Methylene Blue (MB⁺) is a well-known dye in medicine and has been discussed as an easily applicable drug for the topical treatment in photodynamic therapy (PDT). MB⁺ can potentially be used as a redox indicator to detect the important redox reactions that are induced during PDT. MB⁺ induced PDT was successful in the intraluminal treatment of inoperable esophageal tumors and in the topical treatment of psoriasis. In order to improve the therapy, the reaction mechanism of MB⁺ was investigated in vivo by local injection of MB⁺ in a xenotransplanted subcutaneous tumor (adeno-carcinoma, G-3) in female nude mice. The MB⁺ preparation 'MB⁺1%' was applied both undiluted and diluted to 0.1% and 0.01% with isotonic sodium chloride. After an incubation period of 1 h, the tumors were irradiated at 662 nm. Treatment with 1% MB⁺ and subsequent irradiation with 100 J/cm² led to complete tumor destruction in 79% of the treated animals. A decrease of the fluence rate from 100 mW/cm² to 50 mW/cm² significantly increased the phototoxic response, which was attributed to oxygen depletion but also to nonlinear redox reactions. In addition, fractionated light application with 15 s interruption intervals enhanced the effect. When 0.1% MB⁺ was used, complete tumor destruction was observed only in 10% of the treated animals. Below a relatively high threshold dose the therapeutic response was not significant. The efficiency of the therapy was correlated with nonlinear dynamics of MB⁺ on a subcellular level, using laser scanning microscopy. During MB⁺-PDT nonlinear redox- reactions were induced. This could be deduced from local fast changes of the MB⁺-fluorescence as well as the pH-value during irradiation of single cells. The light induced reaction of MB⁺ seems to be correlated with the nonlinear production of reactive oxygen species (ROS). As a consequence below a threshold dose the reducing ability of MB⁺ prevents tissue from oxidative damage. However, above this dose, as a point of no return, MB⁺ acts as an extremely potent oxidant.

Earlier results studying the effect of excited triplet photosensitizer on the zymosan-stimulated and luminol- dependent chemiluminescence of macrophages have been quantitatively re-evaluated and rate constant data indicate that the effect is due to triplet-doublet interactions between sensitizer and free radicals generated. Such interactions, named Type III mechanism, compete with Type I and Type II mechanisms depending on the experimental environment. This suggestion resulted in the synthesis of new sensitizers being the first members of the Antioxidant Carrier Sensitizer (ACS) group of molecules. According to preliminary experiments the PDT treatment of tumor bearing mice seems promising with two of such compounds which demonstrate an inhibitory effect in chemical systems already in their ground state.

Photosensitization and inactivation efficacy of three bacterial species: Prevotella nigrescens, Staphylococcus aureus and Escherichia coli have been investigated. Samples of Staphylococcus aureus and Escherichia coli were treated with the triphenylmethane dye malachite green isothiocyanate and exposed to light from a variety of continuous and pulsed light sauces at a wavelength of approximately 630 nm. Inactivation of the Gram-positive species Staphylococcus aureus was found to increase with radiation dose, whilst Gram-negative Escherichia coli was resistant to such treatment. Samples of the pigmented species Prevotella nigrescens were found to be inactivated by exposure to light alone. The mechanism of photosensitization and inactivation of Staphylococcus aureus with malachite green isothiocyanate is addressed. The possible roles of the excited triplet state of the photosensitizer, the involvement of molecular oxygen, and the bacterial cell wall are discussed. Photosensitization may provide a way of eliminating naturally pigmented species responsible for a variety of infections, including oral diseases such as gingivitis and periodontitis.

Photodynamic therapy with 5-aminolevulinic acid (5-ALA) is a promising alternative treatment for several types of cancer. This work represents a first approach to characterize the transport system for 5-ALA in human cancer cells, using WiDr cells from a primary adenocarcinoma of the rectosigmoid colon as a model system. The transport of 5-ALA in WiDr was found to be dependent on pH and temperature, and partially inhibited by inhibitors of the energetic metabolism. Although WiDr was shown to express System A, a common transport system for small aliphatic amino acids, the transport of 5-ALA in WiDr was not found to be mediated by this system.

PDT relies on the combined action of photosensitizers localized on tumors and activated under laser light. Tumor cells response to PDT is determined by both the photosensitizer and the photoirradiation dose. However, the processes governing the resultant tumour destruction are still unknown and recent advances in the field of cellular biology have stimulated the research interest in this field. In this paper, the genotoxicity and chromosome damage on MIA pancreatic cancer cells, induced by red light and zinc tetrasulfonated phthalocyanines (ZnPcS₄) was studied. The results showed that cells treated with a low concentration (1 (mu) M) of ZnPcS₄ and a light dose of 6 J/cm² indicated more than 70% lethality, 72 hrs after irradiation. The SCE assay, showed that treated cells with various light and drug doses, presented no genotoxic potential, as far as SCE levels were not different from those of the untreated (control) cells. Chromosomal analysis after optimum PDT treatment in various time intervals post irradiation, showed that there was not any significant chromosomal damage on cells, compared to the untreated (control) cells.

Immediate illumination after sensitizer administration is currently often applied in PDT-trials in ophthalmology. The extent of possible damage to healthy ocular tissue after i.v. administration of the photosensitizers bacteriochlorin a (BCA) or mesa (tetrahydroxyphenyl) chlorin (mTHPC) and subsequent illumination with light of the appropriate wavelength and dose was assessed in rabbit eyes. Both hydrophobic drugs were formulated in 30% polyethylene glycol, 20% ethanol and 50% water to obtain an iv injectable suspension. Rabbits destined for BCA-PDT received a single dose of 10 mg/kg.bw. Rabbits destined for mTHPC-PDT received a dose of 0.3 mg/kg.bw. BCA- treated animals were illuminated immediately and 1, 2 and three hours after administration of the dye with an experimental Philips laser diode (760 nm, c.w., 100 mW/cm², 100 J). To illuminate the eyes of the mTHPC- treated animals a Krypton laser was used (648 nm, c.w., 100 mW/cm², 20 J). Illumination of these animals was performed immediately, 24, 48 and 72 hours after administration of the dye. BCA or mTHPC without illumination or illumination without administration of a sensitizing dye did not affect normal ocular tissues as judged by histology. Illumination of the entire eye of BCA-treated animals, immediately after administration of the dye caused a lesion in the macula area with a diameter of 3 mm. At the lesion side the photoreceptors were destroyed, ganglion cells were swollen and the sclera was affected. No skin photosensitivity was observed at anytime. Skin photosensitivity was observed in animals treated with mTHPC. Illumination caused swollen eyelids in all animals except when performed immediately after dye administration.

A series of promising PDT sensitizers having intensive absorption maximum at 710 - 750 nm has been synthesized from the readily available purpurin 18. Modification of an anhydride ring is purpurin 18 afforded two series of cyclic imides. The first group comprises the N-substituted derivatives containing residues of aliphatic alcohols (C₂- C₄) and carboxylic acids (C₁, C₅). The second series is represented by the cyclic N-hydroxyimides containing the -N-OR group, where R is either alkyl or acyl residue. Cyclic imides possess increased resistance to alkali treatment, which enhances their water solubility. They absorb in the region of 706 - 718 nm. Further development of the spectral characteristics was achieved by the oxidation of a 3- vinyl group into the formyl one. The obtained compounds have the absorption maximum in the region of 750 nm, which makes them comparable to those of bacteriochlorophyll derivatives. The presence of a spacer group in the molecule of compounds synthesized affords their attachment to the carriers for the directed transport of a sensitizer into tumor.

It is well known that sulphophthalocyanine derivatives under laser irradiation induce photochemical reaction of II type with generation of cytotoxic agent - singlet oxygen. The combination of phthalocyanine and exogenic reductant - sodium ascorbate may also induce other reactions, involving the formation of free radicals, and thus intensify the antitumor effect. To improve the results of PDT we used the additional injection of sodium ascorbate, the hyperbaric oxygenation and different regimes of laser irradiation. We conducted the experimental study on 100 white mice with Erlich carcinoma. Macroscopic and microscopic data showed that sodium ascorbate significantly increases the effect of PDT in comparison with control group due to the higher tumor damage, vascular alterations, inhibition of cell proliferation and stimulation of antitumor desmoplastic reaction.

Normal skin of nude mice (Balb/c) was treated topically with 5-aminolevulinic acid (ALA) and its methyl ester (ALA-Me) for 24 hours. Approximately 0.1 gram of freshly prepared cream was applied to a spot of 1 cm² on the flank of the mice, which was then covered with a transparent dressing. The ALA induced protoporphyrin IX (PpIX) was studied by means of a noninvasive fiber-optic fluorescence probe connected to a luminescence spectrometer. The excitation wavelength was 407 nm, and the emission wavelength was 637 nm. For the first hour a slight lag in PpIX production was observed for the mice treated with ALA-Me compared to the mice treated with ALA. After approximately 12 hours the ALA and the ALA-Me treated mice showed the same PpIX fluorescence intensity. From 12 hours until 24 hours the PpIX fluorescence intensity decreased for both treatment modalities, even though ALA and ALA-Me were continuously present. At 24 hours ALA-Me-treated mice had less than half the amount of PpIX in their skin compared with ALA- treated mice.

Photodynamic therapy (PDT) is an experimental treatment modality for (pre)malignant oesophageal lesions. 5- Aminolevulinic acid (ALA)-induced, protoporphyrin IX (PpIX)- mediated photo-sensitization could be very useful as ALA- induced porphrin accumulation selectively occurs in the oesophageal epithelium. The present study aimed to optimize the time between illumination and the administration of ALA. 200 mg/kg ALA was given orally to 24 rats (allocated to 6 groups of 4 animals each). Four animals served as controls and received phosphate buffered saline orally. The animals were illuminated at various time-points (either 1, 2, 3, 4, 6, or 12 hours) after ALA administration. Illumination was performed with a cylindrical diffuser placed in a balloon catheter. The device was originally made for percutaneous transluminal coronary angioplasty and consisted of a semi-flexible catheter and an inflatable cylindric optically clear balloon. The diffuser was placed centrally in the catheter. The same illumination parameters (633 nm, 25 J radiant energy/cm diffuser, power output 100 mW/cm diffuser) were used for each animal. During illumination, fluorescence measurements and light dosimetry were performed. The animals were sacrificed at 48 hours after PDT for histological assessment. Highest PpIX fluorescence was found at 2, 3, and 4 hours after ALA administration. Dosimetric measurements showed a 2 - 3 times higher in vivo fluence rate compared to the estimated fluence rate. Histology at 48 hours after PDT showed diffuse epithelial damage at the laser site only in rats illuminated at 2 hours after ALA administration. Illumination at 3, 4, and 6 hours after ALA administration resulted in diffuse epithelial damage in only one of four rats. In none of the rats illuminated at 1 and 12 hours after administration of ALA epithelial damage was found. These results show that illumination at 2 hours after oral ALA administration provides an efficient and safe scheme for ALA-PDT in the normal rat oesophagus. Illumination at other time points results in incomplete epithelial damage.

Preclinical and clinical studies on 5-aminolevulinic acid (5- ALA) induced Protoporphyrin IX (PPIX) are performed in various departments now following promising clinical results for the detection of bladder cancer in urology. This paper provides an overview on the progress of 5-ALA assisted fluorescence diagnosis in urology, pulmonology, neurosurgery, gynecology and ENT coordinated by the Laser Research Laboratory of the Ludwig-Maximilians-University in Munich. 5-ALA can be applied either topically or systematically to induce an intracellular accumulation of fluorescing PPIX. With appropriate dosage of 5-ALA, malignant tissue can be stained selectively, and irradiation with violet light excites a bright red fluorescence of the tumor visible with naked eyes. Optical properties of the tissue tend to hamper the precise identification and demarcation of suspect areas in fluorescence images. Multicolor remission and fluorescence imaging, therefore, should improve tumor localization in future.

The mechanism of the primary sensitization steps in Photodynamic Therapy could be of essential importance concerning the efficiency of the treatment. Besides the well known mechanisms there is a possibility that the interaction between the excited triplet state sensitizer and the doublet state free radicals generated by the cells adds a significant contribution to the overall photodynamic effect. To support this hypothesis the quantification and identification of free radicals would be required. For this purpose chemiluminescence studies and spin trapping seemed to be the most suitable methods due to the extremely short lifetime of free radicals in biological systems. We present data on the kinetics of accumulation of free radicals: (1) by measuring luminol- dependent chemiluminescence of stimulated macrophages both in the absence and in the presence of a free radical inhibitor 3,5 di-tert-butyl-4-hydroxyphenyl propionic acid; (2) by following the production of primary, secondary and tertiary radical adducts using DMPO (5,5-dimethyl-1-pyrroline N-oxide) as spin trapping agent in chemical model systems. The measured data are used to determine the effect of the excited sensitizers on the kinetics of accumulation of free radicals under biological conditions. These results would serve to design new and more effective sensitizers used for therapy based on the triplet-doublet mechanism of action.

Red light (approximately 630 nm) is almost universally used in photodynamic therapy (PDT) as it is the most penetrating of the porphyrin excitation wavelengths. For the treatment of superficial (pre)malignant oesophageal lesions however green light might be more efficient than red in destroying this thin lesions. This study aimed to compare several powers and doses of red and green light for photodynamic therapy with 5- aminolevulinic acid (ALA)-induced photosensibilization of the normal rat oesophagus. Sixty rats received 200 mg/kg ALA orally. Ten untreated rats served as controls. In groups of five rats, at three hours after ALA administration the oesophagi were illuminated with red (633 nm) or green (532 nm) light, using a 1 cm cylindric diffuser, which was placed in the center of an inflated balloon catheter. Several treatment schemes were applied: 33 mW/cm diffuser during 250 and 750 seconds (providing 8.3 and 25 J/cm diffuser), 100 mW/cm diffuser during 83 and 250 seconds (providing 8.3 and 25 J/cm diffuser) and 300 mW/cm diffuser during 83 and 250 seconds (providing 25 and 75 J/cm diffuser). During illumination, fluorescence in the oesophagus was measured. The rats were sacrificed at 48 hours after illumination. During illumination, fluorescence declined in a rate that was dependent on the light-power that was used. A higher power resulted in a faster decline of fluorescence intensity. Both the red-light control group the and green-light control group did not show any damage histologically. In the red light group selective epithelial damage was seen most pronounced in the groups treated with 33 mW/cm diffuser. Illumination during 750 seconds however, also resulted in considerable muscle damage. In the green light group in all subgroups less damage was seen. Only illumination with 300 mW/cm diffuser during 250 seconds resulted in good PDT effect in 2 out of 5 rats. In the rat oesophagus, the efficacy of ALA-PDT treatment depends both on wavelength and illumination scheme. In this study best results were obtained using red light, in a power setting of 33 mW/cm diffuser. The optimal light-dose probably lies between 8.3 and 25 J/cm diffuser.

MDCK II cells in colonies of eight cells and in confluent monolayers were treated with two different sensitizers and blue light. In both cases cooperation between the treated cells were detected. Cells photosensitized with Photofrin died by necrosis after irradiation, while cells photosensitized with tetra(3-hydroxyphenyl)porphyrin died by apoptosis. The cooperative interactions between the cells are contrary to one of the fundamental paradigms of radiation biology, namely that the targets of radiation are independent of each other.

The possibility of the fluorescence spectroscopy for diagnosing a pathology in human tissues is studied now very intensively. However, there are not much and good theoretical models to calculate the fluorescence intensity in scattering and absorbing non-homogeneous biological tissues. Moreover, such models are needed for up-to-date laser medical non- invasive diagnostic equipment and technique which run by principle of an inverse optical task solution. In order to obtain the acceptable model we used the Transport equation and multi-layered plane medium. The Method of Moments was used to solve the Transport equation with calculation of the moment of fluorescence flux at the last step of the solution.

Photodynamic therpay (PDT) used in combination with chemotherapy leads to enhanced efficiency of the treatment. Steady state spectroscopic studies were performed in order to investigate interaction process between photosensitizer Photohem (PH) and chemotherapeutic drug Adriamycin (ADM). Results obtained in aqueous solutions revealed that ADM disrupts equilibrium aggregates of PH and forms complexes with PH. Changes induced by the complexation with ADM in the absorbance of PH in the red spectral region are insignificant, and the amount of the absorbed light quanta at the primary stage of photosensitization in the case of the combined treatment should be very close to that when only PDT is applied. The increased efficiency of PDT in combination with ADM should probably be related to the changes in photophysical and photochemical properties of photosensitizer and this can lead to modified photosensitizing activity. The observed changes in the phototransformations of photosensitizer conditioned by the complexation with antitumor drug may influence sensitization process during PDT and therefore should be taken into account in the estimation of optimal photodynamic doses.

Topical application of 5-aminolevulinic acid (ALA) can be effectively used for photodynamic therapy and diagnosis of superficial bladder cancer. Administration of the heme precursor ALA leads to the selective accumulation of the photosensitizer protoporphyrin IX (PPIX) in certain types of tissue. The aim of this study was to determine the cellular PPIX concentration and the effect of photodynamic therapy mediated by ALA on two bladder cancer cell lines (RT4, J82) and a fibroblast cell line (N1). Following incubation with ALA the kinetics of cellular PPIX were examined using flow cytometry combined with extraction. The cancer cell lines showed considerably higher PPIX concentrations than the fibroblast cell line: RT4 1030, J82 710, and N1 110 ng PPIX/mg protein. Photodynamic therapy was performed with an incoherent light source (580 - 740 nm, 40 mW/cm², 30 J/cm²). In contrast to the fibroblast cell line, which was resistant to photodynamic therapy, the cancer cell lines were effectively killed following the treatment as determined by MTT assay. This study suggests that ALA-mediated photodynamic therapy may be effective in transitional cell carcinoma of the bladder. Based on these findings, this therapeutic method should be further evaluated clinically.

It was found that irradiation of aqueous solution of aluminum sulphophthalocyanine (S_nPcAl) by light with wavelength of 805 nm in the presence of sodium ascorbate results in photodynamic type I effect. S₂PcAl in adsorbed state on proteins or polysaccharide structures was shown to possess rather intense long wavelength absorption band in the NIR spectral region. The comparative tests of S_nPcAl with sodium ascorbate on mice with Ehrlich carcinoma and leucosis P-388 showed that photodynamic efficiency in the case of NIR irradiation (790 nm) is not lower than using visible irradiation (675 nm).

Photodynamic therapy (PDT) is a highly efficient inducer of apoptosis in EY-28 bladder carcinoma cells, resulting in extensive DNA fragmentation. Bladder carcinoma cells EY-28 (Tumorbank Heidelberg, Germany) were incubated for 1 h with 1 (mu) g AamTPPn/ml or 2 (mu) g AamTPPn/ml. After incubation cells were refed with complete medium and irradiated with 0.75 J/cm². To identify apoptotic cells, a in situ cell death detection kit POD (Boehringer Mannheim, Germany) was used. The chromatin condensation characteristic to apoptotic cells was detected by transmission electron microscopy. Using 1 (mu) g AamTPPn/ml and 2 (mu) g AamTPPn/ml (9-Acetamido-2,7,12,17- tetra-n-Porpylporphycene), respectively, and irradiation at 0.75 J/cm², a percentage of 36.9% and 54.7%, respectively, of apoptotic cells was detected.

The clearance of protoporphyrin IX (PpIX) from the skin of hairless BALB/c mice after topical application of 5- aminolevulinic acid (ALA) and its methyl ester (ALA-Me) was investigated. Creams containing 2 or 20% of ALA or ALA-Me were topically applied on spots of approximately 1 cm² for 12 hours. The PpIX fluorescence was detected by the means of a Perkin Elmer LS50B luminescence spectrometer equipped with a fiber-optic probe. The emission spectrum was identical with that of cell-bound PpIX. After 12 hours application of ALA and ALA-Me similar amounts of PpIX were found. After creme removal the ALA-induced PpIX fluorescence decayed with a half-life of about 20 hours (20% ALA cream). The ALA-Me-induced PpIX was faster cleared from the skin than ALA-induced PpIX, and had a half-life of about 7 hours (20% ALA-Me cream).