion and virus penetration are promoted by membrane glycoproteins, we have studied the damage to viral proteins following photodynamic treatment (PDT) of HSV-1 and its relation to inactivation. The effect of AlN 2SB 2POH PDT is assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Major changes are found in the protein profile of PDT-treated HSV-1. A reduced ability of specific antibodies to react with HSV-1 major envelope proteins is detected by employing the Western blot assay. In particular, we demonstrate the related changes of glycoprotein D (gD), a structural protein of the HSV envelope. Since the envelope proteins participate in viral entry into the host cell, these alterations to viral envelope proteins may impair their ability to participate in early events of viral entry, leading to reduced infectivity of HSV-1. In contrast, no significant changes in the proteins' electrophoretic mobility could be seen after PDT with Mc540 or with Pc5. When HSV-1 purified proteins are subjected to combined electrophoretic and electro-osmotic forces on cellulose acetate, there is a shift in their cathode mobility, which may indicate changes in the protein mass and protein net charges following AlN 2SB 2POH photosensitization. There are only minor changes in the virus proteins, assayed as above, when HSV-1 is treated with Pc5.

Acute Effects of Photodynamic Treatment on Elastic Fibre Network in Atherosclerotic Plaques of Rabbit Aorta

Our objective was to demonstrate the acute effects of photodynamic treatment on the elastic framework in the atherosclerotic plaque. The photosensitiser, mono-l-aspartyl chlorin 56 (NP56), was administered intravenously, at a dose of 5 mg/kg of body weight, to cholesterol-fed, atherosclerotic rabbits. Six hours later, visible atherosclerotic lesions of the abdominal aorta were irradiated with a diode laser, wavelength 664 nm. The energy administered ranged from 50 J/cm2 to 200 J/cm2. The abdominal aorta was then excised and digested by a modified hot alkaline method. Scanning electron microscopy revealed the destruction of the architecture of the elastic fibre network in the atherosclerotic plaque, whereas no change was seen in the non-atheromatous aortic wall. Damage to the elastic fibre network was more obvious in plaque that was exposed to higher energy levels. No significant changes were observed in laser-treated plaques in animals that did not receive the NP56 pretreatment. Findings suggest that atherosclerotic plaques of the abdominal aorta can be selectively degraded by laser irradiation following pretreatment with the photosensitiser, NP56.