BODIPY dyes tend to be highly fluorescent, but their emissions can be attenuated by adding substituents with appropriate oxidation potentials. Substituents like these have electrons to feed into photoexcited BODIPYs, quenching their fluorescence, thereby generating relatively long-lived triplet states. Singlet oxygen is formed when these triplet states interact with 3O2. In tissues, this causes cell damage in regions that are illuminated, and this is the basis of photodynamic therapy (PDT). The PDT agents that are currently approved for clinical use do not feature BODIPYs, but there are many reasons to believe that this situation will change. This review summarizes the attributes of BODIPY dyes for PDT, and in some related areas.

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BODIPY dyes in photodynamic therapy.

Singlet molecular oxygen, 02(’Ag) , has been intensively investigated over the last few decades, primarily in relation to photosensitization reactions. In combination with the increasing awareness and interest in singlet oxygen has been the development of various methodologies for the detection and quantification of singlet oxygen production. A large body of literature now exists concerning the singlet oxygengenerating abilities of a large variety of endogenous and exogenous compounds that can be photoactivated in the UV and visible regions of the electromagnetic spectrum. The purpose of this review was to compile data from many different sources to provide a convenient resource for biological scientists with interest in this area. This compilation is essentially an updated subset of the excellent comprehensive review on singlet oxygen yields by Wilkinson et af. (l), which, unlike this compilation, was not limited to biological molecules. The data are current up to the early months of 1999. The following tables include various related parameters in addition to the central parameter of the quantum yield of singlet oxygen generation (QA) for a given photosensitizer. The methodology or technique used to measure the QA value for each entry is given and ranges from direct detection of the luminescence produced on relaxation of singlet oxygen (time-resolved [TRILIT or steady-state [SSIL]), calorimetric

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1751-1097.1999.tb08240.x

A Compilation of Singlet Oxygen Yields from Biologically Relevant Molecules

Current clinical and preclinical photosensitizers for use in photodynamic therapy

Photodynamic therapy (PDT) is an emerging treatment modality for a range of disease classes, both cancerous and noncancerous. This has brought about an active pursuit of new PDT agents that can be optimized for the unique set of photophysical characteristics that are required for a successful clinical agent. We now describe a totally new class of PDT agent, the BF2-chelated 3,5-diaryl-1H-pyrrol-2-yl-3,5-diarylpyrrol-2-ylideneamines (tetraarylazadipyrromethenes). Optimized synthetic procedures have been developed to facilitate the generation of an array of specifically substituted derivatives to demonstrate how control of key therapeutic parameters such as wavelength of maximum absorbance and singlet-oxygen generation can be achieved. Photosensitizer absorption maxima can be varied within the body's therapeutic window between 650 and 700 nm, with high extinction coefficients ranging from 75,000 to 85,000 M(-1) cm(-1). Photosensitizer singlet-oxygen generation level was modulated by the exploitation of the heavy-atom effect. An array of photosensitizers with and without bromine atom substituents gave rise to a series of compounds with varying singlet-oxygen generation profiles. X-ray structural evidence indicates that the substitution of the bromine atoms has not caused a planarity distortion of the photosensitizer. Comparative singlet-oxygen production levels of each photosensitizer versus two standards demonstrated a modulating effect on singlet-oxygen generation depending upon substituent patterns about the photosensitizer. Confocal laser scanning microscopy imaging of 18a in HeLa cervical carcinoma cells proved that the photosensitizer was exclusively localized to the cellular cytoplasm. In vitro light-induced toxicity assays in HeLa cervical carcinoma and MRC5-SV40 transformed fibroblast cancer cell lines confirmed that the heavy-atom effect is viable in a live cellular system and that it can be exploited to modulate assay efficacy. Direct comparison of the efficacy of the photosensitizers 18b and 19b, which only differ in molecular structure by the presence of two bromine atoms, illustrated an increase in efficacy of more than a 1000-fold in both cell lines. All photosensitizers have very low to nondeterminable dark toxicity in our assay system.

In vitro demonstration of the heavy-atom effect for photodynamic therapy.

Methylene blue in photodynamic therapy: From basic mechanisms to clinical applications.

The cytotoxic and photodynamic activities of the commercially-available biological stains methylene blue (MB), 1,9-dimethyl MB (Taylor's Blue) and a newly synthesised compound, 1-methyl MB, were measured against the murine mammary tumour cell line, EMT-6. Both 1-methyl MB and 1,9-dimethyl MB exhibited increased dark toxicity with concomitant higher phototoxicity compared to MB at a light dose of 7.2 J cm-2. While increasing the light dose as a function of the fluence rate increased the photocytotoxicity of MB, this had little effect on the methylated derivatives. In vitro chemical testing proved that successive methylation rendered the phenothiazinium chromophore both more resistant to reduction to its inactive leuco form, and also led to increased levels of singlet-oxygen production, thus providing a possible explanation for the increased toxicities of the methylated derivatives. Comparisons are made with the benzo[a]phenothiazinium photosensitizer, EtNBS.

Increased cytotoxicity and phototoxicity in the methylene blue series via chromophore methylation

The toxicities and phototoxicities of methylene blue (MB), toluidine blue O (TBO) and Victoria blue BO (VBBO) in a murine mammary tumour cell line (EMT6-S) and a multidrug resistant (MDR) sub-line (EMT6-R) were measured and their efficacy against the resistant sub-line was compared to that of doxorubicin and cis-platinum. The MDR cell line was considerably more susceptible to VBBO than to the conventional agent doxorubicin. VBBO was also pho-totoxic whereas illumination did not alter the activity of doxorubicin or of cis-platin. Both TBO and MB showed limited light activation (2-fold) in both the sensitive and resistant cell lines.Pre-treatment with VBBO prior to exposure to doxorubicin caused a twofold increase in doxorubicin toxicity in both cell lines. MB and TBO, however, increased doxorubicin toxicity in EMT6-R cells ×2 and ×3 respectively, but had less effect on the sensitive cell line (increase ×l.4 and ×2 respectively). Thus MB and TBO may act on the MDR cell line via a different mechanis...

Cytotoxicity and adjuvant activity of cationic photosensitizers in a multidrug resistant cell line.

The triarylmethane dye Victoria blue BO (VBBO) is a known photosensitizer which has been shown to induce a cytotoxic response in vitro. Several novel Victoria blue derivatives, with varying physicochemical properties, have been compared to VBBO, with respect both to dark toxicity and phototoxicity, on a mouse mammary tumour cell line, EMT6. Photosensitizer uptake was observed using confocal fluorescence microscopy. The chemical differences, particularly in the naphthyl substitution of the derivatives were shown to alter the light:dark toxicity differential and the uptake of the photosensitizers.

Uptake and cell-killing activities of a series of Victoria blue derivatives in a mouse mammary tumour cell line.

The subcellular localisation of doxorubicin and Victoria Blue BO (VBBO) in a murine mammary tumour cell line EMT6-S, and the resistant sub-lineEMT6-R was studied, using confocal microscopy, in order to investigate their sites of action. In cells treated with doxorubicin (10 μ M) for 90 min, the pattern of intracellular drug distribution differed between the two cell lines. Doxorubicin was found to localise mainly in the nucleus of the sensitive cell line, whereas weak fluorescence was observed in the cytoplasm of the resistant cells, in a punctuate pattern, with no nuclear involvement. The drug also appeared to be effluxed more rapidly by the resistant cell line. The accumulation of doxorubicin at various time intervals over 1h in EMT6-S cells showed that the drug clearly interacted with both the plasma membrane and the nucleus. In contrast to doxorubicin, the intracellular distribution of VBBO in both EMT6-S and EMT6-R was similar, VBBO was clearly localised throughout the cytoplasm, in a punctuate pattern, which may be consistent with the widespread distribution of mitochondria. A more apical pattern of accumulation was noted in the EMT6-R cell line. No interaction with the plasma membrane was evident. These results indicate that the main modes of action for the two drugs differ markedly, suggesting involvement of both the membrane and the nucleus in the case of doxorubicin, but mitochondrial involvement for VBBO.

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Intracellular localisation studies of doxorubicin and Victoria Blue BO in EMT6-S and EMT6-R cells using confocal microscopy

Growth of wild-type Escherichia coli strain MRE600 was severely affected up to 9 h following treatment with the anthracycline doxorubicin (15 μM), however, after 9 h, the cells became resistant. The onset of resistance coincided with some changes in the relative proportions of total saturated, monounsaturated and cyclopropane fatty acids. The anionic lipid content in E. coli strain HDL11 is under lac control and synthesis can be induced by incubation with the lac inducer IPTG. HDL11, with low levels of anionic phospholipid, was unaffected by doxorubicin (100 μM) over 9 h, with only slight inhibition of growth seen over 24 h. When the anionic lipid content of HDL11 was increased, there was a slight increase in the efficacy of doxorubicin, providing evidence for a membrane-based step in doxorubicin action.

Shuna M. Burrow

Papers

Increased cytotoxicity and phototoxicity in the methylene blue series via chromophore methylation

Cytotoxicity and adjuvant activity of cationic photosensitizers in a multidrug resistant cell line.

Uptake and cell-killing activities of a series of Victoria blue derivatives in a mouse mammary tumour cell line.

Intracellular localisation studies of doxorubicin and Victoria Blue BO in EMT6-S and EMT6-R cells using confocal microscopy

Development of a bacterial model for studying anthracycline- membrane interactions