Use of a tetrazolium based colorimetric assay in assessing photoradiation therapy in vitro.

The suitability of a colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] assay to the determination of cell viability following photoradiation therapy (PRT) of human breast and melanoma cell lines has been examined. Results have been shown to correlate with those obtained using a clonogenic assay system. Using the MTT assay system it was shown that differences occur in the susceptibility of both lines to PDT. In addition it has been demonstrated that both lines differ with respect to their ability to develop photosensitivity in the presence of hematoporphyrin derivative (HpD). In the absence of serum this difference is not as obvious. This MTT assay provides a valid, simple and semi-automatable system for assessment of PRT in vitro.

Methotrexate-loaded, photosensitized erythrocytes: a photo-activatable carrier/delivery system for use in cancer therapy.

With a view towards the design of a system incorporating both the use of chemotherapeutics and photodynamic therapy for use in cancer treatment modalities, erythrocytes have been loaded with methotrexate and subsequently photosensitized by exposure to hematoporphyrin derivative. Loading of methotrexate into erythrocytes has been optimized by examining variations in electroporation conditions. Maximum loading indices observed were in the region of 64%. In order to obtain rapid pre-defined release of chemotherapeutic from the system, the erythrocytes were photosensitized. Light-dependent release of methotrexate from the system was examined. In addition, studies measuring the cytotoxic effects of light-activated release from the system using Hela cells as a target, suggested that decreases in cell viability following exposure to light resulted from the combined effects of chemotherapy and photoradiation therapy. Potential applications and advantages associated with this novel system are discussed.

Magnetically responsive photosensitizing reagents for possible use in photoradiation therapy.

The ability of a magnetically responsive material to function as a carrier for photosensitizing agents for use in photoradiation therapy (PRT) has been examined in vitro. The photosensitizer has been attached to the magnetically responsive matrix (Dynabeads) by non-specific adsorption, Intralipid-mediated adsorption and poly-L-lysine mediated adsorption. In these studies, it has been demonstrated that conditions of attachment of photosensitizer to the matrix may be adapted in order to facilitate a diffuse or highly localized photo-toxic effect on target cells in vitro. The authors believe that this system may represent a novel approach to targeting photosensitizing agents to specific areas, thereby circumventing some of the problems associated with conventional photoradiation therapy (PRT), particularly in hollow organs.

Use of real-time confocal laser scanning microscopy to study immediate effects of photodynamic activation on photosensitized erythrocytes.

With a view towards the design of systems capable of combining the use of chemotherapy and photodynamic therapy in the treatment of cancer and other disorders, it has been proposed that photosensitized erythrocytes might be employed as carriers/vehicles for agents such as cancer chemotherapeutics. In studying the light dependent release of entrapped agents from such a system, the efficacy of light induced release is usually studied by measuring release of an entrapped component into centrifugation supernatants following photoactivation. It has hitherto been extremely difficult to examine what occurs upon immediate irradiation at the microscopic level in real-time. In this study we demonstrate that, using real-time confocal laser scanning microscopy, it is possible to directly observe immediate short-term events occurring during direct irradiation with the visualizing beam. Following irradiation of photosensitized erythrocytes with the visualizing beam form the confocal scanning system, it was noticed that some from of cell-disruptive event occurred. In this study we demonstrate a dose dependent response between this relatively immediate, light induced disruptive event with respect to both irradiation exposure and photosensitizer concentration. We suggest that this system may provide a novel means of observing, at a microscopic level, events occurring in real-time during photodynamic therapy.

Contextual control of long-term habituation in rats.

This study examined contextual control of long-term habituation and whether such effects are dependent on the habituating response system. Habituation of the acoustic startle response transferred from the home cage to the testing context, whereas habituation of lick suppression was context specific (Experiments 1 and 2). Contextual control of habituation was demonstrated between 2 experimental contexts for lick suppression to a tone (Experiment 3) and bar-press suppression to a light (Experiment 4). Experiment 5 extinguished habituation of lick suppression and the orienting response to a tone with 27 exposures to the habituation context. Context specificity of both responses also was found. Previous failures to demonstrate contextual control of habituation may be due to the choice of response system and to less sensitive procedures to detect response recovery. The habituation mechanism for startle is independent from the process or processes that underlie habituation in other response systems, but the nature of these mechanisms is not yet known.

The tumoricidal potential of extracorporeal shock wave therapy.

A potential role of extracorporeal shock wave lithotripsy (E.S.W.L.), in the destruction of tumours has been proposed in recent literature. To examine further this potential, we have studied the effects of E.S.W.L. on the sarcoma-derived osteoblast-like cell line MG-63. An in-situ assay of cell viability was used to establish the cellular response to high energy shock wave therapy. A significant tumoricidal effect was confirmed when the cells were grown and tested in conventional monolayer phase. However cells grown in the three-dimensional matrix of alginate beads were significantly less vulnerable to extracorporeal shock wave therapy as earlier studies have suggested.

Encapsulation of the thrombolytic enzyme, brinase, in photosensitized erythrocytes: a novel thrombolytic system based on photodynamic activation.

In order to circumvent many of the problems associated with the systemic administration of agents used in thrombolytic therapy, it was decided to investigate the possibility of using erythrocytes as carriers and delivery vehicles for these agents. The enzyme brinase, a fibrinolytic enzyme produced by Aspergillus oryzae, was loaded into rabbit erythrocytes using electroporation. The loading index for this enzyme was found to be 60% and incorporation appeared to be relatively stable over a period of 4 h. In order to facilitate the predetermined release of the loaded component from the erythrocytes, they were photosensitized using haematoporphyrin derivative (HPD) and release was demonstrated within 5 min of photoactivation. Inclusion of the loaded, photosensitized system into clotting blood and subsequent exposure to light demonstrated almost complete lysis of the clot. We believe that this system exhibits potential for use in thrombolytic therapy.

Short communication: Ethanol production from cellulose at 45°C using a batch-fed system containing alginate-immobilized Kluyveromyces marxianus IMB3.

The thermotolerant yeast, Kluyveromyces marxianus IMB3, was grown in batch culture at 45°C on cellulose-containing media, supplemented with exogenous cellulase activity. At various stages during fermentation, both substrate and enzyme were added in batch mode and fermentation was continued for 220 h. Ethanol production increased to 20 g/l at 200 h, representing 45% of the maximum theoretical yield. In subsequent experiments, the organism was immobilized in calcium alginate beads and these were used in a similar, batch-fed system at 45°C. Again, fermentation was continued for 220 h and ethanol production increased to its maximum, of 28 g/l, within 100 h and this represented in excess of 60% of the maximum theoretical yield.

Studies on the biosorption of uranium by Talaromyces emersonii CBS 814.70 biomass.

Residual biomass, produced by the thermophilic fungus, Talaromyces emersonii CBS 814.70, following growth on glucose-containing media, was examined for its ability to take up uranium from aqueous solution. It was found that the biomass had a relatively high observed biosorption capacity for the uranium (280 mg/g dry weight biomass). The calculated maximum biosorption capacity obtained by fitting the data to a Langmuir model was calculated to be 323 mg uranium/g dry weight biomass. Pretreatment of the biomass with either dilute HCl or NaOH brought about a significant decrease in biosorptive capacity for uranium. Studies on the effects of variation in temperature on the biosorptive capacity demonstrated no significant change in binding between 20 degrees C and 60 degrees C. However, a significant decrease in biosorptive capacity was observed at 5 degrees C. Binding of uranium to the biomass at all temperatures reached equilibrium within 2 min. While the routine binding assays were performed at pH 5.0, adjustment of the pH to 3.0 gave rise to a significant decrease in biosorption capacity by the biomass. The biosorptive capacity of the biomass for uranium was increased when extraction from solution in sea-water was examined.