Investigation into the potential of methylene blue and its derivatives to be used in the photodynamic therapy of non-pigmented and pigmented cells

Rice, Lesley (2001) Investigation into the potential of methylene blue and its derivatives to be used in the photodynamic therapy of non-pigmented and pigmented cells. Doctoral thesis, University of Central Lancashire.

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Photodynamic therapy (PDT) is a novel treatment for malignant disease. The first step is intravenous injection of a light-absorbing, cytotoxic drug (the photosensitizer) that is then allowed time to accumulate in malignant tissue. The second step involves local activation of the photosensitizer with long (red) wavelength light, delivered usually from a laser. Subsequent to irradiation, highly reactive singlet molecular oxygen (Type II mechanism) is likely to be the most damaging cytotoxic species in vivo. The porphyrin molecule, Photofrin®, is the only photosensitizer currently registered for clinical use but is associated with several problems. Most disappointing is the fact that Photofrin® accumulates not only in malignant tissue but also in other organs, such as the liver, kidney and spleen. Its long persistence in the skin commonly causes severe photosensitization reactions in patients for up to three months post-treatment. Photofrin® also has poor light absorption properties within the therapeutic window (600 to 800 nm) for PDT. Furthermore, PDT with Photofrin® has proved of no use in the treatment of malignant melanoma due, possibly, to competition between the photosensitizer and melanin for light absorption.

Second-generation photosensitizers have tended to be porphyrin-based molecules, many of which have reached various stages of clinical trial. Of non porphyrin-based compounds, the cationic dye, methylene blue (MB), used traditionally as a nuclear stain in histology, has proved also to be an efficient photosensitizer, with maximum light absorption properties within the therapeutic window for PDT. Its use as a selective stain for tumour tissue in the bladder led first to its investigation in humans for the PDT of bladder cancer and inoperable tumours of the oesophagus. Radiolabelled MB has also recently been used as a tracer for metastatic melanoma in humans. The disadvantages of MB are an inherent (dark) toxicity and its rapid reduction in vivo to the inactive form, leuco-methylene blue (LMIB).

This study found the cytotoxicity of MB to be enhanced by illumination and that successive methylation of the molecule corresponds to both increased light and dark toxicities in the EMT-6 (murine mammary), the SK-23 (murine melanoma) and SKMEL-28 (human melanoma) cell lines. The increased toxicities may be due to increased resistance to reduction (MBcNMB.cMMB<DMMB), improved singlet oxygen quantum yield (MB.cMMBcDMMBcNMB), increased hydrophobicity (MB.cMMBCDMMBcNMB), improved cellular uptake (MBctvHv1BcDMMBNTvffi) and/or changes in intracellular targeting and localisation. MB is known to target the nucleus but it was proposed that the increased hydrophobicities could lead to the mitochondrial targeting of the derivatives. The intracellular localisation of the photosensitizers following incubation was studied using both fluorescence and confocal microscopy. Here, confocal microscopy showed that none of the four photosensitizers, including MB itself, target the nucleus prior to illumination. DMMB and NIMB in particular appear to be confined to small subcellular bodies within the cytoplasm. However, the precise locations of the photosensitizers, prior to illumination, were not established during the course of this study. Nevertheless, confocal microscopy showedthat, upon illumination, all four photosensitizers rapidly relocalise within the nucleus.

Since photosensitizers that localise in mitochondria are found to be more efficient inducers of apoptosis than photosensitizers that target other subcellular sites, the ability of the photosensitizers, MB, MMB, DMMB and NMB, to induce apoptosis in EMT-6, SK-23 and SK-MEL-28 cells in culture was investigated in this study. The methods used were visual examination of cell morphology, by use of the cyanine dye, JC-1, and the use of the FluorAce® Apopain Assay Kit from Biorad, in cells that had been exposed to the photosensitizers. From these, it was concluded that an apoptotic cell killing mechanism might play an important role in the photocytotoxicity of the photosensitizers, moreso for DMMB and NMB.

The overall purpose of this project was to assess the potential of the derivatives of MB to be used in the PDT of cancer, with a special emphasis on malignant melanoma, since this is a field of cancer treatment where PDT has had no success. Although methyl substitution of MB did not abolish the inherent toxicity of the molecule, it is possible to assess the potential usefulness of the photosensitizers by examination of the light: dark differential. In fact, the light:dark differential was improved for all the derivatives of MB in all three cell lines. Nevertheless, NMB consistently performed the best, achieving maximum photocytotoxicity at concentrations that caused very little toxicity in the dark. The presence of melanin made no difference to the photosensitizing capabilities of the photosensitizers in melanoma cells.

In terms of a clinical application of the current work, PDT employing phenothiazinium photosensitizers is not suggested procedurally for the removal of primary melanoma, since this is routinely performed by excision. However, due to the demonstrated efficacy of MB in tracing microsatellites and its use in sentinel lymph node tracing, it may be of use in the photodynamic treatment of local metastatic lymph infiltration immediately post-surgery, as an alternative to lymphadenectomy. At present, MB is used routinely in various tracing or demarcation procedures, either visible or scintillographic, without reported toxicity. The derivatives used in the present in vitro study were all more effective in terms of the photodynamic effect and it is thus possible that future clinical developments in this direction may be feasible.

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