Increase of the photosensitizing efficiency of the Bacteriochlorin a by liposome-incorporation.

To describe the action mechanisms of Bacteriochlorin a (BCA), a second generation photosensitizer, in phosphate buffer (PB) and in dimyristoyl phosphatidylcholine (DMPC) liposomes we carried out oxygen consumption and ESR measurements. In PB, where BCA was in a monomer-dimer equilibrium, our results suggested that the oxygen consumption was related to the BCA monomers concentration in solution. Incorporation of BCA in DMPC liposomes, by promoting the monomerization of BCA, increased 9-fold the oxygen consumption in comparison to the value in PB. The use of specific singlet oxygen quenchers (Azide and 9,10-Anthracenedipropionic acid) in ESR and oxygen consumption experiments allowed us to assert that BCA was mainly a type II sensitizer when it was incorporated in DMPC. Finally, the cell survival of WiDr cells after a PDT treatment was measured for cells incubated with BCA in cell culture medium and cells incubated with BCA in DMPC. Irrespective of the dye concentration, the cell survival was lower when liposomes were used. This effect could be the result of a better BCA monomerization and/or a different BCA uptake in cells.

The effect of photodynamic therapy with bacteriochlorin a on lens epithelial cells in a capsular bag model.

Photodynamic therapy (PDT) with bacteriochlorin a(BCA) has proved to be a successful treatment for many cancers and to be cytocidal for different cell lines in culture. The present study aimed to investigate in vitro the potential of this treatment for killing lens epithelial cells (LECs) left in the human capsular bag after extracapsular cataract extraction (ECCE). Capsular bags were prepared from donor eyes using an ECCE procedure and incubated in various concentrations of bacteriochlorin a(1.6-50 microg ml(-1)) during various incubation periods (2-10 min). Subsequently, the capsules were illuminated during various exposure times (2-15 min) with a diode laser (wavelength 760 nm). After treatment, the capsular bags were cultured for 7 days in Eagle's minimal essential medium supplemented with 2% fetal calf serum. The specimens were fixed in glutaraldehyde/paraformaldehyde and examined with routine light microscopy, Hoechst staining for DNA and transmission electron microscopy. Proliferation of LECs on the posterior capsule was assessed in flat mounts. Capsular bags receiving BCA without illumination and capsular bags receiving illumination only served as controls.BCA alone or light alone have no effect on structure and proliferative activity of LECs. At a threshold protocol of incubation in BCA at 10 microg ml(-1)for 10 min and subsequent illumination for 15 min, proliferative activity of cells is largely arrested and nearly all LECs on the capsule exhibit severe signs of apoptosis. Photodynamic therapy with bacteriochlorin a induces cell death and suppression of proliferation inlens epithelial cells and could be a promising means of prevention of posterior capsule opacification.

Fluorescence, absorption and electron spin resonance study of bacteriochlorin a incorporation into membrane models.

Analysis of the bacteriochlorin a absorption spectra suggests the existence of a monomer-dimer equilibrium, particularly intense in phosphate buffer and favored by a decrease of the pH. The dye in methanolic solution is predominantly in monomeric form. Fluorescence and electron spin resonance nitroxide spin labeling measurements indicate that incorporation into the lipid phase of dimyristoyl-L-alpha-phosphatidylcholine liposomes induces dye monomerization. Moreover, the molecules are bound in the external surface of the vesicles and a complete incorporation is ensured by a lipid-to-dye ratio greater than 125.

Electron spin resonance evidence of the generation of superoxide anion, hydroxyl radical and singlet oxygen during the photohemolysis of human erythrocytes with bacteriochlorin a.

Photodynamic therapy with bacteriochlorin a (BCA) as sensitizer induces damage to red blood cells in vivo. To assess the extent of the contributuion of reactive oxygen species (ROS) and to determine a possible reaction mechanism, competition experiments with assorted ROS quenching or/and enhancing agents were performed in human erythrocytes as model system and in phosphate buffer. In the erythrocyte experiments, a 2% suspension was incubated with BCA for 1 h, washed with phosphate-buffered saline, resuspended and subsequently illuminated with a diode laser using a fluence rate of 2.65 mW/cm2. Potassium leakage and hemolysis were light and BCA dose dependent. Adding tryptophan (3.3 mM), azide (1 mM) or histidine (10 mM) to the erythrocyte suspension before illumination delayed the onset of K-leakage and hemolysis suggesting a type II mechanism. The D2O did not affect K-leakage nor photohemolysis. Adding mannitol (13.3 mM) or glycerol (300 nM) also caused a delay in the onset of K-leakage and hemolysis, suggesting the involvement of radicals. In phosphate buffer experiments, it was shown using electron spin resonance (ESR) associated with spin-trapping techniques that BCA is able to generate O2-. and OH. radicals without production of aqueous electron. Visible or UV irradiation of the dye in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gave an ESR spectrum characteristic of the DMPO-hydroxyl radical spin adduct DMPO-OH. Addition of ethanol or sodium formate produced supplementary hyperfine splittings due to the respective CH3CHOH. and CO2-. radical adducts, indicating the presence of free OH.. Production of DMPO-OH was partly inhibited by superoxide dismutase (SOD), catalase and desferrioxamine, suggesting that the iron-catalyzed decomposition of H2O2 was partly involved in the formation of one part of the observed OH.. The complementary inhibition of DMPO-OH production by azide and 9,10-anthracenedipropionic acid (ADPA) was consistent with 1O2 production by BCA followed by reaction of 1O2 with DMPO and decay of the intermediate complex to form DMPO-OH and free OH.. All our results seem to indicate that BCA is a 50%/50% type 1/type 2 sensitizer in buffered aqueous solutions and confirmed that the dye-induced hemolysis of erythrocytes was cell caused by a mixed type 1/type 2 mechanism.

Decreased expression of HLA class I on ocular melanoma cells following in vitro photodynamic therapy.

Our objective was to investigate whether photodynamic therapy (PDT) influences the expression of HLA Class I and beta 2-microglobulin molecules on cultured uveal melanoma cells. Uveal melanoma cells were incubated with hematoporphyrin esters (HPE) and illuminated using red light. HLA expression on cells was determined by flowcytometry. PDT treatment induced an immediate reduction in expression of HLA Class I and beta 2-microglobulin, followed by a transient increase in expression after 2 h. Normalization occurred after 6 h. Treatment of ocular melanoma cells with PDT temporally alters the expression of HLA Class I and beta 2-microglobulin, which may affect anti-tumor-immune responses.

Corneal neovascularization in rats as a model for photothrombotic therapy using bacteriochlorin a and an argon laser.

BACKGROUND: The presence of vessels has a negative influence on corneal transplant survival. Closure of such vessels prior to transplantation may improve the transplant results, and this might be achieved by irradiating the vessels with argon laser light after intravenous administration of a photosensitizer, e.g. bacteriochlorin a (BCA). A suture-induced corneal neovascularization model in rats was set up to test this hypothesis. METHODS: Suture-induced vessels in the cornea of male Wistar rats were irradiated with argon laser light after intravenous administration of BCA. We applied irradiation of varying energy levels and duration and assessed the changes in the vessels by slit-lamp examination, fluorescein angiography and histology. RESULTS: Suture-induced corneal vessels in the rat could be used effectively to study photothrombosis therapy. Intravenous administration of BCA prior to irradiation (lambda = 514.5 nm) of the corneal vessels led to vessel closure at lower energy levels and of longer duration than occurred with laser treatment alone. CONCLUSIONS: Suture-induced corneal neovascularization in the rat can be used as a model to study the efficacy of photothrombosis therapy. BCA can be used to enhance the rate and duration of vessel closure.

Non-specific systemic immune suppression induced by photodynamic treatment of lymph node cells with bacteriochlorin a.

Although photodynamic therapy is being used increasingly for the treatment of cancer, its effect on immune responses has received little attention. This aspect was studied in a rat model. Rats were given (intravenously) lymph node cells which were simultaneously exposed to a photosensitizer and light. As a result of this treatment the animals showed a decreased contact hypersensitivity response. Most important is that the generated immunosuppression proved to be non-specific. The consequence of this finding may be that photodynamic therapy of tumours can inhibit the immune response against malignant cells.