Novel methyl helianthrones as photosensitizers: synthesis and biological evaluation

A combination of light, oxygen and a photosensitizer is used to induce death of cancer cells by photodynamic therapy. In this study, we have synthesized several new methyl helianthrone derivatives and compared their phototoxicity with that of hypericin. In contrast to hypericin, methyl helianthrones are soluble in aqueous solutions and have a broad range of light absorbance, which allows the use of polychromatic light. Structural modifications of methyl helianthrone demonstrated that substitution of hydrogen atoms of methyl helianthrone at Positions 2 and 5 with Br atoms or methylation of its phenolic hydroxyls, significantly increases the corresponding singlet oxygen quantum yield and their phototoxicity toward alphaT3-1, M2R and LNCaP cells. The phototoxicity of some of these compounds was similar to that of hypericin. Methyl helianthrones, like hypericin, accumulated mainly in the perinuclear region as evident by confocal microscopy. Irradiation of cells pretreated with methyl helianthrone derivatives generates intracellular reactive oxygen species and lipid free radicals, as shown by a fluorescentic probe and electron paramagnetic resonance methods, respectively. The phototoxicity of these methyl helianthrones as well as their ability to oxidize membrane lipids were significantly decreased on addition of specific Type-II inhibitors, suggesting the involvement of singlet oxygen as the main oxidant.     

Cellular photodestruction induced by hypericin in AY-27 rat bladder carcinoma cells

In a recent clinical study we showed that hypericin accumulates selectively in urothelial lesions following intravesical administration of the compound to patients. In the present study the efficacy of hypericin as a photochemotherapeutic tool against urinary bladder carcinoma was investigated using the AY-27 cells (chemically induced rat bladder carcinoma cells). The uptake of hypericin by the cells increased by prolonging the incubation time and increasing the extracellular hypericin concentration. Photodynamic treatment of the cells incubated with 0.8 and 1.6 microM hypericin concentrations resulted in remarkable cytotoxic effects the extent of which depended on the fluence rates. Photoactivation of 1.6 microM hypericin by 0.5, 1.0 or 2.0 mW/cm2 for 15 min resulted in 3, 30 and 95% of the antiproliferative effect, respectively. Increasing the photoactivating light dose from 0.45 to 3.6 J/cm2 resulted in a five-fold increase in hypericin photodynamic activity. Irrespective of the fluence rates and irradiation times incubation of the cells with 10 microM hypericin induced rapid and extensive cell death in all conditions. The type of cell death (apoptosis or necrosis) induced by photoactivated hypericin depended largely on the hypericin concentration and the postirradiation time. At lower hypericin concentrations and shorter postirradiation times apoptosis was the prominent mode of cell death; increasing the hypericin concentration and/or prolonging the postirradiation time resulted in increased necrotic cell death. Cell pretreatment with the singlet oxygen quencher histidine, but not with the free-radical quenchers, significantly protected the cells from photoactivated hypericin-induced apoptosis, at least when a relatively low concentration (1.25 microM) was used. This result suggests the involvement of a Type-II photosensitization process. However, cells treated with higher hypericin concentrations (2.5-5 microM) were inadequately protected by histidine. Since hypericin is thus shown to be a potent and efficient photosensitizer, and since the conditions used were the same as when hypericin is used clinically to locate early-stage urothelial carcinoma lesions, hypericin may well become very important for the photodynamic treatment of superficial bladder carcinoma.     

Photosensitized luminescence of singlet oxygen in solutions at 1588 nm

Luminescence of singlet oxygen has been observed at 1588 nm in solutions. Singlet oxygen was photosensitized by photoexcitation of some porphyrins in different solvents. The luminescence lifetimes and the quenching rate constant measurements with different quenchers support the suggestion that the detected luminescence is a result of the electronvibrational transition

.     

Studies on the role of peroxy and hydroperoxy groups in polymer photodegradation

Interactions of carbonyl singlet and triplet excited states with peroxides and hydroperoxides were examined in order to ascertain the reasons why the latter two compounds act as nearly diffusion controlled quenchers of ketone-containing polymer photodegradation. From singlet state fluorescence quenching and laser flash photolysis measurements of triplet decays, direct interaction with the carbonyl excited states was ruled out. These processes occurred with rate constants at least one to two orders of magnitude below the diffusion controlled limit. Trapping of the 1,4-biradical precursor to chain scission, thereby preventing its disproportionation was also tentatively eliminated. Results obtained using benzoyl peroxide suggest that quencher photodissociation into alkoxy radicals might represent the key step in the quenching of ketone-containing polymer photodegradation.     

Hypericin phototoxicity induces different modes of cell death in melanoma and human skin cells

Hypericin, the major component of St. John's Wort, absorbs light in the UV and visible ranges whereupon it becomes phototoxic through the production of reactive oxygen species. Although photodynamic mechanisms (i.e. through endogenous photosensitizers) play a role in UVA phototherapy for the treatment of skin disorders such as eczema and psoriasis, photodynamic therapy employing exogenous photosensitizers are currently being used only for the treatment of certain forms of non-melanoma skin cancers and actinic keratoses. There are few reports however on its use in treating melanomas. This in vitro study analyses the phototoxic effect of UVA (400-315 nm) - activated hypericin in human pigmented and unpigmented melanomas and immortalised keratinocytes and melanocytes. We show that neither hypericin exposure nor UV irradiation alone reduces cell viability. We show that an exposure to 1 microM UVA-activated hypericin does not bring about cell death, while 3 microM activated hypericin induces a necrotic mode of cell death in pigmented melanoma cells and melanocytes and an apoptotic mode of cell death in non-pigmented melanoma cells and keratinocytes. We hypothesis that the necrotic mode of cell death in the pigmented cells is possibly related to the presence of melanin-containing melanosomes in these cells and that the hypericin-induced increase in reactive oxygen species leads to an increase in permeability of melanosomes. This would result in toxic melanin precursors (of an indolic and phenolic nature) leaking into the cytoplasm which in turn leads to cell death. Hypericin localisation in the endoplasmic reticulum in these cells shown by fluorescent microscopy, further support a disruption in cellular processing and induction of cell death. In contrast, this study shows that cells that do not contain melanosomes (non-pigmented melanoma cells and keratinocytes) die by apoptosis. Further, using a mitochondrial-specific fluorescent dye, we show that intracellular accumulation of hypericin induces a mitochondrial-associated caspase-dependent apoptotic mode of cell death. This work suggests that UVA is effective in activating hypericin and that this phototoxicity may be considered as treatment option in some cases of lentigo maligna or lentigo maligna melanoma that are too large for surgical resection.     

OXYGEN DEPENDENCE OF HYPERICIN-INDUCED PHOTOTOXICITY TO EMT6 MOUSE MAMMARY CARCINOMA CELLS

The photodynamic effect of hypericin on EMT6 mouse mammary carcinoma cells was investigated in vitro under aerobic and hypoxic conditions. Under aerobic conditions, hypericin-induced photocytotoxicity was dose dependent within a 1-50 microM range. Under hypoxic conditions, cells were resistant to hypericin-induced phototoxicity. In the dark, no cytotoxicity was observed at any hypericin concentration tested either aerobically or hypoxically. Cellular accumulation of hypericin, examined by chemical extraction and spectroscopy, occurred under both hypoxic and aerobic conditions. Fluorescence photomicrographs of cells exposed to hypericin corroborate drug uptake in the plasma membrane and subcellular regions. Our results demonstrate that hypericin cytotoxicity to EMT6 mouse mammary carcinoma cells in vitro is both light and oxygen-dependent. These results suggest that EMT6 cell kill caused by photoactivated hypericin is mediated by an oxygen-dependent mechanism, rather than by a type I oxygen-independent mechanism.     

OXYGEN DEPENDENCE OF HYPERICIN-INDUCED PHOTOTOXICITY TO EMT6 MOUSE MAMMARY CARCINOMA CELLS

Abstract
The photodynamic effect of hypericin on EMT6 mouse mammary carcinoma cells was investigated in vitro under aerobic and hypoxic conditions. Under aerobic conditions, hypericin-induced photocytotoxicity was dose dependent within a 1–50 μ M range. Under hypoxic conditions, cells were resistant to hypericin-induced phototoxicity. In the dark, no cytotoxicity was observed at any hypericin concentration tested either aerobically or hypoxically. Cellular accumulation of hypericin, examined by chemical extraction and spectroscopy, occurred under both hypoxic and aerobic conditions. Fluorescence photomicrographs of cells exposed to hypericin corroborate drug uptake in the plasma membrane and subcellular regions. Our results demonstrate that hypericin cytotoxicity to EMT6 mouse mammary carcinoma cells in vitro is both light and oxygen-dependent. These results suggest that EMT6 cell kill caused by photoactivated hypericin is mediated by an oxygen-dependent mechanism, rather than by a type I oxygen-independent mechanism.     

Reaction of tetramethylpiperidine N-oxides with persistent triplet diphenylcarbenes

Persistent triplet diphenylcarbenes with considerable stability have been shown to be trapped by tetramethylpiperidine N-oxides (TEMPOs) to give the corresponding benzophenones as major products along with tetramethylpiperidine, which indicates that the reaction pattern is essentially identical with that observed for parent triplet diphenylcarbene. The absolute rate constants for the quenching reaction were measured by a laser flash photolysis technique and compared with those for quenching by other typical triplet carbene quenchers. The results showed that the reactivity of TEMPOs toward triplet carbenes was lower than that of oxygen but higher than that of 1,4-cyclohexadiene. The advantages of TEMPOs as a triplet carbene quencher as opposed to the other quenchers are discussed, and TEMPOs are shown to be very convenient reagents to estimate the reactivity of triplet carbenes.     

Light harvesting in microscale metal-organic frameworks by energy migration and interfacial electron transfer quenching

Microscale metal-organic frameworks (MOFs) were synthesized from photoactive Ru(II)-bpy building blocks with strong visible light absorption and long-lived triplet metal-to-ligand charge transfer ((3)MLCT) excited states. These MOFs underwent efficient luminescence quenching in the presence of either oxidative or reductive quenchers. Up to 98% emission quenching was achieved with either an oxidative quencher (1,4-benzoquinone) or a reductive quencher (N,N,N',N'-tetramethylbenzidine), as a result of rapid energy migration over several hundred nanometers followed by efficient electron transfer quenching at the MOF/solution interface. The photoactive MOFs act as an excellent light-harvesting system by combining intraframework energy migration and interfacial electron transfer quenching.     

利用E-V传能模型研究亚稳态分子PCl(b^1∑^+)的猝灭过程

亚稳态分子猝灭机理的研究一直受到人们的极大重视。本文利用E-V传能模型对亚稳态分子PCl(b^1∑^+)的猝灭过程进行了定量处理。结果表明PCl(b^1∑^+)的猝灭是PCl(b^1∑^+→a^1Δ)跃迁和试剂分子端键X-Y振动能级近共振传能的结果, 与PCl(b^1∑^+→a^1Δ)跃迁的Franck-Condon因子及试剂分子端键X-Y振动的非谐性系数有关, 而且也受猝灭试剂分子极化率的影响。     

ω,ω′-Appended nucleo-base derivatives of hypericin

ω,ω′-Disubstituted hypericin derivatives with the nucleo-bases thymine, cytosine, and adenine in these positions were prepared starting from tri-O-methyl-ω-bromoemodin. The most promising derivative proved to be that with a thymine moiety. It displayed the best solubility of the three products together with a potency to produce singlet oxygen and/or reactive oxygen species comparable to the parent compound hypericin. In addition, although no specific interaction with DNA or poly(2′-deoxyadenylic acid) could be detected, it proved to be significantly better accumulating in the nucleus of prostatic cancer LNCaP cells than hypericin making it a promising candidate for a second-generation photodynamic hypericin agent.     

The interaction of boronic acid-substituted viologens with pyranine: the effects of quencher charge on fluorescence quenching and glucose response

The fluorescence sensing of several monosaccharides using boronic acid-substituted viologen quenchers in combination with the fluorescent dye pyranine (HPTS) is reported. In this two-component sensing system, fluorescence quenching by the viologen is modulated by monosaccharides to provide a fluorescence signal. A series of viologen quenchers with different charges were prepared and tested for their ability both to quench the fluorescence of HPTS and to sense changes in glucose concentration in aqueous solution at pH 7.4. Both quenching efficiency and sugar sensing were found to be strongly dependent upon viologen charge. The molar ratio between HPTS and each of the viologen quenchers was varied in order to obtain an optimal ratio that provided a fairly linear fluorescence signal across a physiological glucose concentration range. Both the quenching and sugar sensing results are explained by electrostatic interaction between dye and quencher.     

PHOTODYNAMIC ACTION IN Stentor coeruleus SENSITIZED BY ENDOGENOUS PIGMENT STENTORIN

Abstract— Stentorin acts as the photoreceptor for the step-up photophobic and negative phototactic responses in Stentor coeruleus . The chromophore of stentorin appears to be hypericin which is linked to apoprotein. In addition to the photomovement responses of the organism, S. coeruleus was found to be photodynamically sensitive to light absorbed by the hypericin chromophore, as the apparent action spectrum for the photodynamic killing matches the absorption spectrum of stentorin. The protective effect of β-carotene and crocetin on the photodynamic killing of S. coeruleus suggests that singlet oxygen generated by the stentorin-sensitization plays an important role, according to the so-called Type II mechanism of photosensitization. The generation of singlet oxygen via hypericin triplet was confirmed by in vitro photooxidation of tryptophan as a substrate. The photodynamic killing was more effective in deuterium oxide than in H₂O in both the photosensitization by stentorin (endogenous) and added hypericin (exogenous). These results are consistent with the involvement of singlet oxygen in the photodynamic killing of S. coeruleus .     

Steady-state and time-resolved fluorescence studies on Trichosanthes cucumerina seed lectin

Steady-state and time-resolved fluorescence spectroscopic studies have been carried out on Trichosanthes cucumerina seed lectin (TCSL). The fluorescence emission maximum of TCSL in the native state as well as in the presence of 0.1 M lactose is centered around 331 nm, which shifts to 347 nm upon denaturation with 8 M urea, indicating that all the tryptophan residues of this protein in the native state are in a predominantly hydrophobic environment. The exposure and accessibility of the tryptophan residues of TCSL and the effect of ligand binding on them were probed by quenching studies employing two neutral quenchers (acrylamide and succinimide), an anionic quencher (I(-)) and a cationic quencher (Cs(+)). Quenching was highest with acrylamide and succinimide with the latter, which is bulkier, yielding slightly lower quenching values, whereas the extent of quenching obtained with the ionic quenchers, I(-) and Cs(+) was significantly lower. The presence of 0.1 M lactose led to a slight increase in the quenching with acrylamide and iodide, whereas quenching with succinimide and cesium ion was not significantly affected. When TCSL was denatured with 8 M urea, both acrylamide and succinimide yielded upward-curving Stern-Volmer plots, indicating that the quenching mechanism involves both dynamic and static components. Quenching data obtained with I(-) and Cs(+) on the urea-denatured protein suggest that charged residues could be present in close proximity to some of the Trp residues. The Stern-Volmer plots with Cs(+) yielded biphasic quenching profiles, indicating that the Trp residues in TCSL fall into at least two groups that differ considerably in their accessibility and/or environment. In time-resolved fluorescence experiments, the decay curves could be best fit to biexponential patterns, with lifetimes of 1.78 and 4.75 ns for the native protein and 2.15 and 5.14 ns in the presence of 0.1 M lactose.     

Fluorescence quenching of anthracene by indole derivatives in phospholipid bilayers

The quenching of anthracene fluorescence by indole, 1,2-dimethylindole (DMI), tryptophan (Trp) and indole 3-acetic acid (IAA) in palmitoyloleoylphosphatidylcholine (POPC) lipid bilayers was investigated. A very efficient quenching of the anthracene fluorescence in the lipid membrane is observed. Stern-Volmer plots are linear for DMI but present a downward curvature for the other quenchers. This was interpreted as an indication of the presence of an inaccessible fraction of anthracene molecules. By a modified Stern-Volmer analysis the fraction accessible to the quenchers and the quenching constant were determined. The changes in the fluorescence emission spectrum of indole and DMI have been used to calculate the partition constants of these probes into the membranes, and bimolecular quenching rate constants were determined in terms of the local concentration of quencher in the lipid bilayer. The rate constants are lower than those in homogeneous solvents, which may be ascribed to a higher viscosity of the bilayer. No changes in the emission spectra of Trp and IAA are observed in the presence of vesicles, indicating that these probes locate preferentially in the aqueous phase, or in close proximity to the vesicular external interface in a medium resembling pure water. In these cases quenching rate constants were determined in terms of the analytical concentration. In the quenching by DMI a new, red shifted, emission band appears; it is similar to that observed in non-polar solvents and it is ascribable to an exciplex emission. The exciplex band is absent in the quenching by IAA and Trp and only very weakly present when the quencher is indole. From the position of the maximum of the exciplex emission, a relatively high local polarity could be estimated for the region of the bilayer where the quenching reaction takes place.     

Reduction in hypericin-induced phototoxicity by Hypericum perforatum extracts and pure compounds

Clinical evidence suggests that administration of Hypericum perforatum (Hp) extracts containing the photo-activated hypericin compounds may cause fewer skin photosensitization reactions than administration of pure hypericin. This study was conducted to determine whether the phototoxicity of hypericin in HaCaT keratinocytes could be attenuated by H. perforatum extracts and constituents. Two extracts, when supplemented with 20 microM hypericin: (1) an ethanol re-extraction of residue following a chloroform extraction (denoted ethanol(-chloroform)) (3.35 microM hypericin and 124.0 microM total flavonoids); and (2) a chloroform extract (hypericin and flavonoids not detected), showed 25% and 50% (p<0.0001) less phototoxicity than 20 microM hypericin alone. Two H. perforatum constituents, when supplemented with 20 microM hypericin: (1) 10 microM chlorogenic acid; and (2) 0.25 microM pyropheophorbide, exhibited 24% (p<0.05) and 40% (p<0.05) less phototoxicity than 20 microM hypericin alone. The peroxidation of arachidonic acid was assessed as a measure of oxidative damage by photo-activated hypericin, but this parameter of lipid peroxidation was not influenced by the extracts or constituents. However alpha-tocopherol, a known antioxidant also did not influence the amount of lipid peroxidation induced in this system. These observations indicate that hypericin combined with H. perforatum extracts or constituents may exert less phototoxicity than pure hypericin, but possibly not through a reduction in arachidonic acid peroxidation.     

SINGLET OXYGEN GENERATION FROM LIPOSOMES: A COMPARISON OF TIME-RESOLVED 1270 NM EMISSION WITH SINGLET-OXYGEN KINETICS CALCULATED FROM A ONE DIMENSIONAL MODEL OF SINGLET-OXYGEN DIFFUSION and QUENCHING

Abstract— Time-resolved measurements of 1270 nm singlet-oxygen emission following pulsed-laser excitation were made from unilamellar dimyristoyl 1-α-phosphatidylcholine liposomes labeled with zinc phthalocyanine. The effect of the hydrophobic quenchers, β-carotene and ethyl β-apo-8' trans carotenoate, and the hydrophilic quenchers, histidine and methionine, upon the kinetics of the 1270 nm singlet-oxygen emission was studied. Hydrophobic quenchers principally lowered the intensity of the 1270 nm emission and caused only modest changes in the lifetime of the 1270 nm emission. The decrease in 1270 nm emission caused by hydrophobic quenchers was related to the size of the liposomes. The larger the radius of the liposome, the greater the decrease in 1270 nm emission caused by a given concentration of hydrophobic quencher. In contrast, hydrophilic quenchers principally decreased the lifetime of the 1270 nm emission. The effect of hydrophilic quenchers was independent of the size of the liposomes.
There was good agreement between the experimental results and the kinetics of the singlet-oxygen emission calculated using a one dimensional model of singlet-oxygen quenching and diffusion. The kinetics of singlet-oxygen emission from liposomes without added hydrophobic quenchers closely approximated the theoretical kinetics of singlet oxygen in a homogeneous aqueous solution.     

QUENCHING OF SINGLET MOLECULAR OXYGEN BY PHTHALOCYANINES AND NAPHTHALOCYANINES

Using the direct measurement of the photosensitized luminescence of singlet molecular oxygen (1O2) the rate constants (kq) have been determined for 1O2 quenching by the monomeric molecules of the following phthalocyanines and naphthalocyanines in chloroform: tetra-(4-tert-butyl) phthalocyanine (I); octa-(3,6-butoxy) phthalocyanine (II), tetra-(6-tert-butyl)-2,3 naphthalocyanine (III), aluminium tetra-(1-tert-phenyl)-2,3 naphthalocyanine (IV), tri-(n-hexyl-siloxy) derivatives of silicon- (V), tin- (VI), aluminium- (VII) and gallium- (VIII) 2,3 naphthalocyanine. The following kq values were obtained (kq x 10(-8) M-1 s-1): 2.9 (I), 59 (II), 100 (III), 20 (IV), 3.9 (V), 53 (VI), 33 (VII), 110 (VIII). As most of the quenchers have the low-lying triplet levels, a contribution of the quenching mechanism based on the energy transfer from 1O2 to these levels has been analysed. A formula is proposed describing the relation between kq values caused by this mechanism, and photophysical constants of the quencher triplet state. This formula was applied to phthalocyanines, naphthalocyanines, beta-carotene and bacterochlorophyll a. The data suggest that the energy transfer can fully explain the activity of V and strongly contributes into the activities of II, III and VI-VIII. A charge transfer interaction might be an additional mechanism involved in 1O2 quenching by compounds studied. As some phthalocyanines and naphthalocyanines are strong physical quenchers of singlet oxygen they can be used as efficient inhibitors for photodestructive processes in photochemical systems.     

Quinolone Antibiotic Photodynamic Production of 8-Oxo-7,8-dihydro-2'-deoxyguanosine in Cultured Liver Epithelial Cells

To study the basis for the phototoxicity of quinolones, a class of synthetic antibacterials, the photodynamic ability to mediate 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) formation in cultured cells was measured for lome-floxacin (LMX), which is strongly associated with clinical phototoxicity in humans, and ciprofloxacin (CFX), which has few reports of phototoxicity. Adult rat liver (ARL-18) cells were exposed to the quinolones in the presence of UVA and DNA was extracted and analyzed by HPLC with electrochemical detection. Low levels of 8-oxo-dG were found in the DNA of nonirradiated ARL-18 cells and this was increased up to 6-fold in the presence of either LMX (50–400 uAf) or up to 3.6-fold in the presence of CFX (50–400 µM) and UVA (20 J/cm²) when compared to the UVA control. Comparing separate experiments with LMX and CFX, LMX produced greater levels of 8-oxo-dG either after dark exposure or after UVA exposure at 20 J/cm². Also, LMX and CFX were both shown to photodegrade in the presence of UVA, and it was determined that UVA photoinstability alone does not reflect phototoxic potential. These data suggest that the photodynamic potential of LMX and CFX to produce 8-oxo-dG may relate to their human clinical phototoxicity profile. We suggest that the observed clinical phototoxicity is mediated through a UVA photodynamic effect on the quinolone to form reactive oxygen species in the presence of molecular oxygen. The findings indicate that 8-oxo-dG formation can serve as a marker for the potential phototoxicity of new quinolones.     

Fluorescence quenching of coumarins by halide ions

Fluorescence quenching of 4-methyl-7-methoxy coumarin (1) and 4-methyl-5-ethoxy-7-methoxy coumarin (2) in aqueous solutions have been studied at different concentrations of halide ions (Cl-, Br-, I-), at room temperature approximately 20 degrees C. It is observed that the fluorescence intensity of both the coumarin compounds (1 and 2) decrease with increase in the concentration of Br- and I- ions but remains almost constant in case of Cl- ion. It is observed that the quenching due to halide ions proceeds via both a diffusional and static quenching processes. The rate constants for diffusional as well as static component of quenching process have been calculated using modified Stern-Volmer relation. It is further observed, that I- ion has very high quenching ability than Br- ion and Cl) ion almost behaves as a non-quencher. It is explained in terms of the sphere of action model by showing that the value of radius of sphere of action of the halide quencher is greater than the sum of the radii of the respective coumarin and quencher. Further, pattern of the quenching ability of the halide quenchers is found to be as I- > Br- > Cl- and interestingly this is in the same order as of their ionization energy. Finally, the present quenching process has been attributed to the electron transfer resulting between colliding species.