Johan W.M. Lagerberg

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.

Factors affecting the amount and the mode of merocyanine 540 binding to the membrane of human erythrocytes. A comparison with the binding to leukemia cells

In the presence of albumin Merocyanine 540 (MC540) exhibits a very limited binding to the outer surface of the membrane of normal erythrocytes, whereas pronounced binding is observed to leukemia cells. To find out whether this difference is due to differences in the composition or structural organization of the cell membrane we analyzed effects of a number of covalent and non-covalent perturbations of the red cell membrane on the binding and fluorescence characteristics of membrane-bound MC540. It is shown that exposure of the cells to cationic chlorpromazine, neuraminidase or photodynamic treatment with AlPcS4 as sensitizer caused a limited increase (30-50%) of MC540 binding, together with a red shift of the fluorescence emission maximum and an increase of the relative fluorescence quantum yield of membrane-bound MC540. Other forms of perturbation of the membrane structure, like hyperthermia (48 degrees C) and treatments that produce a decrease of phospholipid asymmetry in addition to accelerated flip-flop, did not result in increased MC540 binding, but did cause a red shift of the fluorescence emission maximum and an increase of the relative fluorescence quantum yield. These changes in fluorescence properties indicate a penetration of the dye into more hydrophobic regions in the membrane. MC540, bound to Brown Norway myelocytic leukemia cells, exhibited a red shift of the fluorescence emission maximum and an increased relative fluorescence quantum yield as compared to MC540 bound to untreated erythrocytes. These changes were of the same order of magnitude as in photodynamically treated red blood cells. Dye binding per surface area, however, was about 3-times higher with these leukemia cells than with photodynamically treated red blood cells. This demonstrates that certain perturbations of the erythrocyte membrane evoked a MC540 binding that became qualitatively comparable to the dye binding to leukemia cells, although dye binding per surface area was still significantly lower.

Stability after thawing of RBCs frozen with the high- and low-glycerol method

BACKGROUND : RBCs can be frozen with either the high‐glycerol method (HGM) or the low‐glycerol method (LGM). To date, the use of frozen RBCs is hampered by a 24‐hour outdating period after thawing. A closed washing system (ACP 215) may solve this problem.

Altered processing of thawed red cells to improve the in vitro quality during postthaw storage at 4°C

BACKGROUND: The use of a functionally closed system (ACP215, Haemonetics) for the glycerolization and deglycerolization of red blood cell (RBC) units allows for prolonged postthaw storage. In this study, the postthaw quality of previously frozen, deglycerolized RBCs resuspended in saline‐adenine‐glucose‐mannitol (SAGM) or additive solution AS‐3 was investigated.

Selective protection of RBCs against photodynamic damage by the band 3 ligand dipyridamole.

BACKGROUND: All studied photosensitizers for virus inactivation impair RBCs. To reduce damage to the RBCs without affecting virucidal activity, selective protection of the RBCs is necessary. The ability of the band 3 ligand, dipyridamole, to react with singlet oxygen and to increase the selectivity of photosterilization was investigated.

In vitro Fluence Rate Effects in Photodynamic Reactions with AIPcS4 as Sensitizer

Abstract— It has been shown previously that the efficiency of photodynamic therapy (PDT) both in vivo and in vitro is dependent on fluence rate. In this study, different in vitro experiments showed that tetrasulfonated aluminum phthalocyanine (AlPcS4) is more efficient in photosensi‐tization if the light is delivered at low fluence rate. Erythrocyte damage, virus inactivation and photooxidation of reduced glutathione (GSH) and histidine were all enhanced if light was delivered at 100 W/m2 as compared to 500 W/m2. Bleaching did not occur under these conditions. Oxygen depletion, shown to be important in fluence rate effects observed in vivo, does not seem to be involved. On theoretical grounds saturation of the triplet state is not likely under these conditions. A possible explanation for the observed fluence rate effects might be found in different reaction pathways, that are favored under high or low fluence rate illuminations. These reactions might involve uni‐ or bimolecular reactions of intermediate products, resulting in less efficiency at higher fluence rate. It proves to be important, under all circumstances, to monitor fluence rate, because a change in fluence rate, even with similar total fluences, might influence photobiological results in an unexpected way.

The effects of cryopreservation on red blood cell rheologic properties

BACKGROUND: In transfusion medicine, frozen red blood cells (RBCs) are an alternative for liquid‐stored RBCs. Little is known about the rheologic properties (i.e., aggregability and deformability) of thawed RBCs. In this study the rheologic properties of high‐glycerol frozen RBCs and postthaw stored in saline‐adenine‐glucose‐mannitol medium were compared to those of conventionally liquid‐stored and fresh RBCs.

Positively charged porphyrins: a new series of photosensitizers for sterilization of RBCs

BACKGROUND:  Photodynamic treatment could be a way to inactivate pathogens in RBCs. The objective of this study was to characterize the virucidal activity and RBC‐damaging activity of a series of cationic porphyrins. Using the most efficacious photosensitizer, various in‐vitro human RBC quality variables and in‐vivo RBC survival in Rhesus monkeys were evaluated.

Photodynamically induced effects in colon carcinoma cells (WiDr) by endogenous photosensitizers generated by incubation with 5-aminolaevulinic acid

Human adenocarcinoma cells of the line WiDr have been treated with 2 mM 5-aminolaevulinic acid (5-ALA) in the presence of 10% foetal calf serum. The treatment induces a linear accumulation of protoporphyrin IX (PpIX) for at least 7.5 h. After 7.5 h of incubation about 45% of the PpIX accumulated is cell-bound, while the rest is found in the medium (25%) or lost from the cells during washing with phosphate-buffered saline (30%). Exposure to white light at an intensity of 30 W/m2 for 18 min results in 95% reduction of clonogenicity in cells treated with 2 mM 5-ALA for 3.5 h. The enzymatic activities of enzymes located in cytosol (glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase) and lysosomes (acid phosphatase and beta-glucuronidase) are not influenced by a 5-ALA and light treatment inactivating about 35% of the cells. The MTT assay, which reflects mitochondrial dehydrogenase activity, but not succinate dehydrogenase, is partly inhibited by the same treatment. Treatment with 5-ALA in the absence of light increases O2 consumption by a factor of two, while the O2 consumption is inhibited when 5-ALA treatment is combined with exposure to light. In addition, 5-ALA and light exposure enhance accumulation of rhodamine 123 by 40% and reduce the intracellular ATP level by 25%. Confocal laser scanning microscopical analysis indicates granular perinuclear localization of the PpIX formed by 5-ALA treatment. In conclusion, photodynamic treatment using 5-ALA as a prodrug induces damage to mitochondrial function without inhibiting lysosomal and cytosolic marker enzymes.

5-Aminolevulinic acid induced lipid peroxidation after light exposure on human colon carcinoma cells and effects of α-tocopherol treatment

Abstract This work relates to studies on modes of phototoxicity by protoporphyrin (PpIX) after incubation of 5-aminolevulinic acid (5-ALA) on cultured cells. Lipid peroxidation in the 5-ALA incubated primary adenocarcinoma cells from the rectosigmoid colon (WiDr cells) was determined by measurement of protein-associated thiobarbituric acid reactive substances (TBARS). TBARS were increased 2-fold in cells treated with 2 mM 5-ALA for 3.5 h in serum enriched medium. After illumination of 5-ALA incubated cells, TBARS were formed in a light dose dependent manner. TBARS analysis were compared with high-performance liquid chromatography (HPLC) analysis of malondialdehyde, and results indicate that 90% of the thiobarbituric reactive substances were due to malondialdehyde. Pretreating WiDr cells with α-tocopherol for 48 h inhibits the cytotoxic effect of 5-ALA and increases 5-fold the light dose needed to kill 50% of the cells. Pretreatment with α-tocopherol shows a considerable decrease (about 80%) on TBARS formation after illumination. The cellular content of α-tocopherol was determined by HPLC and found to be 15.3 pmol/10 6 cells.